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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) Infant BW (g)

0 0 0 0 3.5

75 536 359 202 3.5

237 2070 1385 641 3.5

750 8964 6000 2050 2.8*

*Significantly different from water treated group (negative control), p < 0.05

Table 2. Saillenfait et al. (2003) Fetal BW Dose-Response

Exposure (mg/m3) Max AUC24 (mg-h/L) Average AUC24 (mg-h/L) Cmax (mg/L) Infant BW (g)

0 0 0 0 5.67 ± 0.37

122 96 94 12 5.62 ± 0.36

243 194 191 24 5.47 ± 0.25

487 404 397 50 5.39 ± 0.45*

BMCL05 255 246 30.7 –

*Significant difference from the control (air), P<0.05.

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) Litters/total

0 0 0 0 10/24

122 96 94 12 5/20

243 194 191 24 8/19

487 404 397 50 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) Cmax (mg/L) Litters/total

0 0 0 0 0/20

125 659 574 86 0/21

250 1478 1281 179 0/24

500 3498 3024 380 12/25**

750 5911 5110 591 3/5**

**Significant difference from vehicle control, p < 0.01

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

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Average AUC (mg-hr/L)

Saillenfait et al. (2002), oral

Becci et al. (1982), dermal

Average AUC24 metric

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Average AUC (mg-hr/L)

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Becci et al. (1982), dermal

Max AUC24 metric

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0 300 600 900 1200 1500 1800 2100

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Concentation (mg/L)

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Becci et al. (1982), dermal

Cmax metric
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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 N Effect notes

0 48 22 combined

11.9 20 5 Saillenfait

24.5 19 8 Saillenfait

49.57 25 5 Saillenfait

202.4 22 11 Becci

641.1 23 14 Becci

2049.6 22 18 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 0.125 235.31 180.45 102.61 91.49 49.95

Logistic 0.179 233.77 244.03 169.56 123.48 85.78

LogLogistic 0.140 235 174.78 57.27 98.26 27.13

LogProbit 0.203 233.329 277.44 169.82 192.93 118.09

Multistage 0.205 233.327 162.95 102.49 79.33 49.90

Probit 0.175 233.84 251.66 181.67 127.01 91.67

Weibull 0.125 235.33 168.05 102.5 82.63 49.90

Quantal-Linear 0.205 233.327 162.95 102.49 79.33 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

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Fraction Affected

dose

Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

15:13 09/06 2013

BMDL BMD

 

Log-Logistic

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Fraction Affected

dose

Quantal Linear Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

14:52 09/06 2013

BMDL BMD

 

Quantal Linear
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Table 8. Combined Becci et al., (1982) dermal and Saillenfait et al. (2003) inhalation data for 

vertebrae incomplete ossification, max AUC24 metric

Dose – AUC24 N Effect notes

0 48 22 combined

95.8 20 5 Saillenfait

194.4 19 8 Saillenfait

404.2 25 5 Saillenfait

536.5 22 11 Becci

2070.3 23 14 Becci

8963.7 22 18 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

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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dose

LogProbit Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

22:01 09/05 2013

BMDL BMD

 

LogProbit