File: Lesson 3.doc
NWABR
Aug. 2008
Consumer Awareness: Personal Care Products Safety and Labeling
Lesson 3:
Is it safe? Basic toxicology - Safety testing: MSDS’s and
animal studies - Designing a human study
Summary:
Class discussion about the homework followed by demonstrations, discussion about
basic toxicology principles and dose-response worksheet. Small group activity with
MSDSs. Class review of studies with animals and designing human studies.
Lesson Objectives:
1. Students will be able to identify three basic principles of toxicology.
2. Students will be introduced to material safety data sheets (MSDSs) as a source of cosmetic
ingredient safety information.
3. Students will review the steps in ingredient development and safety evaluation and be able to
explain the role of animals in safety testing.
4. Students will gain an understanding of basic considerations required when designing a human study.
(who, what, when, where; how to obtain reliable results)
Materials & Preparation:
Distribute sample commercial products/containers to lab group benches;
approximately 2-4 containers per group (the containers provided with
"active ingredients" are not useful for this exercise)
Copy S3.1, Dose-Response Worksheet, one for each student
Copy S3.2, Cosmetic Ingredients: Function and Toxicity Chart, one for each student
Optional: TG3.15b, 1 copy per group; cut apart and put activities in appropriate order
Copy (or use from kit) four different MSDS’s, one set of 4 to each group
Create overheads: TG3.1, Sense (or Cents) Behind Safety
TG3.3, Safety warning; reporting
TG3.4, Toxicology definition
TG3.5, Toxicology principles
TG3.7, Dose
TG3.7b, Hazard, risk
TG3.10, Dose response curve
TG3.13, Skin biology
TG3.14, Skin absorption
TG3.15, Cosmetic ingredient development
TG3.16, Drug development process
TG3.17, MSDS purpose and availability
TG3.21, Animal claims (2 pages)
TG3.22, Animal studies – background
TG3.24, IACUC and protocol content
TG3.25, 3 R's of animal studies
TG3.27, Human Study Example (3 pages)
TG3.28, Lotion Study Basics
Copy S3.3, Winning Combination, one for each student (homework)
Engagement: Yesterday we made a lotion, today we are going to talk about whether it can be considered safe
enough to put on the market.
Northwest Association for
Biomedical Research
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Lesson 3
File: Lesson 3.doc
NWABR
Aug. 2008
Has anyone ever had an allergic reaction or skin irritation from a cosmetic?
If so, what did you do about it?
(Quit using the product; contacted manufacturer or FDA;
avoided that product and ones with similar ingredients)
As you answered your homework questions you had a chance to think about both
manufacturers’ and consumers’ responsibilities in keeping cosmetics safe.
(10 min.)
Class discussion about homework assignment
Overhead: “Sense (or Cents) Behind Safety” (S2.3 homework)
Teacher Guide with answers/discussion points
OH TG3.1
TG3.2
It is important to remember that if no safety testing was done, the label
must contain a warning.
The FDA encourages consumers to report problems with cosmetic
products to them, as well as to the manufacturer.
OH TG3.3
Principles of Toxicology
OH TG3.4
The process of determining the safety of cosmetic ingredients falls under the
science of toxicology. Originally it was the science of identifying poisons and
determining how much would kill you. Today, toxicologists are involved in
identifying harmful effects, assessing the probability of harm, and determining safe levels of
many agents on humans, animals and the environment.
You may have heard the statement: “The dose makes the poison.”
Any substance or ingredient can be toxic depending on its amount or dose and individual
sensitivity. Even too much water can dilute the body’s sodium levels and cause hyponatremia –
a potentially series health problem.
There are three basic principles of toxicology that must be considered
OH TG3.5
when determining the safety of any compound, ingredient, or chemical:
Background OH TG3.6
1. Dose-response relationship
2. Hazard vs. risk
3. Individual susceptibility
Dose-Response Relationship
It is not hard to come up with examples of chemicals, foods, or environmental
pollutants that cause increasing harm (or effects) when present at increasing
concentration
Example: UV rays and sun tanning/burning
Caffeine in coffee or coke and stimulation/jitters
Air particulates and breathing/coughing
The term dose refers to the amount of a chemical that actually enters the body,
through lungs (inhalation), mouth (ingestion), or skin (dermal absorption).
The actual dose depends on variations of exposure to the chemical:
Duration and combined exposure from multiple sources
Frequency and time between exposures
Body size
Northwest Association for
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OH TG3.7
Lesson 3
File: Lesson 3.doc
NWABR
Aug. 2008
Dose Determination for Cosmetics
We expect cosmetic manufacturers to determine the safety of their ingredients (and products)
based on an expected “dose” (amount used per application) of the product. Determining the
“dose” of cosmetics can be very difficult. The label doesn’t state a “dose” or “serving size” like
drug or food labels.
Dilemmas for manufacturers planning safety tests:
- The manufacturer expects customers to use the lotion on their hands, but they like it so
much they use it on their hands and legs.
- The manufacturer plans to test the safety of use over one month, since that would be
equivalent to one container of the product, but consumers like the product so much that they
use it continuously for years.
- Some of the ingredients in the manufacturer’s lotion are also found in two or three other
products that consumers use everyday.
- If a product is expected to be used for years, does the manufacturer need to test for the
potential to cause cancer, or birth defects if used by pregnant women?
Hazard vs. Risk
Hazard can be thought of as the potential for a chemical to be harmful, but
it doesn’t mean that it will be harmful under all conditions.
OH TG3.7b
Risk is the likelihood (or probability) that you will be hurt or harmed.
Determining your risk means taking into consideration the
inherent hazard level of the chemical,
the dose-response relationship,
and all aspects of exposure (frequency, duration, body size).
Hazard is not synonymous with risk.
Individual Susceptibility
Individual susceptibility refers to differences in sensitivity to toxic agents.
For some people a bee sting is a nuisance, for others it can be deadly.
For some people peanuts are a great snack, for others they can be deadly.
Susceptibility is influenced by age, gender, health, genetic background and
life style. Sensitivity can develop after repeated exposure. Hair dyes tell you
to do a skin test before every use, even if you haven’t had a problem in the past.
[These basic principles are explained well in the power point slides from UW
TG3.8
Center for Ecogenetics & Environmental Health, Community Outreach & Education Programs.
The slides refer to chemicals in the environment, but the information applies to cosmetics.]
Demo
Dose-Response Demo
TG3.9 pg 1
For most people, the greater the dose of a substance, the greater the response.
This can be seen in a simple demonstration with glasses of water, representing water in the body
or the bloodstream, or with petri dishes representing surface area of the skin. The more lotion
you put on, the more of the ingredients you are exposed to. If your
skin is sensitive to an ingredient or chemical, the greater the dose, the greater
your reaction to it.
TG3.9 pg 2
Northwest Association for
Biomedical Research
-3-
Lesson 3
File: Lesson 3.doc
NWABR
Aug. 2008
But size and weight can also effect the response to a given dose. The amount of caffeine in one
can of cola may have little effect on an adult, but that same can of cola can have a greater effect
on a child because of less body weight or blood volume.
Size and weight also effects response to cosmetic ingredients. If an adult and child use
the same amount of lotion (for example, 1 tablespoon), it may cover a greater
proportion of skin surface area of the child and ingredients that penetrate will be in greater
proportion to the water/blood volume of the child.
Dose Response Curves
Scientists record the experimental response to different doses of a
chemical in a dose-response curve. The x-axis is the dose, measured
in mg/kg, milligrams of chemical per killigram of body weight.
TG3.10
(see R3.2)
The y-axis is the percent of subjects showing a response which may be death or, for cosmetics,
skin reaction (redness, inflammation, blistering).
Knowing the dose-response of a chemical is essential for knowing its cause and
effect profile. A comparison of dose-response curves allows one to compare
one chemical to another.
Three important doses are determined from the curve. (see TG3.10)
The shape (slope) of the curve reflects potency. The more vertical the slope,
the greater the chance of “overdosing” with small changes in dose.
Demo
Variable Response to Dose
All people don’t respond to all chemicals in the same way.
Bee stings and peanuts are tolerated by most, but are deadly to a few.
Teacher or students can demonstrate variability to response.
Discuss factors that are responsible for variable responses.
(genetics, age, size, health, medications, life style)
Worksheet
Dose-Response Worksheet: How much is too much?
You now have the opportunity to generate a dose-response curve and evaluate
several dose-response curves based on what we have just talked about.
TG3.11
S3.1
On the first page you will read about a hypothetical human safety test of a new lotion.
You will be asked to graph the results that are described and then answer some
questions about the graph and the information you learn from such a graph.
On page 2, you are given the results of toxicity tests for two new lotion ingredients.
You are then asked some questions about the differences in toxicity between
these two ingredients.
Answer key
Students may want to measure and see how much lotion represents
1 mg/kg in an average 70 kg human.
TG3.12
Skin Function
Determining the amount of a cosmetic ingredient that gets into or through the skin
and into the blood circulation is important in assessing the ingredients’ safety.
Discuss biology of the skin –
OH TG3.13
Function and layers of the skin
(see R3.3)
Northwest Association for
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Lesson 3
File: Lesson 3.doc
NWABR
Aug. 2008
Normal skin pH is somewhat acidic – in the range of 4.2 to 5.6, varying
from one part of the body to another. The acidity inhibits the growth of foreign
bacteria and fungi.
What are the routes of entry for chemicals on the skin?
Chemicals must reach blood vessels within the dermis to be spread
Metabolism – breakdown within skin cells can change the chemical
Optional:
OH TG3.14
Ingredient Evaluation Process
The process of evaluating the safety and efficacy (function) of new ingredients
for cosmetics and medicines makes use of the principles of toxicology within a
hierarchy of research and development steps.
OH TG3.15
Make copies of TG3.15b, cut apart, and ask students to put them in appropriate
order before showing TG3.15
TG3.15b
Drug development and safety testing is a more structured process based on
regulations and FDA approvals. It includes many of the same initial steps
as in development of new cosmetic ingredients however, there are Food & Drug
Administration (FDA) submission and approval steps and the human testing
step (called clinical trials) is longer and more involved.
OH TG3.16
The results of tests “in vitro”, in cell culture, and in animals must be
presented to the FDA in an “investigative new drug” (IND) application. The IND
also includes information about the manufacturing process, label (for pre-approval
by FDA), and the plan for clinical trials (studies in humans).
(See R3.4, R3.5)
After a drug is approved, most of the information is available to the public through
the “Freedom of Information” Act. Since cosmetic ingredient safety data is not submitted to the
FDA, it is harder for a consumer to find.
Prompt:
Introduction to Material Safety Data Sheets (MSDS)
There is a way for the public to get some information about the safety of cosmetic
ingredients, and specifically, find animal toxicity data. That is what we are going to explore
now through Material Safety Data Sheets (MSDS’s).
Group Activity:
Hand-out
Hand-outs
Divide students into groups of 4 students. Each group should have:
1) 3-4 sample products/containers from the kit
2) “Cosmetic Ingredients: Function and Toxicity Chart” (one per student)
S3.2
3) Four different MSDS sheets
Binder – Back pocket
Option 1 – Each group has 4 different MSDSs but all groups have the same
4 MSDSs.
2 MSDSs will correspond to common ingredients found in cosmetics
(propylene glycol, methyl paraben)
2 MSDSs will be for an ingredient in the lotion made in class
(borax, mineral oil)
Option 2 – From the web site, copy a different MSDS for each student.
NOTE: MSDSs from different companies are similar in format but not
identical in content. Not all MSDSs will contain animal toxicity data.
Northwest Association for
Biomedical Research
-5-
Lesson 3
File: Lesson 3.doc
NWABR
Aug. 2008
Teacher will lead the class discussion.
Each of you within a group has a Material Safety Data Sheet (MSDS) for
a different ingredient. As you look at the information in your MSDS, who
would you say could use this information?
Overhead: Purpose of MSDS’s and public availability
Discussion points
OH TG3.17
TG3.18
Information found in an MSDS
Focus on synonyms
Focus on section 11, “Toxicological Information”
Animal testing, routes of delivery, meaning of LD50
Discussion points
Optional information: mouse LD50 converted to human LD50
Prompt:
TG3.19
TG3.20
Group activity:
S3.2
a) Complete the “Cosmetic Ingredients: Function and Toxicity Chart”
by using the MSDSs to identify what type of animal tests have been done.
b) Study labels on the sample products/containers and determine if any of
those ingredients are found in products labeled “not tested in animals”.
OH TG3.21 pg 1
There are many claims that we can’t check because we don’t have access to
testing data or there is no standard or definition for some claims. As you have discovered, we
CAN determine whether some ingredients have been tested in animals.
Animal-tested ingredients include those used in our class lotion and in many
commercial products.
Several days ago, we talked about labels that claim “not tested in animals”. Today
we find that the ingredients have been tested in animals. How can companies
make that claim?
OH TG3.21 pg 2
(ingredients were tested but not the final lotion; someone else did the
testing; tests were done greater than 5 yrs ago)
Animal Studies
Brief class discussion:
Overhead: Animal studies - Background
Discussion points (also see articles under “Research with Animals”)
OH TG3.22
TG3.23
Although no animal is a perfect imitation of humans, animal models allow us to
judge the effects of multiple cellular interactions and metabolism.
Safety tests on new ingredients for cosmetics and drugs usually include animal
studies. There are no adequate non-animal tests to replace all animal tests but many
previously used animal tests have been replaced.
Safety testing is done for a variety of reasons:
To find the safe dosing range, to determine the potential for skin irritation, allergy, or
potential for harm if the compound gets through the skin and into
the blood stream.
These end-points are hard to achieve in non-animal models.
Cosmetic ingredients that have previously been tested or have a history of safe use
don’t have to be re-tested on animals, although they may require human testing.
Northwest Association for
Biomedical Research
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Lesson 3
File: Lesson 3.doc
NWABR
Aug. 2008
When testing is required on new compounds, animal studies are done
OH TG3.15
after gathering data from non-animal tests. The use of animals in research
is not taken lightly. Federal laws govern the care, research, housing, & transport
and unannounced inspections by US Department of Agriculture are mandated.
By law, all animal study protocols must be approved in advance.
Role of IACUC
TG3.24
When animal studies are needed, the guiding principles are the 3 R’s
Overhead: replacement, reduction, refinement
Discussion points and examples
OH TG3.25
TG3.26
To replace an animal test with a new test requires “validation” and approval
by government agencies. The protocols must be repeatable by multiple labs
and the results must be reproducible.
See R3.6 for description of common safety tests done on cosmetic and
drug ingredients.
R3.6
See R3.7 – a New York Times article on the development of alternatives
to animal testing because of European regulations
R3.7
(Also see “Protecting More than Animals” in Research with Animals and curriculum
summary for “For the Greater Good” in NWABR Curricula
Human Lotion Studies
If ingredients appear safe in in vitro tests and animal studies, the next studies are
conducted in humans. Designing a human safety study, or a lotion quality study,
is like designing any scientific investigation.
Here is an example of a hypothetical human lotion study.
OH TG3.27
What do you think about this description?
Does it meet the criteria for being informative, reliable, and reproducible?
The design of a human lotion study must incorporate all of the information
required in a science lab report.
The hypothetical study description touches on essential aspects of a human
study, but it lacks procedural details which makes it non-reproducible.
OH TG3.27 p 2
OH TG3.27 p 3
An adequate “procedure” must include details:
who, what, when, where, purpose, and how the evaluation will be done.
Within these categories, there are many ways that a human lotion study could
be designed, depending on the purpose(s) of the study.
You should be able to identify the controlled, manipulated, and responding
variables in the study design.
Group
Activity:
Working in small groups, students should discuss possible study design
options under each category of Lotion Study Basics.
Class discussion led by the teacher: Have each group report on one of their
study design categories.
Discussion points
Northwest Association for
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OH TG3.28
TG3.29
Lesson 3
File: Lesson 3.doc
Closure:
NWABR
Aug. 2008
Over the last three lessons you have had an opportunity to investigate cosmetics
on many levels.
- You should have a better understanding of cosmetic regulations.
- You should be more aware of what constitutes puffery vs fact when you read those claims on
cosmetic containers.
- You know where to find safety information about cosmetic ingredients.
- You have a basic appreciation of toxicology principles and the development and
safety evaluation process for cosmetics.
The finale of the unit is to put yourself in the role of the product safety manager, or toxicologist.
You must consider whether animal studies are needed, then design a human lotion study and the
finished label for your lotion.
There are many ways to design a human lotion study but it takes good planning to design a
study that produces results that are reliable and reproducible. If consumers complain to FDA
about your product, the FDA can ask to see your safety test results.
If they feel that you didn’t adequately test the product, then you can be forced to pull
it off the market if a court agrees with the FDA.
Homework Assignment:
Your homework assignment is to
a) assess the need for animal studies on your lotion,
b) using the Lotion Study Basics as a guide, design a human safety or quality study
for your lotion,
c) assume that the lotion passes your safety test; prepare a final label for
your lotion, including claims and other required parts of a label.
“Winning Combination”
Teacher guide: "Winning Combination" evaluation rubric
S3.3
TG3.30
Resources: (yellow pages)
FDA: Animal Testing
Lesson 3 Website resources
Dose-response background
Skin layers
Drug development process
Drug development process – 2
Safety and quality tests
Animal alternatives NYTimes
Northwest Association for
Biomedical Research
FDA website
R3.1
R3.2
R3.3
R3.4
R3.5
R3.6
R3.7
-8-
Lesson 3
File: Sense safety - OH.doc
NWABR
Feb. 2005
Sense (or Cents) Behind Safety
1. Why would a cosmetic company perform safety tests?
2. What do you expect cosmetic manufacturers to do to make products
safe?
3.
What are the consumer’s responsibilities for keeping cosmetics safe to use?
(What does industry expect consumers to do?)
4.
If you were a cosmetic manufacturer, what safety tests would you do before
your product went on the market?
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.1
Overhead
File: Sense safety - TG.doc
NWABR
Feb. 2005
Sense (or Cents) Behind Safety
1. Why would a cosmetic company perform safety tests?
Law requires a product to be safe - but FDA doesn’t review safety test results
before marketing
Company’s reputation for safe products would be at risk if there were a problem
To avoid legal suits (and monetary fines) by consumers that might be hurt by the
product
2. What do you expect cosmetic manufacturers to do to make products safe?
Perform appropriate safety testing
Include cautions/warnings on the labels
Provide directions for use
Use only high quality, non-adulterated ingredients
Accurately label the products so consumers can avoid ingredients that may cause
them allergic reactions.
NOTE: There is no way to guarantee that products will be non-irritating or nonallergenic to all people.
3. What are the consumer’s responsibilities for keeping cosmetics safe to use?
(What does industry expect consumers to do?)
Don’t share – to prevent contamination and spreading bacteria
Prevent bacterial contamination
Keep containers closed and out of sunlight
Discard products if they are discolored
Don’t add water, or saliva, to dried products
4. If you were a cosmetic lotion manufacturer, what safety tests would you do before
your lotion went on the market?
New ingredients require more extensive testing, including animal tests.
Students may say no animal tests are necessary since all of the individual
ingredients are safe and previously tested.
Types of tests that could be done:
Skin irritation, eye irritation, effects of oral ingestion, allergy tests
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.2
File: Safety warning.doc
NWABR
Jan. 2006
Who is responsible for safety testing?
Cosmetic manufacturers
Cosmetic Ingredient Review (CIR) panel = industry safety committee
FDA Requirement:
If a cosmetic product has not been safety tested, FDA requires
that a warning be on the label.
WARNING: The safety of this product has not been determined.
FDA Request:
FDA encourages consumers to report problems with cosmetic
products to them, as well as to the manufacturer.
1. Call your local FDA office
Seattle area - Consumer complaints: 206-553-7001
2. Write:
Call:
Email:
FDA Center for Food Safety and Applied Nutrition
Office of Cosmetics and Colors
200 C St., S.W.
Washington, DC 20204
202-401-9725
CFSAN Adverse Event Reporting System (CAERS)
301-436-2405
CAERS@cfsan.fda.gov
3. Contact the CFSAN outreach and information center:
1-888-723-3366
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.3
Overhead
File: Toxicology intro.doc
NWABR
Jan. 2008
Toxicology
Old Definition:
The study of poisons.
How much will kill you?
Newer Version:
The study of the harmful affects
of chemical or physical agents
on living organisms (humans, animals,
and the environment).
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.4
Overhead
File: Tox principles.doc
NWABR
Jan. 2008
In order to investigate the safety of a new
cosmetic ingredient, one must consider the
Three Principles of Toxicology:
1.
Dose-Response Relationship
2.
Hazard vs. Risk
3.
Individual Susceptibility
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.5
Overhead
File: Tox principlesbkgd.doc
NWABR
Jan. 2008
Principles of Toxicology
1. Dose-Response Relationship:
The larger the dose, the more extreme the response will be.
Dose: Amount of hazard that comes in contact with your body
Exposure: Contact with your body can come from various routes
(skin, lungs, swallowing)
Safety, or harm, from exposure depends on:
Duration
Frequency
Time between exposures
Combined exposure from multiple sources
Body size
Response:
Reaction to the dose;
Can range from no effect to death
2. Hazard vs. Risk
Hazard
= Anything in the environment that could harm you.
Assessment may be based on scientific studies or
personal experience;
Harm may not be immediately obvious
Risk
= The likelihood of harm in defined circumstances.
Assessment based on exposure and degree of hazard
Hazard is not synonymous with Risk
3. Individual Susceptibility: differences in sensitivity to toxic agents
Age, gender, health, genetic background, life style
Sensitivity can develop after repeated exposure
Reference:
Gilbert, Steven G. “A Small Dose of Toxicology”. 2004, CRC Press LLC.
www.asmalldoseof.org
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.6
File: Dose.doc
NWABR
Jan. 2008
Dose can depend on…
Duration of
Exposure:
How long?
Frequency of
Exposure:
How often?
Body Size:
How big or
small are you?
you
M
S
http://whs.wsd.wednet.edu/Faculty/Young/YNHC/ynhc.htm
L
S
Teacher Guide (TG)3.7
Overhead
File: Hazard, risk.doc
NWABR
Jan. 2008
Hazard is not synonymous with risk
Hazards could harm you.
Bacteria & Viruses
Harmful Chemicals
Tobacco & Smoke
Stress
Loud noises
Risk is the likelihood of harm in defined circumstances
Dose-response assessment
What is the relationship
between dose and response?
Hazard identification
Does the agent cause
adverse effects?
Risk
It is not a
simple yes/no
http://whs.wsd.wednet.edu/Faculty/Young/YNHC/ynhc.htm
Exposure assessment
What types, duration,
and levels of exposure
are experienced?
Teacher Guide (TG)3.7b
Overhead
File: Dose-response demo.doc
NWABR
Oct. 2006
Demonstrating the Principles of Dose-Response
Adapted from: “A Small Dose of Toxicology”, by Steven G. Gilbert. 2004, CRC Press LLC.
www.asmalldoseof.org
Demonstrating the Importance of the Amount of the Dose:
The greater the dose, the greater the effect
Materials:
Three large size glasses
Three petri dishes
Food color (blue is best)
One pitcher of water
 Add approximately equal amounts of water to three glasses. This represents the
approximate water content of an individual.
 To visualize skin surface area when thinking of doses of cosmetic ingredients, add
equal, small amounts of water (to cover the bottom) to three petri dishes
 Put one drop of blue food color in the first container (glass or petri dish), three in the
second container and then 6-9 in the last container.
 Stir with a pencil or pen and discuss the change in color as a response to increased dose
of food color in each glass. Discuss how some chemicals, caffeine being one, distribute
throughout the total body water.

Some chemicals from cosmetics can penetrate through the skin and can be
distributed through the blood stream while others remain on or near the
surface of the skin.
Effects of Amount on Response
A Small Dose of Toxicology
Northwest Association for
Biomedical Research.
Dose / Response - A Demonstration 5/25/04
Teacher Guide (TG)3.9
File: Dose-response demo.doc
NWABR
Oct. 2006
Demonstrating the Principles of Dose-Response
Adapted from: “A Small Dose of Toxicology”, by Steven G. Gilbert. 2004. CRC Press
www.asmalldoseof.org
Demonstrating the Importance of Size
The smaller the size, the greater the effect
Materials:
One large and one small size glass
One large and one small petri dish
Food color (blue is best)
One pitcher of water
 Fill the large and small glass (and the large and small petri dish) to approximately ¾ full with water.
The small containers represents a small child in contrast to the adult size containers.
 Put one drop of food color in each container.
The small glass will be much darker and usually look like the high dose glass from the
first demonstration.
 Discuss the importance of size, and the impact weight has on dose, depending on
sophistication of the group. A small child that drinks one can of caffeinated soda will
have a very different response than an adult because of the difference in the dose of
caffeine relative to body size.
Effects of Size on Response
A Small Dose of Toxicology
Northwest Association for
Biomedical Research.
Dose / Response - A Demonstration 5/25/04
Teacher Guide (TG)3.9
File: Dose-response curve.doc
NWABR
Jan. 2008
Dose-Response Curve:
Dose Response Function
All Effected
% Response
100
75
50
Half Effected
25
NO Effect
0
0
10
20
30 40
50
60 70
80
90 100
Dose (mg/kg body weight)
Increasing dose
Principles of Toxicology – 4/12/04
A Small Dose of Toxicology
www.asmalldoseof.org
X axis:
Dose = expressed in mg/kg.
mg = milligrams of substance/chemical
kg = killigrams of body weight
Y axis:
Response = % or number of subjects with pre-defined
end-point (inflammation, death, etc.)
Important information from dose-response curves
Dose at which:
no effect is seen
50% of subjects have the “response”
see maximum response
Shape of curve reflects potency
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.10
Overhead
File: Variable response demo.doc
NWABR
Jan. 2008
Variability in Response Demonstration
Adapted from: Tox-in-a Box, Community Outreach and Education Program
NIEHS CEEH, University of Washington; http://depts.washington.edu/hereuw
Materials and Equipment
Lab coat, safety glasses, and gloves
Distilled water (approximately 400 ml)
Bench cover
50 cc tube containing 35-350 mg bromophenol blue free acid (BPB) or
15 cc tube containing 17-25 mg BPB
0.1 N NaOH (20 ml)
0.3 N HCl (approximately 3 drops)
Spoon for stirring
Pipettes
4-5 clear plastic cups
Blank sheet of white paper
Premise:
The dye/buffer solution will resist color change until the buffer capacity is exceeded. After
the capacity is exceeded, the solution will change color (from blue to yellow) rather quickly with
each additional drop due to the pH sensitivity of BPB.
Preparation:
Note: THE BPB solution is a dye and will stain clothes, tables, etc. Bench covers should be
used to protect the countertop.
1. For a large amount of BPB solution add 20 ml of 0.1 N NaOH to 35-50 mg BPB. For a smaller
amount, add 10 ml of 0.1 N NaOH to 17-25 mg BPB.
Note: The BPB solution is not stable for more than a day or two; do not use tap water in this
experiment, as it is often quite alkaline. Use only distilled water.
2. Fill four or five clear plastic cups with distilled water.
Experiment:
Take four or five glasses each filled with distilled water, one of which as a few drops of 0.3 HCl.
Then add three drops of BPB to each one.
Stir each cup with the spoon provided.
The cup with the HCl will respond differently (it will turn yellow) and represent the “sensitive”
individual.
Holding a blank sheet of white paper behind the cups helps the students see the color changes.
Some people may respond differently to the same dose of a chemical because of genetics, diet, or
other factors. You can brainstorm with the students why some people are more sensitive or resistant
than others to chemicals.
Clean Up:
At these concentrations, these compounds will be similar to table salt and thus are not toxic and will
not harm the environment. The waste, therefore, can be poured down the drain.
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.11
File: Lotion dose response key.doc
NWABR
Aug. 2008
Dose-Response Worksheet: How much is too much?
Name __________________________
Period _________
Date __________
Hypothetical: Human Safety Test of a New Lotion
A local cosmetic manufacturer has developed a new moisturizing lotion. The lotion has passed all of the
animal-based safety tests and is now ready for testing on humans. Healthy males and females have been
recruited for the study. They have been fully informed about the protocol and the risks and have voluntarily
signed informed consent forms.
Ten people are participating in the study. Each person will be given a pre-measured, single use packet of
lotion to rub on their right, inside forearm. Each day for 20 days, they will come to the clinic and be given a
new packet of lotion to apply to their right, inside forearm. A clinical research associate (CRA) will observe
the arm and record skin appearance before application of the next packet of lotion.
Following is a summary of the results of the study based on the CRA’s observations. Please graph the
results below, labeling the axes appropriately.
Day 0: All 10 arm surfaces looked normal and healthy
Days 1-4: After 4 applications, all 10 arm surfaces look normal and healthy
Day 5: After 5 applications, 1 person’s arm had turned green in the area of lotion application
Day 7: After 7 applications, 2 other people’s arms had turned green in the area of lotion application.
Day 10: After 10 applications, 2 more people’s arms had turned green in the area of lotion application.
Day 15: After 15 applications, only one person’s arm still looked normal in the area of lotion application.
Day 18: After 18 applications, the arms of all participants had turned green in the area of lotion application.
Skin Response to Daily Lotion Application
X axis: can be Days, # of Applications, or # of Observations
Y axis: # or % of Study Participants with Green Arm
Questions based on the results of the lotion study.
1. What day would be considered the NOEL? (no observed adverse effect level) Day 4
2. When is the ED50? (effective dose that affects 50% of the people) Day 10
3. When is maximal response obtained? Day 18
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.12
File: Lotion dose response key.doc
NWABR
Aug. 2008
4. The graph you have completed (on page 1) is an “exposure-response” curve. What information do you
need in order to prepare a toxicologist’s true “dose-response” curve?
A toxicologist’s “dose” requires knowing the weight (mg) of the lotion applied and the weight (kg) of each
person to which it was applied.
Answer questions #5-12 based on the graph below.
“Hypothetical toxicity results for two new cosmetic ingredients tested in mice”
5. What is the lethal dose for 50% of the mice (LD50)
for ingredient A?
7 mg/kg
For ingredient B?
7 mg/kg
6. What is the NOEL for ingredient A?
For ingredient B?
2 mg/kg
5 mg/kg
7. Which compound is more toxic below the LD50?
Which compound is more toxic above the LD50?
B
A
8. Does higher LD50 imply lower or higher toxicity?
Lower toxicity
9. Once past the NOEL, how would you describe the response to increasing concentration (represented by
the slope of the curve)? (circle your answer)
For ingredient A:
slow
medium
fast
For ingredient B:
slow
medium
fast
10. If this was a graph of ingredient irritation and both ingredients had the same NOEL, which “slope” would
you prefer to see in a cosmetic and why.
B = there is a broader range of doses before 100% show irritation. Since people use
varied amounts of cosmetics, you want less chance of irritation if they use more than the
recommended amount.
11. If all of the mice received the same amount of ingredient but the mice were of very different ages and
weights, where on the x axis would you find data for the light-weight mice?
To the right on the X-axis. Since all mice receive the same mg of ingredient, there is a higher ratio
(mg/kg) in low weight mice.
12. Would you expect different responses (and therefore a different looking graph) from an ingredient that
penetrated the skin to the dermis vs. one that remained in the epidermis? Explain your answer.
Yes, one would expect a difference. An ingredient that penetrates to the dermis could enter the
bloodstream and be transported to many tissues. Any adverse effects in tissues would be greater in
smaller mice (due higher dose [mg/kg]). If the ingredient stayed in the epidermis, there would be less
effect related to weight. One might even see a straight line response – no difference in effect on mice
of different weights.
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.12
File: Skin biology.doc
NWABR
Jan. 2008
SUMMARY: Skin Biology
Modified from
http://www.cdc.gov/excite/skincancer/mod01.htm#skin
SKIN FUNCTIONS:
Barrier to infection and injury
Regulates temperature, pH and water loss
Protects against UV radiation from the sun
Metabolism: produces Vitamin D, breaks down chemicals
Sensory functions (pressure, pain, temperature)
Three major skin layers:
Epidermis - keratinocytes
Dermis - connective tissue with
blood vessels, glands, follicles
Subcutaneous layer – connective
tissue, fat
Getting Under the Skin
Approx. chemical composition of the skin:
Water
Protein
Lipids
Trace Minerals
Other


70.0%
25.5%
2.0%
0.5%
2.0%
Surface: 10-25% H2O,
Dermis:
70% H2O,
pH 4.2 - 5.6
pH 7.1 - 7.3
Picture from http://www.merck.com/mmhe/print/sec18/ch201/ch201b.html
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.13
Overhead
File: Skin absorption.doc
NWABR
Jan. 2008
Routes of skin absorption:
1. Around the cells – through extracellular lipids (MAJOR ROUTE)
2. Through the cells
3. Through hair follicles, sweat glands, and oil ducts
Epidermal Layers – composed of “keratinocytes”
15-20 cells thick.
Non-living cells.
(no nuclei, no
metabolic func.;
“corneocytes”)
Last layer
of viable cells;
have
metabolizing
enzymes.
Dividing (mitotic)
cells are found
in the basal
layer and move
upward and
differentiate with
age
Melanocytes: lie next to the basal layer; produce pigment and transfer it to the
keratinocytes.
Langerhans cells: immune recognition cells; lie in epidermal layers
Source: www.who.int/ipcs/methods/draftehcdermalabsorption.pdf
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.14
Overhead
File: Skin absorption.doc
NWABR
Jan. 2008
Skin variations that can affect absorption:
1. Age (probably not gender or race)
2. Anatomical site – thickness varies
3. Temperature
4. Hydration of stratum corneum
5. Damage to stratum corneum
6. Metabolism in skin
Extracellular lipids are important!
o Form a barrier to water and polar compounds
o Become a route of entry for oils and non-polar compounds
o The lipid composition varies by anatomical site
Only compounds that make it to the dermis,
can be absorbed into the blood stream.
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.14
Overhead
File: cosmetic ingred devel.doc
NWABR
Jan. 2008
Cosmetic ingredient development:
1.
Literature search – previous safety tests
- similar ingredients
2.
Computer models of similar ingredients
3.
Testing “in vitro” = test tubes, beakers
4.
5.
6.
Northwest Association for
Biomedical Research.
Testing on cells or tissues in culture
Research with animals to determine
safe levels.
Human safety and quality testing
Teacher Guide (TG)3.15
Overhead
File: cosmetic devel activity.doc
NWABR
Literature search - previous safety tests
- similar ingredients
Computer models of similar ingredients
Testing “in vitro” = test tubes, beakers
Testing on cells or tissues in culture
Research with animals to determine
safe levels.
Human safety and quality tests
(TG) 3.15b
File: Drug devel process.doc
NWABR
Jan. 2008
Drug development process:
1.
Literature search – previous safety tests
- similar ingredients
2.
Computer models of similar ingredients
3.
Testing “in vitro” = test tubes, beakers
4.
Testing on cells or tissues in culture
5.
Research with animals to determine safe levels
Manufacturing plan, label, clinical trial plan
IND submitted to FDA for approval (30 days)
6.
Human safety and efficacy testing = Clinical Trials
Phase I, II, III
NDA submitted to FDA for approval (~1.5 yrs)
Phase IV – post-marketing studies
Northwest Association for
Biomedical Research
(TG)3.16
Overhead
File: MSDS info - OH.doc
NWABR
Jan. 2008
Material Safety Data Sheet (MSDS)
1. Purpose:
an MSDS provides safety information to anyone who
works with or comes in contact with the chemical
2. Availability: from chemical manufacturers
web: http://hazard.com/msds
3. Information in an MSDS:
Section
1
Title
Chemical Product and Company Identification
NOTE: synonyms (one reason it is so difficult to identify
ingredients on product labels.)
2
Composition, Information on Ingredients
3
Hazards Identification
4
First Aid Measures
5
Fire Fighting Measures
6
Accidental Release Measures
7
Handling and Storage
8
Exposure Controls, Personal Protection
9
Physical and Chemical Properties
10
Stability and Reactivity
11
Toxicological Information
NOTE: results from animal studies
12
Ecological Information
13
Disposal Considerations
14
Transport Information
15
Regulatory Information
16
Additional Information
Northwest Association for
Biomedical Research
(TG)3.17
Overhead
File: MSDS info.doc
NWABR
Jan. 2008
Material Safety Data Sheet (MSDS)
1. Purpose: An MSDS provides safety information to anyone who works with or
comes in contact with chemicals:
Chemists and other scientists in laboratories
Chemical manufacturers
Transporters of chemicals
Any occupation that could come in contact with chemicals
janitors, repairmen, firemen
They are especially important for the manufacturers of the chemical
because large quantities are handled and transported.
The amount of information is overwhelming.
2. Availability: chemical manufacturers
http://hazard.com/msds
Any company that sells a chemical, must supply an MSDS to the consumer.
Company MSDSs are available to the public from companies or the web.
3. Information in an MSDS:
MSDSs from different companies are similar in format but the information may be
different. Some have more information than others.
Section
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Title
Chemical Product and Company Identification – section of interest
NOTE: synonyms (one reason it is so difficult to identify ingredients in
products.)
Composition, Information on Ingredients
Hazards Identification
First Aid Measures
Fire Fighting Measures
Accidental Release Measures
Handling and Storage
Exposure Controls, Personal Protection
Physical and Chemical Properties
Stability and Reactivity
Toxicological Information – section of interest
NOTE: results from animal studies
Ecological Information
Disposal Considerations
Transport Information
Regulatory Information
Additional Information
Northwest Association for
Biomedical Research
(TG)3.18
File: MSDS tox info.doc
NWABR
Jan. 2008
Information about Section 11 in an MSDS
Within Section 11, you will see the results of animal testing.
1. Species and routes of administration
Results include animal species, route of administration and results
(Format and results may be slightly different between different MSDSs.)
The safety of chemicals are usually tested by exposing animals to the chemical in various
ways. (orally, topically on the skin, injection)
The routes of administration reflect the many ways that humans might be exposed to a
chemical – through the skin, the mouth, the lungs, or an injection. Remember that in
chemical manufacturing plants, workers could get exposed to large quantities so
understanding the limits of safe concentrations is important.
2.
Toxicity results are usually presented as LD50 or LC50
LD50 – the lethal dose at which 50% of animals die
LC50 – the lethal concentration at which 50% of animals die
The original LD50 tests have been replaced with tests that use fewer animals.
“Limit tests” are done instead. Toxicologists start at low doses and test a few animals at
a time. If animals survive, then they go to increasingly higher doses on small numbers of
animals at a time.
A low LD50 means the compound is highly toxic.
LD50 doesn’t tell you about non-lethal toxic effects, such as redness, irritation, swelling, etc.
Draize eye test: a substance is placed in the eye of an albino rabbit to measure the reaction
induced after 24 hours.
The original Draize test protocol has been modified. Compounds are not tested at
concentrations that are expected to be toxic. Analgesics may be used. This in vivo test is
used after compounds have been screened in other non-whole animal assays. (see R3.6)
3.
Extrapolation to humans
Animals and humans share many infectious diseases, as well as chronic, non-infectious
diseases because of similar physiology. Different species are better models for different
diseases. Tests are done in animal species that are most similar to humans for that particular
test.
Example: Pigs are used for heart studies and skin penetration studies
Rabbits are used for glaucoma studies (inherited in NZW rabbits)
Transgenic or gene-knockout animals may be used because they
closely mimic human diseases
Test results from animals can be extrapolated to humans. There are conversion factors
to adjust for the fact that metabolism rates or skin penetration rates differ between animals and
humans.
Northwest Association for
Biomedical Research
(TG)3.19
File: LD50 conversion.doc
NWABR
Jan. 2008
Converting Animal Results to Humans
Results from toxicity tests in animals can be extrapolated to humans to give estimated
toxicities. For greater accuracy, species-specific conversion factors are available to account for
the fact that the basic biology (e.g., metabolism rates, skin penetration, etc.) differs between
animals and humans.
EXAMPLE: LD50 value from a mouse
Substance:
Triethanolamine (TEA)
Results:
Oral delivery to a mouse
LD50 = 5846 mg/kg
MATHEMATICAL CONVERSION of mouse LD50 to a human LD50
based on weight, without use of species-specific conversion factors:
1 kilogram (kg) = 1000 grams (g) = 2.2 pounds
1 milligram (mg) = 0.001 gram
Average mouse = 20-25 grams = 0.02-0.025 kg
Average human = 154 pounds = 70 kg
Average rat = 0.25-0.40 kg
Average rabbit = 4-6 kg
For a 20 g mouse: oral LD50 = 5846 mg/kg x 0.02 kg/mouse = 116.92 mg / mouse
For a 70 kg human:
5846 mg/kg x 70 kg/human = 409,220 mg / human
1000 mg / gram, therefore 409,220 mg = 409.22 grams / human
How many pounds is 409.22 grams?
1 pound (avdp) = 453.59 grams
409.22 grams / 453.59 grams/pound = 0.9 pounds
Therefore, by straight conversion based on species’ weight:
Oral LD50 for triethanolamine (TEA)
Human (70 kg) = 409,220 mg = 0.9 pounds
Mouse (20 g) =
117 mg = 0.00026 pounds
Therefore, 0.9 pounds of TEA would be lethal to 50% of the humans who ate it.
That is a lot of TEA!
Northwest Association for
Biomedical Research.
Teacher Guide (TG)3.20
File: Animal claims.doc
NWABR
Aug. 2008
Lotion Ingredient
Ingredient
on labels
MSDS
Claim on label:
“Not tested in animals”
Animals used;
≠
multiple delivery
modes
Do consumers really understand
what that claim means?
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.21
Overhead
File: Animal claims.doc
NWABR
Aug. 2008
“Not tested in animals.”
How can companies make this claim?
1. Individual ingredients were tested but not
the final product.
2. Someone else did the testing.
3. Animal tests were done more than 5 years ago.
4. No new, unique ingredients therefore new
animal safety testing is not needed.
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.21
Overhead
File: Animal studies bkgd - OH.doc
NWABR
Jan. 2008
Animal Studies - Background
1. New ingredients for cosmetics and drugs are required to be safe.
At this time, some aspects of safety can only be obtained through
responsible studies with animals.
2. When testing is required on new compounds, animal studies are
done after gathering data from other sources
3. When animal studies are needed, the guiding principles are the 3 R’s
Replacement
Reduction
Refinement
4. Federal regulations: govern care, research, housing, transport
a. Animal Welfare Act, 1966; amended 1970, 1985, 1990
b. Public Health Service Policy based on Health Research Extension
Act, 1985
5. Animal study proposals must be approved by site committees before
they can be started
6. To replace an animal test with a new test requires “validation”
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.22
Overhead
File: Animal studies discussion - OH.doc
NWABR
Jan. 2008
Animal Studies - Background
1. New ingredients for cosmetics and drugs must be safe. Animal studies are needed.
The cosmetic safety studies do not have to be approved by FDA before marketing
No alternative tests exist for some animal safety tests and for understanding
ingredient distribution, metabolism, and excretion.
2. When testing is required on new compounds, animal studies are done after gathering data
from other sources:
Literature searches for information about similar compounds
Mathematical and computer based modeling from available data bases
In vitro (in glass) testing (for example, effect on a specific target enzyme)
Cell and tissue culture (non-human or human cells or tissues; normal or cancerous
cells)
3. When animal studies are needed, the guiding principles are the 3 R’s
Replacement = using non-animal models when appropriate
Reduction = using the minimum number of animals necessary
Refinement = enhancing animal welfare and ensuring the best conditions possible
4. Federal regulations:
a. Animal Welfare Act, 1966; amended 1970, 1985, 1990.
Regulates transport, sale, and handling of all warm-blooded animals (not, mice or rats)
Requires registration with, and inspections by, USDA
Requires review and approval of all proposed animal studies
c. Public Health Service Policy based on Health Research Extension Act, 1985
Applies to publicly funded research
Covers all vertebrate animals, including fish, reptiles, rats, mice
5. Animal study proposals must be approved before they can be started
Institutional Animal Care and Use Committee (IACUC) = composed of
veterinarian, researcher, non-scientist professional, community member
Reviews protocols and inspects facilities
Protocol review includes:
Literature search to show that a similar study has not been done
Justification for using animals and the species chosen
Use of appropriate number of animals - to get statistically significant result
Use of appropriate procedures to reduce pain
Personnel appropriately trained
6. To replace an animal test with a new test requires “validation”
Multiple US agencies must evaluate the new procedure
A new test must be repeatable by multiple labs and results must be
reproducible
The results must correlate with previously acquired data
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.23
File: IACUC.doc
NWABR
Jan. 2008
Animal study protocols
must be approved in advance by the
Institutional Animal Care and Use Committee (IACUC)
WHO are they: The committee must include at least 3-5 members, including:
Veterinarian
Researcher
Non-scientist professional
Community Member
– experienced in research with animals
– such as lawyer, clergy
– not affiliated with the institution
FUNCTION:
 Review protocols and personnel qualifications
 Post-approval monitoring
 Facility review
The animal study protocol must include:
1. Literature search - to show that a similar study has not been
done
2. Justification for using animals and the species chosen
3. Use of appropriate number of animals – to get statistically
significant result
4. Use of appropriate procedures to reduce pain
5. Documentation that shows personnel have been appropriately
trained
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.24
Overhead
File: 3Rs - OH.doc
NWABR
Jan. 2008
Research with Animals: Practices and Procedures
People who work with animals:
 Are passionate about high standards of care for animals
 Take animal studies very seriously
 Use animals in research only after institutional approval
The guiding principles for using fewer animals are summed up in the
3 R’s
Replacement – whenever possible, replace animal testing with
methods that don’t use whole animals.
Reduction – use methods that reduce the number of animals
necessary to get the same information
Refinement – use procedures to eliminate or minimize discomfort
and improve animal well-being
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.25
Overhead
File: 3Rs - disc.doc
NWABR
Jan. 2008
Research with Animals: Practices and Procedures
The goal of all people who work with animals is to use as few animals as possible and to
replace animal testing with non-animal testing when possible. Maintaining high standards of
animal care ensures that study results will be more accurate and reproducible.
Currently, in vitro (in test tube or non-animal) tests are not able to replace all animal safety
tests. Only a live animal can provide the dynamic environment of multiple cell types interacting
and responding to the environment.
The guiding principles for using fewer animals are summed up in the 3 R’s.
Replacement – whenever possible, replace animal testing with methods that don’t use whole
animals.
EXAMPLES:
a. In the past, rabbits were used for pregnancy tests. Technicians injected a urine extract from a
woman into a rabbit and later tested the rabbit to see if the injection had caused the rabbit to
ovulate, indicating a positive test for pregnancy. Now, test strips can test for hormones in human
urine.
b. Tests for bacteria that could cause fevers were done by injecting the test compound into rabbits
and taking their temperature 24 hours later. Now, the “blood” of horseshoe crabs is used to test for
fever-inducing bacteria (LAL assay)
c. Human blood samples are incubated with the test compound and then tested for the production of
proteins called cytokines, which signal the brain to initiate a fever.
Reduction – use methods that reduce the number of animals necessary to get the same
information
EXAMPLES:
a. By referring to data bases or using computer modeling, predictions can be made about the safety
of some compounds. Very acidic compounds are known to be irritating to skin so there is no need
to test those compounds at concentrations that are known to be problematic.
b. Biophotonics uses light to detect and measure changes in biological tissues (such as tumor
growth) without sacrificing animals.
c. Species lower on the evolutionary ladder are used in assays when possible = yeast, fish, fruit
flies, or worms (nematodes).
Refinement – use procedures to eliminate or minimize discomfort and improve animal well-being
EXAMPLES:
a. New and more effective anesthetics and analgesics are being developed.
b. Animals are housed in groups since that is less stressful.
c. Animals are provided “toys” to alleviate boredom and make their environment more natural.
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.26
File: Human study example.doc
NWABR
Jan. 2008
Description of a human lotion study:
I gave the lotion to my friends to use.
They thought it was fine so I believe it is
OK to put on the market.
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.27
Overhead
File: Human study example.doc
NWABR
Jan. 2008
A well-designed study (scientific investigation)
 Must be informative - with a clear purpose,
- with 1 variable that is changed
(manipulated or independent variable)
- with other variables kept the same
(constants or controlled variables)
- with variable(s) that are measured
(responding or dependent variable(s))
 Must be reliable - objective data collection and
measurable results
 Must be reproducible
Scientific Method: Information found in a lab report
Purpose
Hypothesis
Testable and measurable
Methods
Materials
Procedure ** enough details for
reproducibility
Results
Observations
Measurable data
Data analysis
Conclusion
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.27
Overhead
File: Human study example.doc
NWABR
Jan. 2008
Details of a human lotion study:
WHAT
WHO
WHEN
WHERE
I gave the lotion to my friends to use.
They thought it was fine so I believe it is
OK to put on the market.
HOW evaluate
PURPOSE
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.27
Overhead
File: Lotion study basics - OH.doc
NWABR
Jan. 2008
Lotion Study Basics
An informative, reliable, and reproducible lotion study must be well
designed.
Use these basic categories to help you design a human safety or quality
study for your lotion. Identify the controlled, the manipulated (independent)
and responding variables.
PURPOSE:
WHO:
WHAT:
WHEN:
WHERE:
HOW will the results be evaluated?
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.28
Overhead
File: Lotion study discuss.doc
NWABR
Jan. 2008
Designing a Lotion Study
PURPOSE:
What aspect of safety is being studied?
What qualities of the lotion are desirable or undesirable?
Study Design Considerations
WHO:
Who can be in your study? Age, gender, race, health
Is a consent form needed?
How many people will participate?
WHAT:
What will be tested?
Will subjects compare your product against a commercially available product?
Will subjects know which product they are testing?
Will the investigator know which product subjects are testing?
If the study is testing 2 products, does each subject get to test both products?
Sequentially? Simultaneously (for example: one product per arm)?
What method of application and by whom – investigator or subject?
What amount should be applied and where?
WHEN:
How often will the test article be applied?
What time of day will the test article be applied?
How long does the test article have to be left on (before being washed off)?
How long will the study last?
HOW will the results be evaluated:
Who will record the results and how?
Verbal interview
Daily diary
Written evaluation at end using rating scales, multiple choice, narrative
Pictures - before and after
When will the results be recorded – daily, periodically, or end of study?
Who will tabulate and summarize the results?
Do you have to repeat the study for confidence (statistical significance) in the
results?
Did you include enough people for the results to be representative of
your intended consumers?
Identify the controlled variables and manipulated (independent) and responding variables in
your study.
Northwest Association for
Biomedical Research.
Teacher Guide (TG) 3.29
File: Lotion dose response wksht.doc
NWABR
July 2008
Evaluation Rubric: Winning Combination: Superb Lotion Study and Eye-Catching Label
Name:
Date:
Period:
Concept
Exceeds Expectations
Meets Expectations
Developing; Some
Expectations Met
Below Expectations
Drug vs.
Cosmetic
Product is correctly
identified as a cosmetic or
drug. The justification for
the designation chosen
draws upon exceptional
understanding of product
regulation.
Product is correctly
identified as a cosmetic
or drug, with an accurate
justification of
designation chosen.
Product is correctly
identified as a cosmetic
or drug, but the
justification of
designation chosen is
unclear or incomplete.
Product is incorrectly
identified as a
cosmetic or drug,
and/or the justification
of the designation
chosen is rudimentary
or absent.
Animal Test
Describes the relation of
the product to animal
testing with insight,
demonstrates exceptional
understanding of current
regulations and the need
for testing.
Describes the relation of
the product to animal
testing, demonstrates
understanding of current
regulations and the need
for testing.
Describes the relation of
the product to animal
testing, but relation may
be unclear or
incomplete.
Demonstrates some
understanding of current
regulations and the need
for testing.
Is unable to describe
the relation of the
product to animal
testing. Does not
reference current
regulations or the
need for testing.
Claims
Provides 2 or more claims.
Demonstrates in-depth
understanding of claims
that can be made on labels.
Label claims are accurate.
Provides 2 claims.
Demonstrates
understanding of claims
that can be made on
labels. Label claims are
accurate.
Demonstrates some
understanding of claims
that can be made on
labels. Label claims are
mostly accurate.
Demonstrates little
understanding of
claims that can be
made on labels. Label
claims are inaccurate
or irrelevant
Northwest Association for
Biomedical Research.
Student (S)3.1
Score
File: Lotion dose response wksht.doc
NWABR
July 2008
Human
Lotion Test
Describes 2 or more
features of each of the
study design elements
(Who, What, When,
Reliable Results) Study
design is exceptionally
thoughtful, clear, and
logical.
Describes 2 features of
each of the study design
elements. Study design
is clear and logical.
Describes fewer than 2
features for some of the
study design elements
below. Study design
may be incomplete or
unclear.
Few of the study
design elements are
described. Study
design is rudimentary
or absent.
Label
Information
Label information is
clearly and accurately
presented, with product
name, ingredients listed in
proper order (from those
present in largest volume
to least), manufacturing
address, and reasonable
estimate of quantity
included.
Label information is
accurately presented,
with product name,
ingredients listed in
proper order,
manufacturing address,
and a reasonable estimate
of quantity included.
Label information is
partially presented.
Label ingredients are
included but may not be
correctly ordered. The
manufacturing address
may be incomplete or
missing. Product name
may be missing.
Estimated quantity may
be misjudged.
Label information is
missing most required
elements: Label
ingredients,
manufacturing
address, and product
name, estimated
quantity.
Label
Design
The label is exceptionally
well-designed, with all
elements attractively
presented.
The label has all the
required elements clearly
presented.
The required elements
are not presented in a
clear way.
The label design
detracts from the
elements, or the
elements are not
presented in the
design.
Northwest Association for
Biomedical Research.
Student (S)3.1
File: Lotion dose response wksht.doc
NWABR
July 2008
Dose-Response Worksheet: How much is too much?
Name __________________________
Period _________
Date __________
Hypothetical: Human Safety Test of a New Lotion
A local cosmetic manufacturer has developed a new moisturizing lotion. The lotion has passed all of the
animal-based safety tests and is now ready for testing on humans. Healthy males and females have been
recruited for the study. They have been fully informed about the protocol and the risks and have voluntarily
signed informed consent forms.
Ten people are participating in the study. Each person will be given a pre-measured, single use packet of
lotion to rub on their right, inside forearm. Each day for 20 days, they will come to the clinic and be given a
new packet of lotion to apply to their right, inside forearm. A clinical research associate (CRA) will observe
the arm and record skin appearance before application of the next packet of lotion.
Following is a summary of the results of the study based on the CRA’s observations. Please graph the
results below, labeling the axes appropriately.
Day 0: All 10 arm surfaces looked normal and healthy
Days 1-4: After 4 applications, all 10 arm surfaces look normal and healthy
Day 5: After 5 applications, 1 person’s arm had turned green in the area of lotion application
Day 7: After 7 applications, 2 other people’s arms had turned green in the area of lotion application.
Day 10: After 10 applications, 2 more people’s arms had turned green in the area of lotion application.
Day 15: After 15 applications, only one person’s arm still looked normal in the area of lotion application.
Day 18: After 18 applications, the arms of all participants had turned green in the area of lotion application.
Skin Response to Daily Lotion Application
____________________________________________
Questions based on the results of the lotion study.
1. What day would be considered the NOEL ? (no observed adverse effect level)
2. When is the ED50? (effective dose that affects 50% of the people)
3. When is maximal response obtained?
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Student (S)3.1
File: Lotion dose response wksht.doc
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July 2008
4. The graph you have completed (on page 1) is an “exposure-response” curve. What information do you
need in order to prepare a toxicologist’s true “dose-response” curve?
Answer questions #5-12 based on the graph below.
“Hypothetical toxicity results for two new cosmetic ingredients tested in mice.”
5. What is the LD50 for ingredient A? ____________
For ingredient B? _________
6. What is the NOEL for ingredient A? ___________
For ingredient B? _________
7. Which compound is more toxic below the LD50?
Which compound is more toxic above the LD50?
8. Does higher LD50 imply lower or higher toxicity?
9. Once past the NOEL, how would you describe the response to increasing concentration (represented by
the slope of the curve)? (circle your answer)
For ingredient A:
For ingredient B:
slow
slow
medium
medium
fast
fast
10. If this was a graph of ingredient irritation and both ingredients had the same NOEL, which “slope” would
you prefer to see in a cosmetic and why.
11. If all of the mice received the same amount of ingredient but the mice were of very different ages and
weights, where on the x axis would you find data for the light-weight mice?
12. Would you expect different responses (and therefore a different looking graph) from an ingredient that
penetrated the skin to the dermis vs. one that remained in the epidermis? Explain your answer.
Northwest Association for
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Student (S)3.1
File: Func, tox chart.doc
NWABR
Feb. 2005
Cosmetic Ingredients: Function & Toxicity Chart
Name:
Date:
Period:
Instructions:
1. From the ingredients listed on page 2, circle the ingredient that matches the MSDS that you have been given.
2. Fill in "MSDS Toxicity Tests" (column 4, page 2) using information from Section 11 of your MSDS.
List all animals in which tests were done and how the ingredient was administered.
Sample routes of administration:
Subcutaneous (sc) = under the skin
Oral = by mouth or delivered directly into the stomach
Intraperitoneal (i.p.) = injected into the body cavity but not into any organs
Skin = applied to shaved or unshaved area
Draize = eye or skin tests in rabbits
Eyes
3. Record the toxicity test information for ingredients from other MSDSs in your group.
Each of you should end up with toxicity test information recorded for 4 ingredients.
4. Study the labels on the sample products your group has been given. Record whether any of the ingredients
that were tested in animals can be found on labels of products that claim "not tested in animals".
Northwest Association for Biomedical Research.
Student (S)3.2 Handout
File: Func, tox chart.doc
NWABR
Feb. 2005
Cosmetic Ingredients: Function & Toxicity Chart
page 2
Date:
Name:
Cosmetic
Function
Emulsifier
(detergent)
Circle ingredient(s)
Class Lotion
Commercial Product
Ingredients
Ingredients
Borax
Cetyl alcohol
Lecithin
Xanthan gum
Sodium lauryl sulfate
Triethanolamine (TEA)
Di propylene glycol
Polysorbate
Cocamidopropyl betaine
Preservative
Period:
Fill in tox tests for circled ingredient(s)
Yes or No
MSDS Toxicity Tests (Section 11)
Found in products labeled
Animal?
How administered?
"Not tested in animals"
Methylparaben
Propylparaben
Moisturizer, Occlusive
Mineral oil
Beeswax
Coconut oil
Vegetable oil
Soybean oil
Petrolatum
Dimethicone
Caprylic/capric triglyceride
Acetylated lanolin alcohol
Moisturizer, Humectant
Glycerin
Propylene glycol
Sorbitol
Urea
Xylose
Solvent
Polyethylene glycol (PEG)
Water
Buffer (controls pH)
Function Unknown
Northwest Association for Biomedical Research.
Citric acid
rat - oral, intraperitoneal, subcutaneous
rabbit - skin, eyes
mouse - oral, intraperitoneal, intravenous
Yes
Stearic acid
Sorbitol
DMDM Hydantoin
Student (S)3.2 Handout
File: Func, tox chart.doc
NWABR
Feb. 2005
This page was intentionally left blank.
Northwest Association for Biomedical Research.
Student (S)3.2 Handout
File: Winning combination.doc
NWABR
Jan. 2008
Winning Combination: Superb Lotion Study and Eye-Catching Label
Name: __________________________
Date: _______________ Period: _______
1. Evaluate your lotion:
Is the lotion a cosmetic or a drug? Explain your answer.
Is animal testing required on your lotion? Explain your answer.
2. List at least two claims for your label. Explain why you can make these claims
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Students (S)3.3
Homework
File: Winning combination.doc
NWABR
Jan. 2008
Winning Combination: Superb Lotion Study and Eye-Catching Label
3. Design a human study for the safety or quality of the skin care lotion that you made in class. The
study design should address the following points: Except for “Purpose”, include at least 2 features of
your study design under each point.
Purpose:
Who:
What:
When:
Where:
How will results be evaluated?
4. On a separate page, design a label for your lotion that includes claims and information required for
all cosmetic labels: product name, ingredients listed in decreasing order of quantity,
manufacturer’s address, product quantity (estimated)
Northwest Association for
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Students (S)3.3
Homework
File: L3 websites.doc
NWABR
July 2008
Lesson 3 Website Resources
Skin Biology
1. http://www.cdc.gov/excite/skincancer/mod01.htm#skin
Basic explanations of skin biology
2. http://www.who.int/ipcs/methods/draftehcdermalabsorption.pdf
“Dermal Absorption”, International Programme on Chemical Safety Environmental Health Criteria,
Draft Feb. 2005
Excellent descriptions of skin function and dermal absorption testing
3. http://www.nature.com/nature/supplements/insights/skin/index.html
Skin Biology Supplement: melanocytes, skin development, pigmentation, sensory transduction,
psoriasis, tissue engineering.
4. http://www.nlm.nih.gov/medlineplus/medlineplus.html
“Medline Plus” from NIH and US National Library of Medicine
Sections on health topics, medical encyclopedia, other resources
Material Safety Data Sheets
1. http://hazard.com/msds
Find MSDS’s by company or compound name. Toxicology reports searchable by compound name.
2. http://www.msdssearch.com
Background information about MSDS’s. Directory of MSDS’s by ingredient and manufacturer.
3. http://www.ilpi.com/msds/
References to MSDS information sites. Contains a glossary of terms found in MSDS’s.
4. http://www.householdproducts.nlm.nih.gov
Selected personal care/use products and ingredients have summaries taken from MSDS
sheets provided by manufacturers. Specific animal toxicology testing information is not
included.
Toxicology Information
1. http://toxnet.nlm.nih.gov
TOXNET contains databases on toxicology, hazardous chemicals, environmental health, and toxic
releases
2. http://toxipedia.org/conf/display/toxipedia/Welcome+to+Toxipedia
Toxipedia – Great site devoted to providing a better understanding of many aspects of toxicology; includes a
toxic substances dictionary, teaching resources, lists of agencies and organizations involved in health and
environmental protection.
3. http://www.atsdr.cdc.gov/COM/exposure.pdf
The Agency for Toxic Substances and Disease Registry is a federal public health agency of the US
Dept. of Health and Human Services. This is a two page explanation of “exposure”. Although
primarily focused on environmental health, the main web site has a nice glossary of terms for the
broad area of toxicology.
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File: L3 websites.doc
NWABR
July 2008
4. http://whs.wsd.wednet.edu/Faculty/Young/YNHC/ynhc.htm
The Youth Network for Healthy Communities program was developed by UW Center for
Ecogenetics and Environmental Health. See Resources -> Orientation Power Point and Resource
List
Product Safety Testing
1. http://www.kids4research.org/productsafety.html
Product safety testing information and glossary; animals in research information
2. http://www.nabr.org/issues.htm
“The use of animals in product safety testing”, 1997
See copy of article under Tab 8.
3. http://www.cosmeticsdesign-europe.com/news/ng.asp?n=78158-ecvam-eu-animal-sccp-colipa
News article about animal testing in Europe and REACH effects, 7-16-07
4. http://www.fda.gov/oc/history/historyoffda/default.htm
History of the Food and Drug Administration
5. http://www.cosmeticsdesign-europe.com/news/ng.asp?n=78158-ecvam-eu-animal-sccp-colipa
News article about animal testing in Europe and REACH effects, 7-16-07
6. http://www.fda.gov/oc/history/historyoffda/default.htm
History of the Food and Drug Administration
Alternatives to Animal Testing
1. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM);
responsible for testing the accuracy and reliability of test protocols.
National Toxicology Program, Dept. of Health and Human Services
Alternative animal tests for dermal corrosivity and irritation
http://iccvam.niehs.nih.gov/methods/dermal/dermal.htm
Alternatives to animal testing for ocular toxicity
http://iccvam.niehs.nih.gov/methods/ocutox/ocutox.htm
In vitro pyrogen test methods
http://iccvam.niehs.nih.gov/methods/pyrogen/pyrogen.htm
Alternatives to animal testing for acute oral toxicity
http://iccvam.niehs.nih.gov/methods/acutetox/acutetox.htm
2. http://altweb.jhsph.edu/
Johns Hopkins School of Public Health
3. http://caat.jhsph.edu/
Johns Hopkins Center for Alternatives to Animal Testing
4. http://ecvam.jrc.cec.eu.int/index.htm
European Centre for the Validation of Alternative Methods (ECVAM)
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http://pmep.cce.cornell.edu/profiles/extoxnet/TIB/dose-response.html Viewed 8-10-07
PMEP Home
Page
Pesticide Active Ingredient
Information
EXTOXNET: The Extension
Toxicology Network
Toxicology Information
Briefs
Dose-Response Relationships In
Toxicology
E X T O X N E T
Extension Toxicology Network
A Pesticide Information Project of Cooperative Extension Offices of Cornell University, Michigan State University,
Oregon State University, and University of California at Davis. Major support and funding was provided by the
USDA/Extension Service/National Agricultural Pesticide Impact Assessment Program.
Toxicology
Information
Brief
Dose-Response Relationships In
Toxicology
Publication Date: 9/93
"The right dose differentiates a poison and a remedy." -Paracelsus
INTRODUCTION
The science of toxicology is based on the principle that there is a relationship between a toxic
reaction (the response) and the amount of poison received (the dose). An important assumption in
this relationship is that there is almost always a dose below which no response occurs or can be
measured. A second assumption is that once a maximum response is reached any further increases
in the dose will not result in any increased effect.
One particular instance in which this dose-response relationship does not hold true, is in regard to
true allergic reactions. Allergic reactions are special kinds of changes in the immune system; they
are not really toxic responses. The difference between allergies and toxic reactions is that a toxic
effect is directly the result of the toxic chemical acting on cells. Allergic responses are the result of
a chemical stimulating the body to release natural chemicals which are in turn directly responsible
for the effects seen. Thus, in an allergic reaction, the chemical acts merely as a trigger, not as the
bullet.
For all other types of toxicity, knowing the dose-response relationship is a necessary part of
understanding the cause and effect relationship between chemical exposure and illness. As
Paracelsus once wrote, "The right dose differentiates a poison from a remedy." Keep in mind that
the toxicity of a chemical is an inherent quality of the chemical and cannot be changed without
changing the chemical to another form. The toxic effects on an organism are related to the amount
of exposure.
MEASURES OF EXPOSURE
Exposure to poisons can be intentional or unintentional. The effects of exposure to poisons vary
with the amount of exposure, which is another way of saying "the dose." Usually when we think of
dose, we think in terms of taking one vitamin capsule a day or two aspirin every four hours, or
something like that. Contamination of food or water with chemicals can also provide doses of
chemicals each time we eat or drink. Some commonly used measures for expressing levels of
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contaminants are listed in table 1. These measures tell us how much of the chemical is in food,
water or air. The amount we eat, drink, or breathe determines the actual dose we receive.
Concentrations of chemicals in the environment are most commonly expressed as ppm and ppb.
Government tolerance limits for various poisons usually use these abbreviations. Remember that
these are extremely small quantities. For example, if you put one teaspoon of salt in two gallons of
water the resulting salt concentration would be approximately 1,000 ppm and it would not even
taste salty!
Table 1. Measurements for Expressing Levels of Contaminants in Food and Water.
Dose
Abbrev.
Metric equivalent
Abbrev.
Approx. amt. in water
parts per million ppm
milligrams per kilogram mg/kg
1 teaspoon per 1,000 gallons
parts per billion ppb
micrograms per kilogram ug/kg
1 teaspoon per 1,000,000 gallons
DOSE-EFFECT RELATIONSHIPS
The dose of a poison is going to determine the degree of effect it produces. The following example
illustrates this principle. Suppose ten goldfish are in a ten-gallon tank and we add one ounce of 100proof whiskey to the water every five minutes until all the fish get drunk and swim upside down.
Probably none would swim upside down after the first two or three shots. After four or five, a very
sensitive fish might. After six or eight shots another one or two might. With a dose of ten shots, five
of the ten fish might be swimming upside down. After fifteen shots, there might be only one fish
swimming properly and it too would turn over after seventeen or eighteen shots.
The effect measured in this example is swimming upside down. Individual sensitivity to alcohol
varies, as does individual sensitivity to other poisons. There is a dose level at which none of the fish
swim upside down (no observed effect). There is also a dose level at which all of the fish swim
upside down. The dose level at which 50 percent of the fish have turned over is known as the ED50,
which means effective dose for 50 percent of the fish tested. The ED50 of any poison varies
depending on the effect measured. In general, the less severe the effect measured, the lower the
ED50 for that particular effect. Obviously poisons are not tested in humans in such a fashion.
Instead, animals are used to predict the toxicity that may occur in humans.
One of the more commonly used measures of toxicity is the LD50. The LD50 (the lethal dose for 50
percent of the animals tested) of a poison is usually expressed in milligrams of chemical per
kilogram of body weight (mg/kg). A chemical with a small LD50 (like 5 mg/kg) is very highly
toxic. A chemical with a large LD50 (1,000 to 5,000 mg/kg) is practically non-toxic. The LD50
says nothing about non-lethal toxic effects though. A chemical may have a large LD50, but may
produce illness at very small exposure levels. It is incorrect to say that chemicals with small LD50s
are more dangerous than chemicals with large LD50s, they are simply more toxic. The danger, or
risk of adverse effect of chemicals, is mostly determined by how they are used, not by the inherent
toxicity of the chemical itself.
The LD50s of different poisons may be easily compared; however, it is always necessary to know
which species was used for the tests and how the poison was administered (the route of exposure),
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since the LD50 of a poison may vary considerably based on the species of animal and the way
exposure occurs. Some poisons may be extremely toxic if swallowed (oral exposure) and not very
toxic at all if splashed on the skin (dermal exposure). If the oral LD50 of a poison were 10 mg/kg,
50 percent of the animals who swallowed 10 mg/kg would be expected to die and 50 percent to live.
The LD50 is determined mathematically, and in actual tests using the LD50, it would be unusual to
get an exact 50% response. One test might produce 30% mortality and another might produce 70%
mortality. Averaged out over many tests, the numbers would approach 50%, if the original LD50
determination was valid.
The potency of a poison is a measure of its strength compared to other poisons. The more potent the
poison, the less it takes to kill; the less potent the poison, the more it takes to kill. The potencies of
poisons are often compared using signal words or categories as shown in the example in table 2.
The designation toxic dose (TD) is used to indicate the dose (exposure) that will produce signs of
toxicity in a certain percentage of animals. The TD50 is the toxic dose for 50 percent of the animals
tested. The larger the TD the more poison it takes to produce signs of toxicity. The toxic dose does
not give any information about the lethal dose because toxic effects (for example, nausea and
vomiting) may not be directly related to the way that the chemical causes death. The toxicity of a
chemical is an inherent property of the chemical itself. It is also true that chemicals can cause
different types of toxic effects, at different dose levels, depending on the animal species tested. For
this reason, when using the toxic dose designation it is useful to precisely define the type of toxicity
measured, the animal species tested, and the dose and route of administration.
Table 2. Toxicity Rating Scale and Labeling Requirements for Pesticides.
Category
Signal word required on
label
LD50 oral
mg/kg(ppm)
LD50 dermal
mg/kg(ppm)
Probable oral lethal
dose
I
highly toxic
DANGER-POISON
(skull and crossbones)
less than 50
less than 200
a few drops to a
teaspoon
II
moderately toxic
WARNING
51 to 500
200 to 2,000
over 1 teaspoon to 1
ounce
III
slightly toxic
CAUTION
over 500
over 2,000
over 1 ounce
IV
practically nontoxic
none required
Toxicity assessment is quite complex, many factors can affect the results of toxicity tests. Some of
these factors include variables like temperature, food, light, and stressful environmental conditions.
Other factors related to the animal itself include age, sex, health, and hormonal status.
The NOEL (no observable effect level) is the highest dose or exposure level of a poison that
produces no noticeable toxic effect on animals. From our previous fish example, we know that there
is a dose below which no effect is seen. In toxicology, residue tolerance levels of poisons that are
permitted in food or in drinking water, for instance, are usually set from 100 to 1,000 times less than
the NOEL to provide a wide margin of safety for humans.
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The TLV (threshold limit value) for a chemical is the airborne concentration of the chemical
(expressed in ppm) that produces no adverse effects in workers exposed for eight hours per day five
days per week. The TLV is usually set to prevent minor toxic effects like skin or eye irritation.
Very often people compare poisons based on their LD50's and base decisions about the safety of a
chemical based on this number. This is an over-simplified approach to comparing chemicals
because the LD50 is simply one point on the dose-response curve that reflects the potential of the
compound to cause death. What is more important in assessing chemical safety is the threshold
dose, and the slope of the dose-response curve, which shows how fast the response increases as the
dose increases. Figure 1 shows examples of dose-response curves for two different chemicals which
have the same LD50. Which of these chemicals is more toxic? Answer this question for doses
below the LD50 and it is chemical A which is more toxic, at the LD50 they are the same, and above
the LD50, chemical B is more toxic. While the LD50 can provide some useful information, it is of
limited value in risk assessment because the LD50 only reflects information about the lethal effects
of the chemical. It is quite possible that a chemical will produce a very undesirable toxic effect
(such as reproductive toxicity or birth defects) at doses which cause no deaths at all.
A true assessment of chemical toxicity involves comparisons of numerous dose-response curves
covering many different types of toxic effects. The determination of which pesticides will be
Restricted Use Pesticides involves this approach. Some Restricted Use Pesticides have very large
LD50s (low acute oral toxicity), however, they may be very strong skin or eye irritants and thus
require special handling.
The knowledge gained from dose-response studies in animals is used to set standards for human
exposure and the amount of chemical residue that is allowed in the environment. As mentioned
previously, numerous dose-response relationships must be determined, in many different species.
Without this information, it is impossible to accurately predict the health risks associated with
chemical exposure. With adequate information, we can make informed decisions about chemical
exposure and work to minimize the risk to human health and the environment.
Revised 9/93
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File: Skin layers.doc
NWABR
Aug. 2007
Modified from http://www.cdc.gov/excite/skincancer/mod01.htm#skin
EPIDERMIS
The epidermis is made up of several different types of cells. Two of these cell types are important in
studying skin cancer: keratinocytes and melanocytes. Langerhans’ cells are involved in protecting
the body from foreign substances and they initiate the skin’s allergic reactions.
Keratinocytes
The main cells that make up the epidermis are called keratinocytes. These cells are arranged
into five layers, or "strata."
 basal layer (stratum basale), with dividing keratinocytes
Deepest layer
 "prickle-cell" layer (stratum spinosum),
 granular layer (stratum granulosum),
 lucid layer (stratum lucidum), and
 cornified layer (stratum corneum).
Top, surface
Basal Layer
The basal layer is the deepest layer. Cells in this layer can divide and reproduce themselves.
They are the most immature, or least specialized, of the keratinocytes. These cells mature and
move toward the surface of the skin. As they move upward, they become flattened and lose
most of their water content.
Cornified Layer
The stratum corneum is the outermost (top) layer, composed mainly of dead keratinocytes,
hardened proteins (keratins) and lipids, forming a protective crust. It has the flattest cells,
arranged in a basket weave pattern. Keratin in these cells helps the cornified layer protect
against moisture, light, and infection. The keratin works together with lipids (fats) and tight
inter-cellular connections called "desmosomes." The cells of the stratum corneum are the most
specialized of the keratinocytes. These dead cells are continually sloughed off the surface of the
skin and replaced by cells that mature from the basal layer. The skin completely renews itself
every 3-5 weeks.
Melanocytes
Melanocytes are another kind of cell found in the epidermis. These cells are in the basal
layer. Melanocytes make the pigment called melanin. The melanin is then transferred to nearby
keratinocytes. Some of this melanin then works its way up with the keratinocytes as they move to
the top layer.
Skin color depends on how much melanin is made and how much is carried toward the
surface. All people have about the same number of melanocytes, no matter what their skin color.
Those with darker skin have more melanin in the upper layers of the skin. Sunlight raises the rate at
which melanin is made and transferred. This is what causes tanning.
Melanin can protect the cells from ultraviolet (UV) radiation and its harmful effects. It does
this by absorbing harmful UV rays. It also cleans up toxins that come from UV damage to skin
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cells. Within cells, melanin tends to form caps above the nuclei. The caps protect the cells' genetic
material from UV damage.
Langerhans’ Cells
These epidermal cells are part of the skin’s immune system. They help detect foreign
substances and play a role in the development of skin allergies.
DERMIS
The dermis (2-3 mm thick) lies under the epidermis and is much thicker than the epidermis
(< 1mm). It is made of loosely arranged connective tissue. The surface of the dermis is covered
with rows of projections much like fingers. These are called dermal papillae. They help hold the
epidermis and dermis together by forming ridges and grooves.
The lower part of the dermis is a network of interlacing fibers. These fibers give the skin
strength and allow it to stretch. The dermis contains a lot of blood vessels, nerve endings, muscle
fibers, sweat and oil (sebaceous) glands, and hair follicles. The roots of hairs are located deep in
follicles. Hair shafts stick out from the follicles. Small muscles raise and lower the hairs. These
muscles extend from the dermis to the side of the hair follicles. Sebaceous glands open into hair
follicles and secrete oil called sebum. Sebum oils the skin and hair.
SUBCUTANEOUS LAYER
Subcutaneous tissue lies under the dermis. It is a thick layer of connective tissue and fat. The
fat helps control body temperature and stores energy from food. The subcutaneous tissue also acts
as a shock absorber. It protects the tissues below from injury.
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Summary of the drug development process:
Timeline for
Drug
Development
Discovery:
Literature, synthesis,
lab screening;
2-10 years
Compound Success
Rates by Stage
Preclinical
Development
Lab & animal
testing; 4-5 yrs
Phase 1: 1.5 yrs
Phase II: 2 yrs
Phase III: 4-5 yrs
FDA Review &
Approval: 1.5 yrs
Post-Marketing
Testing
5,000–10,000
Screened
250 Enter
Preclinical
Testing
5 Enter
Clinical
Testing
1
Approved
by the FDA
Avg of 15 yrs
Source: Tufts Center for the Study of Drug Development
*Variations for biopharmaceuticals
(e.g., “biologicals” = antibodies, cellular proteins):
Cost: $1.2 B for biotechnology product: $615 M, preclinical costs
$626, clinical costs
Time: Longer development timeline
8% longer to get through clinical testing and FDA review
Approval rate:
30% (1 of 3 biologicals that enter clinical testing)
vs. 21% (1 of 5 pharmaceuticals that enter clinical testing)
*http://csdd.tufts.edu/NewsEvents/NewsArticle.asp?newsid=69
Nov. 2006
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Drug Development Process
Science – Regulations – Business Decisions
Chemistry:
Making the drug
Pre-clinical Testing






>500,000
Compounds
Northwest Association for
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Clinical Testing
Marketing
Dosage
Adverse effects
Regulatory approval – FDA
Recovery
Fertility damage
Carcinogenicity
Formulation development
$0.8-1.2 B
10-15 years
1 Drug
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File: Safety and Quality Tests.doc
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Aug. 2008
Safety and Quality Tests
Test Purpose
Skin Penetration
Or Absorption
Name
Franz Cell
The Franz Cell is a vertical diffusion chamber that is used to determine the rate and extent of
penetration of a test compound through the skin. Skin is positioned with a donor chamber above
and a receptor chamber below. The skin may be from Yorkshire pigs or fresh or frozen human skin
(from elective surgeries or cadavers). The test compound is applied to the top surface of the skin,
while the skin’s dermis is in contact with a fluid, at body temperature, in the lower, receptor
chamber. The skin may be covered, or not covered. After a pre-determined amount of time, the test
is stopped and the amount of test compound is determined at the skin’s surface, within particular
layers of the skin, and in the solution in the receptor chamber.
Allergy / Irritation
Local Lymph Node Assay (LLNA)
The Local Lymph Node Assay (LLNA) uses mice to test for sensitization (allergic) potential,
replacing a previous test in guinea pigs that required more animals. (Pre-test: Various
concentrations of the test compound are applied to the ear to determine a concentration that will not
cause irritation/swelling.) Concentrations of test compound that do not cause irritation are applied
to the ears of mice once a day for three days, then on day 6, the mice are injected with a radioactive
label (BrdU = bromodeoxyuridine). If the test compound causes sensitization (activation of
immune system cells), there will be lymphocyte proliferation seen in the lymph nodes that drain the
ears. The dividing lymphocytes will incorporate BrdU into their DNA. The lymph nodes are
removed and analyzed for the number and type of cells incorporating BrdU. The test requires 20-25
mice.
Eye Irritation,
Corrosion
a. Bovine Corneal Opacity and Permeability (BCOP) test
b. Isolated Chicken Eye (ICE) test
c. Draize eye test
http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox.htm
ICCVAM = Interagency Coordinating Committee on the Validation of Alternative Methods;
responsible for testing the accuracy and reliability of test protocols.
June 2008: ICCVAM approved the BCOP Test Method and the ICE Test Method for use in a tiered
testing, screening strategy to determine ocular hazards, with specific limitations for certain chemical
classes and/or physical properties. Substances that test positive in these assays can be classified as
ocular corrosives or severe irritants without further testing in animals. A substance that tests
negative in the in vitro ocular toxicity test would need to be tested in the in vivo ocular test to
identify possible in vitro false negatives and to identify moderate and mild ocular irritants.
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a. Bovine Corneal Opacity and Permeability (BCOP) test
This test measures the effect of a compound on the opacity and permeability of an isolated bovine
cornea.
PROTOCOL: Eyes from young adult cattle from slaughterhouses are kept at 4 degrees C and used
within 5 hours after collection. Undamaged corneas are removed and put into O-ring holders with
chambers containing saline solution bathing the front and back of the cornea. Corneas are held at
32 degrees C. for one hour. (a) The opacity of the cornea is measured. The saline is removed from
the chamber next to the front of the cornea, the test compound is added to the chamber and gently
rocked for 10 minutes; the test compound is removed by rinsing three times with saline and the
opacity is measured. (b) Permeability of the cornea is determined by sodium-fluorescein staining
for 90 minutes in the chamber on the front of the cornea, then removing the saline solution from the
chamber on the under side of the cornea and measuring the amount of fluorescein dye present.
(c) The corneas are fixed in a formalin solution for later histology if needed. The outcomes of the
test are used to predict the in vivo ocular irritation potential.
b. Isolated Chicken Eye (ICE) test
This test measures the effect of a compound on corneal swelling, opacity, permeability (fluorescein
dye retention), and morphological damage to the corneal surface on a chicken eye in an isolated
system.
PROTOCOL: Chicken heads from spring chickens, approximately 7 weeks old, male or female,
with a weight range of 2.5-3.0 kg, are received from poultry slaughterhouses within two hours after
humane killing. Eye lids are removed, corneas are stained with fluorescien for 10-20 seconds,
rinsed, and microscopy is used to ensure the cornea is not damaged. Eyes are dissected from the
eye socket with a visible portion of optic nerve remaining attached. The eyes are vertically
mounted on a clamp in a superfusion chamber. Saline solution at 32 degrees C is pumped over the
cornea. The corneas are examined for damage and corneal thickness is measured with a slit-lamp
microscope.
For compound testing, the eye is removed from the clamp so that the cornea is horizontal.
The test compound is applied and left for 10 seconds. The cornea is rinsed with saline and
remounted in the superfusion chamber. The eye is examined at 30, 75, 120, 180, and 240 minutes
(4 hrs) after treatment. At each time point, corneal opacity, corneal thickness, and morphological
effects are evaluated. Fluorescein retention is examined at 30 minutes only. (Morphological effects
include pitting, loosening of the epithelium, roughening of the corneal surface, and sticking of test
compound to the corneal surface.) Photographs are recommended.
After the final time point, the eye is preserved in formaldehyde for possible later
examination (histopathology).
Two additional ocular irritancy tests are being validated by ICCVAM:
Isolated Rabbit Eye (IRE) test
Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM)
c. The Draize eye test
This test involves placing drops of a test compound in one eye of a rabbit and 24 hours later
examining it for reddening, blistering, corneal clouding or corrosion. The healing can be observed
after exposure. The original Draize test has been modified. Compounds known to be corrosive or
irritating are not tested in the Draize test and anesthetics are used whenever possible.
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a. EPISKIN TM
b. Transcutaneous Electrical Resistance (TER) Assay
c. Corrositex ®
http://iccvam.niehs.nih.gov/methods/dermal/dermal.htm
http://ecvam.jrc.cec.eu.int/index.htm
Skin Corrosion,
Irritation
European Centre for the Validation of Alternative Methods (ECVAM) validated and approved
(April 2007) the use of EPISKIN as a full replacement for animal testing for assessing skin
irritancy.
a. EPISKIN™ (from L’Oreal) is a three-dimensional human skin model comprised of a
reconstructed epidermis and a functional stratum corneum. The test material is topically
applied to the skin for 15 minutes with subsequent assessment of the effects on cell viability
after 42 hrs using the colorimetric MTT assay. Release of IL-1a is a recommended adjunct
to the MTT assay to increase the sensitivity of the test.
o www.iivs.org/images/EPISKIN_statement.pdf
b. The transcutaneous electrical resistance (TER) assay uses rat skin to identify corrosive
materials by the ability to produce a loss of normal stratum corneum integrity and barrier
function, which is measured as a reduction of the inherent transcutaneous electrical
resistance below a predetermined threshold level.
Not approved as a full replacement test, but useable in a tier-testing strategy.
c. Corrositex® is based on the ability of a corrosive chemical or chemical mixture to pass
through a biobarrier, by diffusion and/or destruction/erosion, and to elicit a color change in
the underlying liquid Chemical Detection System (CDS). Corrositex® is useful as a standalone assay for evaluating the corrosivity or noncorrosivity of acids, bases, and acid
derivatives. For other product classes, Corrositex® may be used as part of a tiered
assessment strategy.
Moisture Evaporation
Transepidermal Water Loss (TEWL)
One example: Delfin Technologies VapoMeter for measuring water evaporation. It is a closed
chamber system that calculates the evaporation rate from the increase of relative humidity in the
measurement chamber, compared to ambient humidity.
http://www.delfintech.com/prod_vapo_faq.html
Mutagenesis/ Carcinogenesis
Ames Test
The Ames test makes use of bacterial strains to determine if a compound causes mutations, which
theoretically could lead to cancer in humans. Special strains of Salmonella typhimurium are used
that require a mutation in order to grow into colonies on specific growth media. The special
bacteria require different types of mutations (frameshift and point) in order to grow. The bacterial
strains are grown in the presence of test substance. Only if mutations are induced will bacteria
grow into visible colonies. The tests may also be done in the presence of rat liver extract to
simulate in vivo metabolism of the compound, since some compounds are mutagenic only after
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being metabolized. The greater the number of colonies, the greater the mutagenic potential of the
test compound.
Fever induction
a. Limulus amebocyte lysate (LAL) test
b. Cytokine Production
a. The Limulus amebocyte lysate (LAL) test; http://www.fda.gov/CBER/gdlns/lal.pdf
The LAL has been adopted to replace injections in rabbits to determine if a fever is produced in 24
hrs. It is required for medical devices and injected compounds that come in contact with blood or
cerebral spinal fluid. The hemolymph (essentially, the blood cells) of the horseshoe crab react to
bacterial endotoxins, lipopolysaccharide, and (1,3)-β-D-glucan. These are the same compounds that
cause inflammation (and fever) in humans.
LAL is an aqueous extract of the blood cells of horseshoe crabs which forms a clot, becomes turbid,
or changes color, depending on the technique, in the presence of bacterial endotoxin. The test
sample is compared to a standard series of endotoxin dilutions. The endpoint or reaction times of
these dilutions are used to calculate the amount of endotoxin present in the sample. All tests are
performed in at least duplicate. Before performing the assay, one must determine if there is anything
in the test sample/solution that will interfere (inhibit or enhance) the LAL assay.
b. Cytokine Production
Human blood can be used to determine whether or not a test compound causes white blood cells
(leukocytes) to release cytokines (particular proteins) that are capable of signaling the initiation of a
fever. A variety of commercial testing kits are available for quantifying multiple cytokines at once
through colorimetric assays. Dilutions of the test compound are assayed and the results are
quantified by comparison to known dilutions of cytokines.
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The New York Times, November 20, 2007
http://www.nytimes.com/2007/11/20/business/worldbusiness/20cosmetics.html?_r=2&oref=slogin&pagewanted=print&ore
f=slogin
A New Science, at First Blush
By DOREEN CARVAJAL
GRASSE, France — The delicate hybrids thriving in the balmy climes of Provence, southern France’s traditional perfume
region, include sweet jasmine, May roses — and fresh layers of artificial human skin.
Scientists here are working feverishly to develop new technologies to test cosmetics before a European Union ban on
animal testing begins in March 2009.
These advanced materials — including reconstructed eye tissue and tiny circles of skin developed from donor cells
harvested from cosmetic operations — are a vital part of the industry’s future as it faces rapidly tightening European
regulations, rules that apply to any company wishing to sell in the 27-nation European Union.
The looming European ban is not only forcing multinational companies to adopt new practices. It is also bringing together
regulators in Brussels with agencies from the world’s other large cosmetics markets — the Food and Drug Administration
in the United States and the Ministry of Health in Japan — to harmonize regulation.
Even more surprising, the new standards are pushing longtime secretive rivals to cooperate, grudgingly and sometimes with
prodding from regulators and politicians.
The European commissioner for science, Janez Potocnik, appeared this month at a meeting for multinational companies and
chided them for slowing the search for alternatives by failing to share information.
The stakes are high: Europe is the world’s leading cosmetics market, and it also exports more than $23.4 billion worth of
cosmetics every year. Cosmetics exported from the United States to Europe amount to nearly $2 billion a year, about 7
percent of the European market. After the United States, Japan is the second leading provider of cosmetics to Europe .
“Without question these regulations are having an impact,” said Dr. Alan Goldberg, director of the Center for Alternatives
to Animal Testing at Johns Hopkins University in Baltimore. “What company is going to want to eliminate 450 million
customers by not complying?”
The cosmetics giant L’Oréal has devoted more than $800 million in the last 20 years to the development of alternatives to
animal testing, while its American rival, Procter & Gamble, maker of the Cover Girl line, has spent almost $225 million.
“For the cosmetics industry, it’s a race,” said Hervé Groux, 45, a French immunology scientist who presides over a year-old
research lab in Grasse that aids smaller companies lacking the resources of titans like L’Oréal and Procter & Gamble. “The
rules are pushing everyone to move faster and to put more money into research.”
The European Commission itself is spending almost 25 million euros ($36.5 million) yearly on the search for animal
alternatives, while many countries are seeding programs with annual budgets of 15 million to 20 million euros.
Mr. Groux’s lab, Immunosearch, had its official debut party Wednesday in a boxy industrial park, where Mr. Groux and his
wife, a molecular biologist, and other newly recruited veteran researchers are striving to shape a new world of beauty
research — and at the same time spare the lives of thousands of rabbits, mice, rats and guinea pigs.
As the 2009 deadline approaches, European regulators issue periodic tallies of the number of laboratory animals potentially
spared by alternatives to animal tests, across all kinds of industrial uses. Part of the pressure for alternatives also stems from
additional legislation, known as Reach, requiring companies to develop safety data on 30,000 chemicals over the next 11
years — research that could raise the prospect of increased animal testing.
In fact, the actual number of animals tested for cosmetics is small compared with medical or educational uses, according to
a new European Commission report. But from 2002 to 2005 the tally grew 50 percent in Europe, to 5,571 animals.
Much of that testing was taking place here in France, the country that leads Europe in testing and vigorously fought the ban,
ultimately appealing, in vain, to the European Court of Justice.
But it is also in Provence — a region fabled for its fragrances and the professional “noses” who create them — where
scientists are gathering to work on alternative testing research in vitro, literally “in the glass.”
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In nearby Nice, SkinEthic, a 15-year-old company, is developing and manufacturing a line of cellular tools that includes a
wide range of human tissues. Last year, SkinEthic was purchased by L’Oréal, which propelled the parent company into a
dominant position in the testing field, with two critical patents on reconstructed skin. SkinEthic produces its own form of
reconstructed skin, RHE, while L’Oréal holds the patent to Episkin, which its scientists developed in Lyon.
Episkin was validated this year by European regulators as a test tool that could fully replace animals. Its closest competitor,
EpiDerm — developed by MatTek, a company in Ashland, Mass. — received only qualified approval for research use
because the artificial skin reacted too sensitively, producing different results than natural skin would.
To make Episkin, donor keratinocyte cells, collected after breast and abdominal plastic surgery, are cultured in tiny wells of
collagen gel, immersed in water, amino acids and sugars, and then air-dried for 10 days or aged to mimic mature skin by
exposure to ultraviolet light.
Cosmetics are tested by smothering the almost babylike skin with the cosmetic material. The skin is checked for dying cells
by adding a yellow chemical, MTT, which turns blue against living tissue, and then checked again for irritation.
“We have finally succeeded in showing that artificial skin can fully replace rabbit testing,” said Thomas Hartung, the head
of the European Center for the Validation of Alternative Methods in Italy.
His agency, which is part of the European Commission, is also in the midst of evaluating 12 methods for testing eye
irritation, to replace the classic Draize test on rabbits that dates back to the mid-1940s.
“If there’s a holy grail that we’re searching for, it’s a test for eye irritation,” said Mr. Hartung. “The issue has a very strong
emotional factor.”
Other alternatives include using tiny membranes within chicken eggs for testing chemicals, because the blood vessels
mimic human eye membranes. The center has also approved the use of cow and chicken eyeballs cast off from
slaughterhouses.
The mighty research budgets of the large multinational companies threaten to sweep aside smaller players with limited
resources and different needs. But out of necessity, historic rivals in the secretive fragrance industry have joined to back
Immunosearch, their homegrown research project, as a “deterrent force” to L’Oréal and P.& G.
Robertet, founded in 1850, and Mane Fils, dating back to the late 19th century, are supporting Immunosearch’s ambition of
adapting L’Oréal’s Episkin to be more suitable for testing natural ingredients. Immunosearch is also benefiting from
millions of dollars in private investments and government grants, as well as alliances with France’s National Science
Research Center, nearby, and its National Institute for Research in Computer Science.
Robertet and Mane Fils, perfume companies run for four generations by rival families, acted out of self-interest. Both said
they were alarmed that European regulations would force them to submit complex natural ingredients, like lavender
essence, to the same tests intended for chemicals.
“Our industry has poorly defended natural ingredients because our essences have been classified as chemicals,” said
Philippe Maubert, president of Robertet. “It’s unfortunate because these ingredients have existed for centuries.”
The companies worry that costly new European testing regulations could spell the end of many essential oils used in
perfumery because the substances are a blend rising out of a distillation process that could fail existing chemical tests for
safety.
“Those tests were developed to make tests on pure substances with few impurities,” said Eric Angelini, the regulatory
affairs manager at Mane. “But our essential oils are a natural blend of materials coming from the plant that is part of the
distillation process.”
At the Champagne party on Wednesday to celebrate Immunosearch’s debut, city and regional officials crowded into the
pristine laboratory, where kits of artificial skin layers that are sold by the dozen were stocked in a special machine warmed
to human body temperature.
Jean-Pierre Leleu, the mayor of Grasse, toasted the future with a reminder that the industry has come full circle.
In the 16th century, Grasse was a leather-tanning town that specialized in perfumed gloves. In the 18th century, the glove
makers and perfumers split from the tanners to concentrate on perfumery.
Now, Mr. Leleu said, the industry has turned its attention back to skin, albeit human.
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