Using Internal Dose-Response Comparisons to Identify Dose Metrics
Which Best Correlate With Toxicity
(Version 09/16/13)
An internal dose metric such as a measure of toxicant concentration in the blood is expected to be a
better predictor of response than the applied dose (e.g., concentration in air) since it is closer to the site
of damage and chemical activity which causes that damage. Further, a good internal dose metric should
correlate with or be predictive of toxicity irrespective of the route of exposure by which it occurs.
However this is only true if the metric is in fact a measure of the likelihood of a toxic response, or
intensity of a toxic effect. For example, if the parent compound is the toxic moiety while a metabolite is
relatively inert, then the concentration of the metabolite is a measure of exposure, but one that would
likely be a worse predictor than the applied dose.
For NMP the existing toxicity data identify the parent (NMP) rather than the primary metabolite
(5HNMP) as the proximate toxicant. But toxicity could depend on peak concentration (Cmax,
irrespective of the number of days or hours over which it occurs), the area-under-the curve (AUC) of
concentration during the vulnerable period of fetal development (lasting a week or more in rats, likely
longer in humans), or some intermediate measure. Here we consider the highest 24-hour AUC (Max
AUC24) in addition to the two metrics mentioned above for developmental effects; i.e., the highest AUC
for a 24-hour period, which would reflect a need for more than momentary exposure for a toxic effect,
but that a significant developmental effect could result from a single day exposure during a critical
period of vulnerability.
Below we first analyze dose-metrics related to reductions in fetal body weight (BW) as a toxic endpoint
and then skeletal aberrations. By comparing these metrics and observed responses for two (or more)
routes of exposure, we can determine if a metric is at least consistent in its prediction of a toxic
response across routes. For example, if a much higher Cmax occurs for oral exposures for which the
toxic endpoint is not triggered than for inhalation exposures for which the response is triggered, that
would be an inconsistent metric relative to the response, indicating that some other metric would be
better. Specifically, we compare dose-response relationships based on the following internal doses
estimated using the EPA-revised PBPK model: a) Max AUC24, the maximum 24-hr AUC as described
above; b) Average AUC24, the average daily AUC over the entire study period (total AUC divided by the
number of days), GD6-20 for Becci et al. (1982) and GD5-21 for Saillenfait et al. (2003); and c) Cmax.
Fetal BW
Becci et al. (1982) observed a reduction in fetal BW at the highest dose administered by dermal
exposure (Table 1), while Saillenfait et al. (2003) observed a significant reduction in fetal BW at their
highest inhalation exposure (Table 2), though with a clearer trend across intermediate doses. Becci et
al. (1982) applied their dermal doses on GD 6 to GD 15 and evaluated the impact on the developing pups
at GD 20. Saillenfait exposed by inhalation from GD 6to GD 20 (6 h/d) and evaluated the pups on GD 21.
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Table 1. Becci et al. (1982) Fetal BW Dose-Response
Exposure (mg/kg)
Max AUC24 (mg-h/L) Average AUC24 (mg-h/L) Cmax (mg/L)
0
0
0
0
75
536
359
202
237
2070
1385
641
750
8964
6000
2050
*Significantly different from water treated group (negative control), p < 0.05
Infant BW (g)
3.5
3.5
3.5
2.8*
Table 2. Saillenfait et al. (2003) Fetal BW Dose-Response
Exposure (mg/m3)
Max AUC24 (mg-h/L) Average AUC24 (mg-h/L)
0
0
0
122
96
94
243
194
191
487
404
397
BMCL05
255
246
*Significant difference from the control (air), P<0.05.
Cmax (mg/L)
0
12
24
50
30.7
Infant BW (g)
5.67 ± 0.37
5.62 ± 0.36
5.47 ± 0.25
5.39 ± 0.45*
–
The first thing to be noted is that all of the internal doses for the Saillenfait et al. (2003) study are well
below the corresponding doses for the Becci et al. (1982). Further, the Benchmark Concentration Lower
(95%) confidence limit for a 5% response (BMCL05) values obtained using each of the internal metrics
from Saillenfait et al. (2003) with their observed BW response are all below the lowest corresponding
internal doses for Becci et al. (1982), where there is no indication of any toxicity.
The internal doses estimated with the PBPK model indicate that the lack of responses in fetal BW at 75
and 237 mg/kg by dermal application did not occur because a smaller amount of NMP penetrated to the
maternal circulation than occurred from the inhalation exposures of Saillenfait et al. (2003) where an
effect occurred. Other factors must be considered for the dose-response comparison.
How is it possible that the Saillenfait et al. (2003) animals appear to have responded at much lower
internal doses than the animals dosed by Becci et al. (1982)? A key difference in the two studies is the
days when dosing vs. observation occurred:
Saillenfait exposed GD 6-20 and sacrificed on GD 21;
Becci exposed GD6-15 and sacrificed on GD 20.
A possible explanation is that the 5 days between administration of the last dose and animal sacrifice in
the Becci et al. study allowed for a partial recovery with regard to this endpoint. NMP’s
pharmacokinetics are such that it is almost entirely cleared within 24 hours. So the animals in the Becci
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et al. study would have had 5 days of development with essentially zero internal dose. On the other
hand, given the very high internal doses associated with the Becci et al. (1982), it seems unlikely that a
larger effect on fetal BW would not have occurred, if they had continued dosing until GD 19 (one day
before sacrifice in that study) or GD 20 (the last day of exposure in the Saillenfait et al., 2003 study).
Thus it appears that the fetal BW decrement depends on continued exposure, rather than intermittent
exposure. Thus, a higher exposure occurring earlier in gestation is not the principal determinant for this
adverse effect on BW. Therefore, the average AUC24 is likely the best internal dose metric for this
endpoint, in particular when evaluating the risks of workplace exposures which can occur repeatedly
over longer periods of gestation.
If this explanation is valid, then human internal doses from dermal as well as inhalation exposure should
be evaluated against internal dose BMCL05 values obtained from the Saillenfait et al. (2003) study,
particularly for workplace scenarios.
Note that BMD calculations for the fetal BW endpoint were not conducted for Becci et al., (1982)
because the mean fetus weight values were reported without standard deviations. Consequently,
BMDL05 comparisons for skeletal effects, but not the fetal BW endpoint, were conducted for the dermal
and inhalation internal doses reported in Becci et al., (1982) and Saillenfait et al. (2003).
Skeletal Effects
Becci et al. (1982) observed a number of significant skeletal abnormalities at their highest dose. Here
the incomplete ossification of vertebrae (Table 3) is specifically considered because the dose-response is
amenable to benchmark analysis, and Saillenfait et al. (2003) also reported this endpoint (Table 4). As
seen in Tables 3 and 4 (same internal dose metrics as those for fetal BW), the results of Saillenfait et al.
(2003) are consistent with those of Becci et al. (1982), irrespective of dose metric, for skeletal effects. In
particular Saillenfait et al. (2003) did not see a significant effect at the lower internal dose levels from
their inhalation exposures, compared to the effect seen by Becci et al. (1982). Thus all of the internal
metrics appear to be consistent between the Becci et al. (1982) dermal exposures and Saillenfait et al.
(2003) studies. Note that the highest dose (750 mg/kg) in Becci et al., (1982) showed a significant
decrease in fetal BW and significant increased incidence for vertebrae incomplete ossification. But
changes in fetal BW appear to be reversible and depend on continued exposure that would be best
expressed as the average AUC24. On the other hand, the type of fetal skeletal effects analyzed here (i.e.,
vertebrae/ thoracic vertebral centra, incomplete ossification) may be less reversible and may result from
acute -dose effects. For vertebrae incomplete ossification, consistency of the two dose-response data
sets does not preclude Cmax or Max AUC24 as metrics.
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Table 3. Becci et al. (1982) “Vertebrae, incomplete ossification
Exposure (mg/kg)
Max AUC24 (mg-h/L) Average AUC24 (mg-h/L) Cmax (mg/L)
Litters/total
0
0
0
0
12/24
75
536
359
202
11/22
237
2070
1385
641
14/23
750
8964
6000
2050
18/22*
BMCL05†
110
74
31.2
–
*Significantly different from water treated group (negative control), p < 0.05
†
Only LogLogistic model tested for AUC metrics; was selected for a proportional average AUC metric
Table 4. Saillenfait et al. (2003) “Thoracic vertebral centra, incomplete ossification”
Exposure (mg/m3)
Max AUC24 (mg-h/L) Average AUC24 (mg-h/L) Cmax (mg/L)
0
0
0
0
122
96
94
12
243
194
191
24
487
404
397
50
Litters/total
10/24
5/20
8/19
5/25
Further indication of which internal metric is best may come from also analyzing the oral toxicity study
of Saillenfait et al. (2002), for which the dose-response is given in Table 5. Unfortunately incomplete
ossification of the vertebrae was not reported in this study, but the total incidence of skeletal
abnormalities was provided.
Table 5. Saillenfait et al. (2002) “Litters with skeletal malformations”
Exposure (mg/kg)
Max AUC24 (mg-h/L) Average AUC24 (mg-h/L)
0
0
0
125
659
574
250
1478
1281
500
3498
3024
750
5911
5110
**Significant difference from vehicle control, p < 0.01
Cmax (mg/L)
0
86
179
380
591
Litters/total
0/20
0/21
0/24
12/25**
3/5**
The Becci et al. (1982) dermal results appear roughly consistent with the Saillenfait et al. (2002) oral
studies; the skeletal responses become significant in the same range of doses. Hence examining these
more closely may give insight as to which is the best dose metric.
However, the incomplete ossification characterized by Becci et al. (1982) had a 50% background rate,
while the skeletal malformations scored by Saillenfait et al. (2002) had a zero background rate. In order
to put these on a more similar scale, the extra risk was calculated:
Extra risk = (response – background)/(100% – background) .
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For Saillenfait et al. (2002), this is just the fraction of litters effected since the background is zero, but for
Becci et al. (1982) it is the increase over background relative to the fraction of unaffected litters in the
control group. Plots of the extra risk for each study vs. the three metrics follow (Figure 1).
1) The average AUC metric (first plot) appears slightly inconsistent in that there is an increase in the
Becci et al. (1982) response at 1385 mg-hr/L, but no response in the Saillenfait et al. (2002) study at
1281 mg-hr/L. Some caution is warranted, though, since the change from control seen by Becci et
al. (1982) is only 2 additional litters affected and by itself is not statistically significant.
2) Both the max AUC24 and Cmax metrics are consistent in where the dose response changes from
control. It appears more so for the Cmax metric, but this may be a result of the dose spacing.
3) Given that total skeletal abnormalities (Saillenfait et al., 2002) should be inclusive of the specific
vertebrae delayed ossification (Becci et al., 1982), that the response at higher doses is greater for
the Saillenfait et al. (2002) study when based on the Cmax metric (last plot) does not necessarily
indicate an inconsistency for this metric.
Hence both the Cmax and the max AUC24 metrics appear reasonable for further evaluation.
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Figure 1. Plots of “extra risk” vs. dose for Becci (1982) dermal exposures vs. Saillenfait et al. (2002)
oral studies
0.7
Average AUC24 metric
0.6
Extra risk
0.5
0.4
0.3
0.2
Saillenfait et al. (2002), oral
0.1
Becci et al. (1982), dermal
0
0
0.7
1000
2000
3000
4000
5000
Average AUC (mg-hr/L)
6000
Max AUC24 metric
0.6
Extra risk
0.5
0.4
0.3
0.2
Saillenfait et al. (2002), oral
0.1
Becci et al. (1982), dermal
0
0
1500
3000
4500
6000
7500
Average AUC (mg-hr/L)
0.7
9000
Cmax metric
0.6
Extra risk
0.5
0.4
0.3
0.2
Saillenfait et al. (2002), oral
Becci et al. (1982), dermal
0.1
0
0
300
600
900
1200 1500 1800 2100
Concentation (mg/L)
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Combined inhalation and oral dose-response analysis
Since the Saillenfait et al. (2003) inhalation data and the Becci et al. (1982) dermal data for vertebrae
delayed ossification appear consistent relative to the internal dose metrics, the two studies are analyzed
together for both the Cmax metric and the max AUC24 metric, since both of those also appear consistent
with the oral response of Saillenfait et al. (2002), as shown just above (Tables 6-9; Figures 2 and 3).
Combining the data sets should provide additional statistical power for identifying the BMD(L) and
provide a more robust dose-response (low to high).
Table 6. Combined Becci et al., (1982) dermal and Saillenfait et al. (2003) inhalation data for
vertebrae incomplete ossification, Cmax metric
Dose -- Cmax
0
11.9
24.5
49.57
202.4
641.1
2049.6
N
48
20
19
25
22
23
22
Effect
22
5
8
5
11
14
18
notes
combined
Saillenfait
Saillenfait
Saillenfait
Becci
Becci
Becci
Table 7. Benchmark dose analysis of combined Becci et al. (1982) dermal and Saillenfait et al. (2003)
inhalation data for vertebrae incomplete ossification, Cmax metric
BMD Estimation Results
Model
P-Value
AIC
BMD
(BMR=10%)
BMDL
(BMR=10%)
BMD
(BMR=5%)
BMDL
(BMR=5%)
Gamma
Logistic
LogLogistic
LogProbit
Multistage
Probit
Weibull
Quantal-Linear
0.125
0.179
0.140
0.203
0.205
0.175
0.125
0.205
235.31
233.77
235
233.329
233.327
233.84
235.33
233.327
180.45
244.03
174.78
277.44
162.95
251.66
168.05
162.95
102.61
169.56
57.27
169.82
102.49
181.67
102.5
102.49
91.49
123.48
98.26
192.93
79.33
127.01
82.63
79.33
49.95
85.78
27.13
118.09
49.90
91.67
49.90
49.90
Note: Multistage model and Quantal-Linear model have the lowest AIC. But the BMDLs exceed 3-fold
range, so the BMDL from the Log logistic model should be selected. The Log logistic and the best fitted
model Quantal-linear is shown below.
The BMDL05 is essentially identical to that obtained from the analysis of the BW data using the same
metric (30.9 mg/L) and for the analysis of the Becci et al. data alone (31.2 mg/L).
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Figure 2. Plots for log-logistic model and quantal linear model, BMDL05 (Cmax) for vertebrae
incomplete ossification
Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
1
Log-Logistic
Fraction Affected
0.8
0.6
0.4
0.2
0
BMDL
BMD
0
500
1000
1500
2000
dose
15:13 09/06 2013
Quantal Linear Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
1
Quantal Linear
Fraction Affected
0.8
0.6
0.4
0.2
0
BMDL
0
BMD
500
1000
dose
14:52 09/06 2013
~8~
1500
2000
Table 8. Combined Becci et al., (1982) dermal and Saillenfait et al. (2003) inhalation data for
vertebrae incomplete ossification, max AUC24 metric
Dose – AUC24
0
95.8
194.4
404.2
536.5
2070.3
8963.7
N
48
20
19
25
22
23
22
Effect
22
5
8
5
11
14
18
notes
combined
Saillenfait
Saillenfait
Saillenfait
Becci
Becci
Becci
Table 9. Benchmark dose analysis of combined Becci et al. (1982) dermal and Saillenfait et al. (2003)
inhalation data for vertebrae incomplete ossification, max AUC24 metric
BMD Estimation Results
Model
P-Value
AIC
BMD
(BMR=10%)
BMDL
(BMR=10%)
BMD
(BMR=5%)
BMDL
(BMR=5%)
Gamma
0.07
236.82
853.56
439.84
444.50
214.13
Logistic
0.11
235.22
1079.48
733.36
545.60
370.63
LogLogistic
0.08
236.39
783.86
247.37
447.77
117.18
LogProbit
0.13
234.57
1187.74
698.74
825.93
485.89
Multistage
0.12
234.86
718.01
438.26
349.56
213.36
Probit
0.11
235.28
1114.7
790.95
562.06
298.77
Weibull
0.07
236.85
788.37
438.71
395.28
213.58
Quantal-Linear
0.12
234.86
718.01
438.26
349.56
213.36
Note: Models that cannot fit the data properly are not considered. LogProbit model, which has the
lowest AIC value is selected. Figure for LogProbit model is shown below (next page).
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Figure 3. Plots for log-logistic model and quantal linear model, BMDL05 (max AUC24) for vertebrae
incomplete ossification
LogProbit Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
1
LogProbit
Fraction Affected
0.8
0.6
0.4
0.2
0
BMDL
0
BMD
1000
2000
3000
4000
5000
6000
7000
8000
9000
dose
22:01 09/05 2013
References
Becci, P. J., Knickerbocker, M. J., Reagan, E. L., Parent, R. A., and Burnette, L. W. (1982). Teratogenicity
study of N-methylpyrrolidone after dermal application to Sprague-Dawley rats. Fundam. Appl. Toxicol.
2(2), 73-76.
Saillenfait, A. M., Gallissot, F., and Morel, G. (2003). Developmental toxicity of N-methyl-2-pyrrolidone in
rats following inhalation exposure. Food Chem. Toxicol. 41(4), 583-588.
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