**~ 1**  **~**



## 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 th e 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) rat her 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 vulnera ble 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 AUC 24 ) in addition to the two metrics mentioned above for developmental effects; i.e., t he 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 fi rst 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 co nsistent 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 in consistent 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 AUC 24 , the maximum 24
- hr AUC as described above; b) Average AUC 24 , 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. (198 2) 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 t he pups on GD 21.**~ 2**  **~**



### Table 1. **Becci et al. (1982) Fetal BW Dose -**  **Response Exposure (mg/**  **kg )**  **Max AUC 24**  **(mg -**  **h/L) Average AUC**  **24 (mg**  **- h/L)**  **Cmax (mg/L) Infant BW (g)**  0 0 0 0 3.5 75 536 3 59 202 3.5 237 2070 1 385 641 3.5 750 8964 60 00 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/m 3**  **) Max AUC**  **24 (mg**  **- h/L)**  **Average AUC 24**  **(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 in ternal 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 interna l 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. T he internal doses estimated with the PBPK model indicate that the lack of responses in fetal BW at 75 and 23 7 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 o ccurred. O ther factor s must be considered for the dose
- response comparison . H ow is it possible that the Saillenfait et al. (2003) animals appear to have respond ed at much lower internal doses than the animals dosed by Becci et al. (1982)? A key differenc e 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**~ 3**  **~**

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 averag e AUC 24 is likely the best internal dose metric for this endpoint, in particular when evalu ating 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 o btained 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 dev iations. 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) a lso 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 effect s . 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 be tween 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) show ed a significant decrease in fetal BW and significant increas ed incidence for vertebrae incomplete ossification
. But c hanges in fetal BW appear to be reversible and depend on continued exposure that would be best expressed as the average AUC 24 . On the other hand, the type of fetal skeletal effects analyzed here (i.e., vertebrae/ thoracic vertebral cen tra, incomplete ossification) may be less reversible and may result from acute - dose effect s. For vertebrae incomplete ossification , consistency of the two dose
- response data sets do es not preclude Cmax or Max AUC 24 as metrics.**~ 4**  **~**

**Table 3. Becci et al**  **. (1982) “Vertebrae, incomplete ossification Exposure (mg/kg)**  **Max AUC 24**  **(mg -**  **h/L) Average AUC**  **24 (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 o ssification”**  **Exposure (mg/m 3**  **) Max AUC**  **24 (mg**  **- h/L)**  **Average AUC 24**  **(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 p rovided. **Table 5. Saillenfait et al. (2002) “Litters with skeletal malformations”**  **Exposure (mg/kg) Max AUC**  **24 (mg**  **- h/L)**  **Average AUC 24**  **(mg -**  **h/L) Cmax (mg/L)**  **Litters/total** 0 0 0 0 0/20 125 659 574 86 0 /2 1 250 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 skeleta l 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) .** ~** **5 ~** 

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. So me caution is warranted, though, since the change from control seen by Becci et al. (1982) is only 2 additional litters a ffected and by itself is not statistically significant. 2) Both the max AUC 24 and Cmax metrics are consistent in where the dose respon se 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 AUC 24 metrics appe ar reasonable for further evaluation.**~ 6**  **~**

**Figure 1. Plots of “extra risk” vs. dose for Becci**  **(1982) dermal exposures v s. Saillenfait**  **et al. (2002) oral studies** 

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 1000 2000 3000 4000 5000 6000**E x** **t r** **a r** **i s** **k**



## Ave **ra ge AUC (mg**  **- hr/L)**  Saillenfait et al. (2002), oral Becci et al. (1982), dermal 

## Average AUC24 metric

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 1500 3000 4500 6000 7500 9000**E x** **t r** **a r** **i s** **k**



## Ave **ra ge AUC (mg**  **- hr/L)**  Saillenfait et al. (2002), oral Becci et al. (1982), dermal 

## Max AUC24 metric

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 300 600 900 1200 1500 1800 2100**E x** **t r** **a r** **i s** **k**



## Concentation (mg **/L)** Saillenfait et al. (2002), oral Becci et al. (1982), dermal 

## Cmax metric**~ 7**  **~**

**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 stu dies are analyzed together for both the Cm ax metric and the max AUC 24 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 dat a 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 ossif ication, Cmax metric**  Dose -- Cmax N E ffect 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 l ogist i c 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).** **~ 8**  **~**

**Figure 2. Plots for log -**  **logistic model and quantal linear model, BMDL05 (Cmax)**  **for vertebrae** 

**incomplete ossification**

0 0.2 0.4 0.6 0.8 1

0 500 1000 1500 2000FractionAffected

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

0 0.2 0.4 0.6 0.8 1

0 500 1000 1500 2000FractionAffected

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**~ 9**  **~**

**Table 8. Combined Becci et al., (1982) dermal and Saillenfait et al. (2003) inhalation data for vertebrae**  **incomplete ossification, max AUC**  **24 metric**  Dose – AUC24 N E ffect 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 AUC 24**  **metric** 

### BMD Estimation Result **s** 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).**~ 10**  **~**

**Figure 3. Plots for log -**  **logistic model and quantal linear model, BMDL05 (max AUC 24**  **) for vertebrae** 

**incomplete ossification**

**Refere nces**  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. 0 0.2 0.4 0.6 0.8 1

0 1000 2000 3000 4000 5000 6000 7000 8000 9000FractionAffected

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