N,N-dimethylformamide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication, which meets basic scientific principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

published 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication, which meets basic scientific principles

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

- Principle of test: Continuous breeding protocol (NTP): a dose range-finding phase (optional), an F0 cohabitation and lactation phase, a crossover mating trial of the F0 generation (conducted if F0 reproductive performance is affected), and finally fertility assessment of the Fl generation (born and reared during the F0 lactation phase).

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

(CD-l® (ICR)BR outbred Swiss albino mice; Charles River Laboratories, Inc., Raleigh, NC), 6 weeks old upon arrival, During quarantine (2 to 5 weeks), animals were housed singly.
Throughout the study, solid-bottom polypropylene or polycarbonate cages (111⁄2“ 3 7” 3 5“) with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ) and Ab-Sorb-Dri® bedding (Laboratory Products, Garfield, NJ) were used.
Cages were changed at least once weekly.
Deionized/filtered water and pelleted feed (NIH-07 Rodent Chow, Zeigler Brothers, Gardners, PA) were made available ad libitum.
Temperature and humidity were maintained at mean values of 72 +/- 0.2 °F and 58 +/- 0.1 % RH, and light/dark cycles were automatically controlled at 14-h light/10-h dark (Barber- Colman Network 8000 System, Barber-Colman Company, Loves Park, IL).
All animals were identified using a tail tattoo (AIMS, Inc., Piscataway, NY).

Route of administration:

oral: drinking water

Vehicle:

other: deionized/filtered drinking water

Details on exposure:

In the dose range-finding trials, DMF was dosed as 0, 2500, 5000, 7500, 10,000, and 15,000 ppm.
For each study, 48 males and 48 females at 8 weeks of age were randomly assigned to six treatment groups (eight/sex/ group) by stratified randomization using body weight.
In the remainder of the RACB study, DMF was administered at 0, 1000, 4000, and 7000 ppm.

F0 cohabitation and lactation (definitive studies). For each study, 100 male and 100 female Swiss CD-1® mice, 11 weeks of age, were assigned to one of four dose groups by stratified randomization on the basis of body weight. The control group consisted of 40 males and 40 females, and each FORM- or DMF-treated group consisted of 20 males and 20 females. Body weights and feed and water consumption were monitored during treatment weeks 1, 8, and 16.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Confirmation of purity and identity was conducted at Research Triangle Institute (NTP/NIEHS Contract No. N01-ES-45061) and involved infrared spectrometry and nuclear magnetic resonance spectrometry. Purity of the bulk compound was reconfirmed periodically during the study by capillary column gas chromatography. Stability, homogeneity, and dose simulation studies were also completed (24,25), and dosing regimens were based on those studies.
Dosing formulations were tested to verify concentrations five times during each of the studies and were within 90 to 110 % of targeted concentrations.

Details on mating procedure:

F0 cohabitation and lactation:
During Week 1 of exposure to chemicals, animals were individually housed. During Weeks 2 through 15, animals were housed in breeding pairs within dose groups, and new-born litters were sacrificed immediately after evaluation. Data collected during the F0 cohabitation were the litter interval, number, sex, weight of pups per litter, number of litters per breeding pair, and the Postnatal Day (PND) 0 dam body weight. Because fertility was severely affected at Litter 5 for FORM and throughout the 14-week cohabitation period for DMF, crossover mating trials were conducted for both studies using the control and high-dose groups, and an F1 fertility assessment phase using the control, low-, mid-, and high-dose groups was conducted (see below). Starting at Week 16 of exposure, the breeding pairs were separated, and F0 females were allowed to deliver and rear the final litter until PND 21. On PND 0, 4, 7, 14, and 21 of the lactation phase, pups were sexed, counted, and weighed. On PND 21, randomly selected Fl pups from each dose group were weaned and housed in same-sex pairs by dose and saved for the Fl fertility assessment phase.

After completion of the cohabitation and lactation phase, all F0 animals were maintained on FORM or DMF dosed water until scheduled sacrifice after the completion of the crossover mating phase.

Cross-over mating trial:
The crossover mating trial was conducted using the control and high-dose animals for both the FORM and the DMF studies. Three breeding groups of F0 animals were created: (1) control male 3 control female, (2) high-dose male 3 control female, and (3) control male 3 high-dose female.
Beginning at Week 23 of treatment, animals were cohabited until a vaginal copulatory plug was observed or for 1 week, whichever occurred first. During the week of cohabitation, no treatments were administered to avoid possible exposure of control animals to FORM or DMF or their metabolites. At the start of Week 24, all animals were singly housed, and dosing was resumed.

Duration of treatment / exposure:

Premating exposure period (males & females): 7 d; test duration up to the F2 generation

Frequency of treatment:

Continuously in the drinking water, the actual doses delivered were calculated periodically, considering consumption and body weights, and are reported (24,25) and summarized in the Results section.

Duration of test:

up to 21 day of lactation phase of F1 animals

Dose / conc.:

1 000 ppm (nominal)

Remarks:

ca. 219 mg/kg/d

Dose / conc.:

4 000 ppm (nominal)

Remarks:

ca. 820 mg/kg/d

Dose / conc.:

7 000 ppm (nominal)

Remarks:

ca. 1455 mg/kg/d

No. of animals per sex per dose:

20/sex/ dose

Control animals:

yes, concurrent vehicle

Details on study design:

F1 fertility assessment.
At weaning (PND 21), randomly selected Fl pups from the control, low-, mid-, and high-dose groups were housed two per cage by sex within dose group for both studies. Although FORM or DMF was administered directly to the weanlings beginning on PND 22, prior to direct dosing, indirect exposure to FORM or DMF likely occurred to gametes, in utero, or during lactation. At 74 6 10 d of age, males and females in the control or treated groups were cohabited as non-sibling breeding pairs until a vaginal copulatory plug was observed or for 1 week, whichever occurred first. Because of reduced survival in some of the dose groups, especially the high-dose group of DMF, 20 non-sibling pairs were not available, so 15 pairs were cohabited, some of which were siblings. Litter data resulting from the Fl cohabitation were collected as described above for F0 adults in the crossover mating trial.

Maternal examinations:

Upon delivery of each litter, lethality, gestation length, sex, number, weight of the pups, and dam weight were determined. After all litters had been delivered, vaginal smears were collected from F0 females for 12 d. At Week 29 of FORM or DMF exposure, immediately following CO2 asphyxiation, all F0 males and females were weighed and necropsied. Liver and paired kidney (with attached adrenal) weights were collected for both sexes. The right ovary with attached oviducts was weighed in females. All tissues, except ovaries, were fixed in 10 % neutral buffered formalin. Male reproductive tissues were embedded in glycol methacrylate, sectioned at 2.5-mm thickness, and stained with hematoxylin/PAS (Bio-Tek Research Consultants, Durham, NC). Ovaries were fixed in Bouin’s fixative for 24 h, then rinsed and held in 70 % ethanol until embedding into paraffin. All other tissues were embedded in paraffin.

Ovaries and uterine content:

After all litters had been delivered, vaginal smears were collected from F0 females for 12 d.
The right ovary with attached oviducts was weighed in females.
Ovaries were fixed in Bouin’s fixative for 24 h, then rinsed and held in 70 % ethanol until embedding into paraffin. All other tissues were embedded in paraffin.

Fetal examinations:

Upon delivery of each litter, lethality, gestation length, sex, number, weight of the pups, and dam weight were determined. All new-born litters were killed humanely following evaluation.

After delivery of the F2 litters, vaginal smears were collected for 12 days from Fl females. Body weight and feed and water consumption were recorded at 74 +/- 10 (mating), 84 +/- 10, and 112+/- 10 d of age during the Fl fertility assessment period. Maternal body weight was also recorded upon discovery of an F2 litter.

Statistics:

Most hypotheses were tested using the Williams’ modification of Dunn’s or Shirley’s nonparametric multiple comparisons procedures. Jonckheere’s test was used to ascertain whether there was sufficient evidence of a dose-related response to apply Shirley’s test. If the P-value from Jonckheere’s test was less than 0.10, Shirley’s test was used; otherwise, Dunn’s test was applied. For data expressed as a proportion, the Cochran-Armitage test was used to test for a dose-related trend, and pairwise comparisons were performed using a Chi-square test. To adjust for the potential effect of the number of pups per litter on the average pup weight, a parametric analysis of covariance was performed. The covariate used was average litter size, including live and dead pups. Least square estimates of dose group means, adjusted for litter size, were computed and tested for overall equality using an F-test and pairwise equality using Dunnett’s test. Unadjusted weights were analysed with Shirley’s or Dunn’s test. An arcsine transformation was performed on vaginal cytology data, then a multivariate analysis of variance was conducted. Cycle length was analysed using Shirley’s or Dunn’s test.

Clinical signs:

no effects observed

Description (incidence and severity):

No dose-related clinical signs or increased incidence of mortality were observed for the F0 animals.

Mortality:

no mortality observed

Description (incidence):

No dose-related clinical signs or increased incidence of mortality were observed for the F0 animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At necropsy body weight was significantly depressed in the females of the 7000 ppm group.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At all dose levels in F0 mice, there was increased liver weight for males and increased absolute and relative liver weights and increased relative kidney plus adrenal weights in females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy body weight was significantly depressed in the females of the 7000 ppm group.
Although liver histopathology was only examined in DMF-treated mice exhibiting gross hepatic lesions (2/10 high dose males and 2/10 mid-dose females), all those examined exhibited centrilobular hepatic hypertrophy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At F0 necropsy, sperm parameters, and estrous cycle length were not adversely affected, with the exception of a decreased number of females in the high dose group having normal cycles and of a slight decrease in sperm concentration at the low and the high dose. However, microscopic evaluation of the reproductive organs revealed no histopathology due to DMF treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced; F1 pup postnatal survival at 4000 and 7000 ppm was reduced during the pre- and post- weaning periods.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The crossover mating trial was not able to determine the gender responsible for the decrease in fertility observed in the continuous breeding phase of the study. However, females treated with 7000 ppm produced somewhat smaller litters compared to control pairs or the treated males and pup weights were lower from treated females compared to those sired by treated males. These data suggest that the female was the sex affected by DMF exposure. In addition, 7000 ppm females mated to control males produced pups with malformations similar to those observed in Task 2. Further examinations of pups from 7000 ppm task 3 females revealed abnormal ossification of the cranial plates and abnormal or incomplete formation of the sternebrae.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: body weight reduction, on females yes, on males no

Dose descriptor:

LOAEL

Effect level:

<= 1 000 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced; F1 pup body weight was reduced at the mid and high doses.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced; F1 pup postnatal survival at 4000 and 7000 ppm was reduced during the pre- and post- weaning periods. Because of decreased fertility, increased prenatal death and postnatal cannibalism in the high dose group a slight reduction in the percentage of litters with malformed pups was seen in comparison to the mid-dose group.

Changes in sex ratio:

not specified

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced; F1 pup postnatal survival at 4000 and 7000 ppm was reduced during the pre- and post- weaning periods. Because of decreased fertility, increased prenatal death and postnatal cannibalism in the high dose group a slight reduction in the percentage of litters with malformed pups was seen in comparison to the mid-dose group.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Surviving F1 pups in the mid- and high-dose groups exhibited craniofacial malformations. The proportion of litters with one or more pups with an abnormal appearance was 10.5 %, 90.0 % and 77.8 % for the 1000, 4000 and 7000 ppm groups, respectively, compared to 7.9 % for the control group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Surviving F1 pups in the mid- and high-dose groups exhibited craniofacial malformations. The proportion of litters with one ore more pups with an abnormal appearance was 10.5 %, 90.0 % and 77.8 % for the 1000, 4000 and 7000 ppm groups, respectively, compared to 7.9 % for the control group.

Visceral malformations:

not specified

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: yes.
Remark: reduced body weight, malformations

Dose descriptor:

LOAEL

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Basis for effect level:

fetal/pup body weight changes

Dose descriptor:

NOAEL

Based on:

test mat.

Basis for effect level:

other: 27.7 % malformations already at the lowest dose tested (compared to 0 % malformations in the controls).

Remarks on result:

not determinable

Abnormalities:

not specified

Developmental effects observed:

not specified

Reproductive performance of the second generation: DMF was a reproductive toxicant in F1 mice. It caused a significant reduction in the mating index at 7000 ppm (data not shown) and in fertility (number pregnant) at 4000 and 7000 ppm. The average days to litter was increased, and the number of live pups per litter, pup body weight, and the proportion of pups born alive was decreased at the mid- and high-dose levels. Live pup weight was also decreased in low-dose F2 pups. F2 pups born to DMF-treated Fl pairs exhibited malformations similar to those observed for Fl litters of F0 pairs. The proportion of litters with one or more externally malformed pups was 0, 27.7, 60, and 75 % in the control, 1000, 4000, and 7000 ppm groups. Fl estrous cycles were monitored with vaginal smears for 12 consecutive days following birth of the F2 litter. Females in the high-dose group had significantly longer cycles and tended to be in either metestrus or diestrus longer than control animals. At necropsy, Fl male and female body weight was reduced at mid- and high-dose DMF. Absolute and relative liver weight were significantly increased in all DMF-treated groups for both sexes. In addition, female relative kidney plus adrenal weight was increased at the mid- and high-dose levels. Histopathologic evaluation of livers exhibiting gross lesions from animals in the low- and high-dose groups revealed treatment-related centrilobular hepatocellular hypertrophy. These findings indicate a general toxicity at 1000 ppm DMF.

Male and female mice were exposed at doses of 1000, 4000 and 7000 ppm. Average doses in 1000 ppm males ranged from 182 +/- 6.9 mg/kg bw/d on week 1 to 187.9 +/- 27.7 mg/kg bw/d on week 27. Females consumed 256 +/- 38 to 193 +/- 11.1 mg/kg bw/d for the same period. Doses for 4000 ppm ranged 545 +/- 29 to 845 +/- 39 mg/kg bw/d in F0 males and females. At 7000 ppm 1026 +/- 42 to 1578 +/- 104 mg/kg bw/d were consumed. For F1
animals (week 12 - 16) average doses ranged from 213 +/- 16 to 315 +/- 13 mg/kg bw/d at 1000 ppm, 1006 +/- 30 to 1172 +/- 36 mg/kg bw/d at 4000 ppm and from 1684 +/- 113 to 2160 +/- 72 mg/kg bw/d at 7000 ppm. In general, females consumed more DMF per kg body weight than did males, most likely due to pregnancy. No dose-related clinical signs or increased incidence of mortality were observed for the F0 animals. At all dose levels in F0 mice, there was increased liver weight for males and increased absolute and relative liver weights and increased relative kidney plus adrenal weights in females. Moreover at necropsy body weight was significantly depressed in the females of the 7000 ppm group. Although liver histopathology was only examined in DMF-treated mice exhibiting gross hepatic lesions (2/10 high dose males and 2/10 mid-dose females), all those examined exhibited centrilobular hepatic hypertrophy. Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced; F1 pup postnatal survival at 4000 and 7000 ppm was reduced during the pre- and post- weaning periods, and F1 pup body weight was reduced at the mid and high doses. Surviving F1 pups in the mid- and high-dose groups exhibited craniofacial malformations. The proportion of litters with one ore more pups with an abnormal appearance was 10.5 %, 90.0 % and 77.8 % for the 1000, 4000 and 7000 ppm groups, respectively, compared to 7.9 % for the control group. Because of decreased fertility, increased prenatal death and postnatal cannibalism in the high dose group a slight reduction in the percentage of litters with malformed pups was seen in comparison to the mid-dose group. At F0 necropsy, sperm parameters, and estrous cycle length were not adversely affected, with the exception of a decreased number of females in the high dose group having normal cycles and of a slight decrease in sperm concentration at the low and the high dose. However, microscopic evaluation of the reproductive organs revealed no histopathology due to DMF treatment.

The crossover mating trial was not able to determine the gender responsible for the decrease in fertility observed in the continuous breeding phase of the study. However, females treated with 7000 ppm produced somewhat smaller litters compared to control pairs or the treated males and pup weights were lower from treated females compared to those sired by treated males. These data suggest that the female was the sex affected by DMF exposure. In addition, 7000 ppm females mated to control males produced pups with malformations similar to those observed in Task 2. Further examinations of pups from 7000 ppm task 3 females revealed abnormal ossification of the cranial plates and abnormal or incomplete formation of the sternebrae.

The selected males and females of the F1 generation for inclusion in the reproductive performance evaluation of the F1 generation showed reduced body weights in the mid- and high-dose group (from PND 74 to necropsy). In the F1 mating trial, the mating index was reduced at 7000 ppm, while the pregnancy index, litter size and proportion of pups born alive/litter were reduced at 4000 and 7000 ppm. Live pup weight was reduced at all doses. Malformations of F2 pups were similar to those observed for F1 litters of F0 pairs. The proportion of externally malformed pups was 0, 27.7, 60 and 75 % in the control, low-, mid- and high- dose groups. The F1 animals of all DMF-treated groups had an increase in liver weight in males and females associated with centrilobular hepatocellular hypertrophy. F1 estrous cycles were significantly longer in the 7000 ppm females compared to the control females. Evaluation of F1 reproductive tissues revealed some significant reproductive effects for males but not for females. Relative prostate weight was decreased at all doses as was absolute prostate weight in males of the mid- and high-dose group and epididymidal spermatozoa concentration was decreased at the high dose. At necropsy, F1 animals from each DMF dose group and the control group (5 females/5 males, each) selected for skeletal evaluation exhibited malformations persistent from birth at 4000 ppm and above. In summary, the MTD for generalized toxicity was 1000 ppm for both the F0 and F1 generation.

According to the authors, the NOAEL for generalized toxicity could not be determined for either the F0 or F1 generation. Significant reproductive and developmental toxicity was observed at 4000 ppm for the F0 and F1 generation in the presence of some general toxicity.

Conclusions:

Chronic exposure to DMF in drinking water at 0, 1000, 4000, and 7000 ppm (200 to 1300 mg/kg/d) reduced fertility by the first litter at 4000 ppm, reduced body weight in F0 females at 7000 ppm, and increased liver weights at all doses in both sexes. A crossover mating at 7000 ppm identified F0 females as the affected sex. F1 postnatal survival was reduced at > 4000 ppm DMF. F1 mating reduced F2 litter size and live pup weight at > 1000 ppm. At necropsy, body weight of F1 males and females was reduced at > 4000 ppm. DMF-treated pups (both Fl and F2) and Fl adults had cranial and sternebral skeletal malformations. Only DMF caused overt developmental toxicity. A No-Observed-Adverse-Effect-Level for DMF was not established.

Executive summary:

Chronic exposure to DMF in drinking water at 0, 1000, 4000, and 7000 ppm (200 to 1300 mg/kg/d) reduced fertility by the first litter at 4000 ppm, reduced body weight in F0 females at 7000 ppm, and increased liver weights at all doses in both sexes. A crossover mating at 7000 ppm identified F0 females as the affected sex. F1 postnatal survival was reduced at > 4000 ppm DMF. F1 mating reduced F2 litter size and live pup weight at > 1000 ppm. At necropsy, body weight of F1 males and females was reduced at >4000 ppm. DMF-treated pups (both Fl and F2) and Fl adults had cranial and sternebral skeletal malformations. Only DMF caused overt developmental toxicity. A No-Observed-Adverse-Effect-Level for DMF was not established.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Continuous breeding protocol (NTP): a dose range-finding phase (optional), an F0 cohabitation and lactation phase, a crossover mating trial of the F0 generation (conducted if F0 reproductive performance is affected), and finally fertility assessment of the Fl generation (born and reared during the F0 lactation phase).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethylformamide

EC Number:

200-679-5

EC Name:

N,N-dimethylformamide

Cas Number:

68-12-2

Molecular formula:

C3H7NO

IUPAC Name:

N,N-dimethylformamide

Details on test material:

N,N-dimethylformamide, purity > 99 %,
DMF (CAS No. 68–12-2; Aldrich Chemical Co., Milwaukee, WI) was mixed in deionized/filtered drinking water for dosing.
Fresh dosing solutions were provided every 7 d or more frequently to ensure 10 % compound loss (24, 25).
The formulations were stored at room temperature for up to 14 d.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: (CD-l® (ICR)BR outbred Swiss albino mice; Charles River Laboratories, Inc., Raleigh, NC),
- Age: 6 weeks old upon arrival
- Housing: During quarantine (2 to 5 weeks), animals were housed singly. Throughout the study, solid-bottom polypropylene or polycarbonate cages (111⁄2“ 3 7” 3 5“) with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ) and Ab-Sorb-Dri® bedding (Laboratory Products, Garfield, NJ) were used. Cages were changed at least once weekly.
- Diet (e.g. ad libitum) and Water (e.g. ad libitum): Deionized/filtered water and pelleted feed (NIH-07 Rodent Chow, Zeigler Brothers, Gardners, PA) were made available ad libitum.
- Acclimation period: Quarantine (2 to 5 weeks)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +- 0.2 °F
- Humidity (%): 58 +- 0.1 %
- Photoperiod (hrs dark / hrs light): 14-h light/10-h dark (Barber- Colman Network 8000 System, Barber-Colman Company, Loves Park, IL).

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

other: deionized/filtered drinking water

Details on exposure:

In the dose range-finding trials, DMF was dosed as 0, 2500, 5000, 7500, 10,000, and 15,000 ppm. For each study, 48 males and 48 females at 8 weeks of age were randomly assigned to six treatment groups (eight/sex/ group) by stratified randomization using body weight. In the remainder of the RACB study, DMF was administered at 0, 1000, 4000, and 7000 ppm.

F0 cohabitation and lactation (definitive studies). For each study, 100 male and 100 female Swiss CD-1® mice, 11 weeks of age, were assigned to one of four dose groups by stratified randomization on the basis of body weight. The control group consisted of 40 males and 40 females, and each FORM- or DMF-treated group consisted of 20 males and 20 females. Body weights and feed and water consumption were monitored during treatment weeks 1, 8, and 16.

Details on mating procedure:

F0 cohabitation and lactation:
During Week 1 of exposure to chemicals, animals were individually housed. During Weeks 2 through 15, animals were housed in breeding pairs within dose groups, and newborn litters were sacrificed immediately after evaluation. Data collected during the F0 cohabitation were the litter interval, number, sex, weight of pups per litter, number of litters per breeding pair, and the Postnatal Day (PND) 0 dam body weight. Because fertility was severely affected at Litter 5 for FORM and throughout the 14-week cohabitation period for DMF, crossover mating trials were conducted for both studies using the control and high-dose groups, and an F1 fertility assessment phase using the control, low-, mid-, and high-dose groups was conducted (see below). Starting at Week 16 of exposure, the breeding pairs were separated, and F0 females were allowed to deliver and rear the final litter until PND 21. On PND 0, 4, 7, 14, and 21 of the lactation phase, pups were sexed, counted, and weighed. On PND 21, randomly selected Fl pups from each dose group were weaned and housed in same-sex pairs by dose and saved for the Fl fertility assessment phase.

After completion of the cohabitation and lactation phase, all F0 animals were maintained on FORM or DMF dosed water until scheduled sacrifice after the completion of the crossover mating phase.

Cross-over mating trial:
The crossover mating trial was conducted using the control and high-dose animals for both the FORM and the DMF studies. Three breeding groups of F0 animals were created: (1) control male 3 control female, (2) high-dose male 3 control female, and (3) control male 3 high-dose female.
Beginning at Week 23 of treatment, animals were cohabited until a vaginal copulatory plug was observed or for 1 week, whichever occurred first. During the week of cohabitation, no treatments were administered to avoid possible exposure of control animals to FORM or DMF or their metabolites. At the start of Week 24, all animals were singly housed, and dosing was resumed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Confirmation of purity and identity was conducted at Research Triangle Institute (NTP/NIEHS Contract No. N01-ES-45061) and involved infrared spectrometry and nuclear magnetic resonance spectrometry. Purity of the bulk compound was reconfirmed periodically during the study by capillary column gas chromatography. Stability, homogeneity, and dose simulation studies were also completed (24,25), and dosing regimens were based on those studies.
Dosing formulations were tested to verify concentrations five times during each of the studies and were within 90 to 110 % of targeted concentrations.

Duration of treatment / exposure:

Premating exposure period (males & females): 7 d; test duration up to the F2 generation

Frequency of treatment:

continuously in the drinking water, the actual doses delivered were calculated periodically, considering consumption and body weights, and are reported (24, 25) and summarized in the Results section.

Details on study schedule:

F1 fertility assessment.
At weaning (PND 21), randomly selected F1 pups from the control, low-, mid-, and high-dose groups were housed two per cage by sex within dose group for both studies. Although FORM or DMF was administered directly to the weanlings beginning on PND 22, prior to direct dosing, indirect exposure to FORM or DMF likely occurred to gametes, in utero, or during lactation. At 74 +/- 10 d of age, males and females in the control or treated groups were co-habited as non-sibling breeding pairs until a vaginal copulatory plug was observed or for 1 week, whichever occurred first. Because of reduced survival in some of the dose groups, especially the high-dose group of DMF, 20 non-sibling pairs were not available, so 15 pairs were co-habited, some of which were siblings. Litter data resulting from the F1 cohabitation were collected as described above for F0 adults in the crossover mating trial.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm (nominal)

Remarks:

ca. 219 mg/kg/d in water

Doses / concentrations 2

Dose / conc.:

4 000 ppm (nominal)

Remarks:

ca. 820 mg/kg/d in water

Doses / concentrations 3

Dose / conc.:

7 000 ppm (nominal)

Remarks:

ca. 1455 mg/kg/d in water

No. of animals per sex per dose:

F0 cohabitation and lactation (definitive studies): each study 100 male and 100 female
Crossover mating trial: three breeding groups of F0 animals were created: (1) control male 3 control female, (2) high-dose male 3 control female, and (3) control male 3 high-dose female.
F1 fertility assessment: 20 non-sibling pairs (in case of DMF high dose group only 15 pairs, in part siblings).

Control animals:

yes, concurrent vehicle

Details on study design:

1 fertility assessment.
At weaning (PND 21), randomly selected Fl pups from the control, low-, mid-, and high-dose groups were housed two per cage by sex within dose group for both studies. Although FORM or DMF was administered directly to the weanlings beginning on PND 22, prior to direct dosing, indirect exposure to FORM or DMF likely occurred to gametes, in utero, or during lactation. At 74 6 10 d of age, males and females in the control or treated groups were cohabited as non-sibling breeding pairs until a vaginal copulatory plug was observed or for 1 week, whichever occurred first. Because of reduced survival in some of the dose groups, especially the high-dose group of DMF, 20 non-sibling pairs were not available, so 15 pairs were cohabited, some of which were siblings. Litter data resulting from the Fl cohabitation were collected as described above for F0 adults in the crossover mating trial.

Examinations

Parental animals: Observations and examinations:

Upon delivery of each litter, lethality, gestation length, sex, number, weight of the pups, and dam weight were determined. After all litters had been delivered, vaginal smears were collected from F0 females for 12 d. At Week 29 of FORM or DMF exposure, immediately following CO2 asphyxiation, all F0 males and females were weighed and necropsied. Liver and paired kidney (with attached adrenal) weights were collected for both sexes. The right ovary with attached oviducts was weighed in females. All tissues, except ovaries, were fixed in 10 % neutral buffered formalin. Male reproductive tissues were embedded in glycol methacrylate, sectioned at 2.5-mm thickness, and stained with hematoxylin/PAS (Bio-Tek Research Consultants, Durham, NC). Ovaries were fixed in Bouin’s fixative for 24 h, then rinsed and held in 70 % ethanol until embedding into paraffin. All other tissues were embedded in paraffin.

Oestrous cyclicity (parental animals):

After all litters had been delivered, vaginal smears were collected from F0 females for 12 d. The right ovary with attached oviducts was weighed in females. Ovaries were fixed in Bouin’s fixative for 24 h, then rinsed and held in 70% ethanol until embedding into paraffin. All other tissues were embedded in paraffin.

Sperm parameters (parental animals):

Male reproductive tissues were embedded in glycol methacrylate, sectioned at 2.5-mm thickness, and stained with hematoxylin/PAS (Bio-Tek Research Consultants, Durham, NC).
Sperm evaluations from the right testis and epididymis included manual assessments of motility, concentration, and morphology. Homogenization resistant spermatid heads were counted from the left testis.

Litter observations:

Upon delivery of each litter, lethality, gestation length, sex, number, weight of the pups, and dam weight were determined. All new-born litters were killed humanely following evaluation.
After delivery of the F2 litters, vaginal smears were collected for 12 days from Fl females. Body weight and feed and water consumption were recorded at 74 +/- 10 (mating), 84 +/- 10, and 112 +/- 10 d of age during the Fl fertility assessment period.
Maternal body weight was also recorded upon discovery of an F2 litter.

Postmortem examinations (parental animals):

Gross necropsy was performed and histopathologic evaluations were conducted on all livers, right and left kidneys and adrenals, the right testis and epididymis, prostate, seminal vesicles, ovary, and any gross lesions noted during the necropsy (PATHCO, Inc., Research Triangle Park, NC).

Postmortem examinations (offspring):

At the necropsy (119 +/- 10 d of age), following CO2 asphyxiation, F1 males and females were weighed and data collected, as described previously for F0 animals. Histopathologic evaluations were conducted on all livers, right and left kidneys and adrenals, the right testis and epididymis, ovary, and any gross lesions noted during the necropsy.

Special or triggered technical procedures. Since craniofacial malformations were observed in some F1 pups born during F0 cohabitation of the DMF study, selected F2 litters were preserved on PND 1 and evaluated for whole body skeletal malformations and soft tissue malformations of the head. Selected adult Fl males and females in the DMF study were evaluated for skeletal malformations (28).

Statistics:

Most hypotheses were tested using the Williams’ modification of Dunn’s or Shirley’s non-parametric multiple comparisons procedures. Jonckheere’s test was used to ascertain whether there was sufficient evidence of a dose-related response to apply Shirley’s test. If the P-value from Jonckheere’s test was less than 0.10, Shirley’s test was used; otherwise, Dunn’s test was applied. For data expressed as a proportion, the Cochran-Armitage test was used to test for a dose-related trend, and pairwise comparisons were performed using a Chi-square test. To adjust for the potential effect of the number of pups per litter on the average pup weight, a parametric analysis of covariance was performed. The covariate used was average litter size, including live and dead pups. Least square estimates of dose group means, adjusted for litter size, were computed and tested for overall equality using an F-test and pairwise equality using Dunnett’s test. Unadjusted weights were analysed with Shirley’s or Dunn’s test. An arcsine transformation was performed on vaginal cytology data, then a multivariate analysis of variance was conducted. Cycle length was analysed using Shirley’s or Dunn’s test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

no dose-related signs or increased incidence of mortality

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

no dose-related signs or increased incidence of mortality

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight of females was significantly depressed at the high dose on Weeks 8 and 16 reflecting, at least in part, the non-pregnant status in 20 to 40 % of these animals. However, for those animals that delivered litters, body weight was affected by treatment at all doses by Week 16.
There was no effect of treatment on male body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Relative maternal feed consumption (g/kg/d) was significantly depressed only at 7000 ppm on PND 0 through 4, at 4000 ppm mid-lactation, and at 1000 ppm on PND 14 through 21.
No effect of treatment on male feed / water intake.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Relative maternal water consumption: like feed concumption but more pronounced effect.
No effect of treatment on male feed / water intake.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Although liver histopathology was only examined in DMF-treated mice exhibiting gross hepatic lesions (2/10 high dose males and 2/10 mid-dose females), all those examined exhibited centrilobular hepatic hypertrophy.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Description (incidence and severity):

At 7000 ppm DMF, fertility was reduced in the first litter to 90 %, compared to 100 % in controls.

Details on results (P0)

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced; F1 pup postnatal survival at 4000 and 7000 ppm was reduced during the pre- and post- weaning periods, and F1 pup body weight was reduced at the mid and high doses. Surviving F1 pups in the mid- and high-dose groups exhibited craniofacial malformations. The proportion of litters with one ore more pups with an abnormal appearance was 10.5 %, 90.0 % and 77.8 % for the 1000, 4000 and 7000 ppm groups, respectively, compared to 7.9 % for the control group. Because of decreased fertility, increased prenatal death and postnatal cannibalism in the high dose group a slight reduction in the percentage of litters with malformed pups was seen in comparison to the mid-dose group. At F0 necropsy, sperm parameters, and estrous cycle length were not adversely affected, with the exception of a decreased number of females in the high dose group having normal cycles and of a slight decrease in sperm concentration at the low and the high dose. However, microscopic evaluation of the reproductive organs revealed no histopathology due to DMF treatment. The crossover mating trial was not able to determine the gender responsible for the decrease in fertility observed in the continuous breeding phase of the study. However, females treated with 7000 ppm produced somewhat smaller litters compared to control pairs or the treated males and pup weights were lower from treated females compared to those sired by treated males. These data suggest that the female was the sex affected by DMF exposure. In addition, 7000 ppm females mated to control males produced pups with malformations similar to those observed in Task 2. Further examinations of pups from 7000 ppm task 3 females revealed abnormal ossification of the cranial plates and abnormal or incomplete formation of the sternebrae.

Effect levels (P0)

1

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: based on significantly female but not male body weight reduction

2

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: based on reduced fertility at 7000 ppm

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

F1 estrous cycles were significantly longer in the 7000 ppm females compared to the control females.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of F1 reproductive tissues revealed some significant reproductive effects for males but not for females. Epididymidal spermatozoa concentration was decreased at the high dose.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

In the F1 mating trial, the mating index was reduced at 7000 ppm, while the pregnancy index, litter size and proportion of pups born alive/litter were reduced at 4000 and 7000 ppm.

Details on results (P1)

Live pup weight was reduced at all doses. Malformations of F2 pups were similar to those observed for F1 litters of F0 pairs. The proportion of externally malformed pups was 0, 27.7, 60 and 75 % in the control, low-, mid- and high- dose groups. The F1 animals of all DMF-treated groups had an increase in liver weight in males and females.

Effect levels (P1)

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: based on reduced fertility and fecundity at doses above 1000 ppm

Results: F1 generation

General toxicity (F1)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The selected males and females of the F1 generation for inclusion in the reproductive performance evaluation of the F1 generation showed reduced body weights in the mid- and high-dose group (from PND 74 to necropsy).

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The F1 animals of all DMF-treated groups had an increase in liver weight in males and females associated with centrilobular hepatocellular hypertrophy.
Evaluation of F1 reproductive tissues revealed some significant reproductive effects for males but not for females. Relative prostate weight was decreased at all doses as was absolute prostate weight in males of the mid- and high-dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, F1 animals from each DMF dose group and the control group (5 females/5 males, each) selected for skeletal evaluation exhibited malformations persistent from birth at 4000 ppm and above.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

treatment-related centrilobular hepatocellular hypertrophy.

Details on results (F1)

Reproductive performance of the second generation: DMF was a reproductive toxicant in F1 mice. It caused a significant reduction in the mating index at 7000 ppm (data not shown) and in fertility (number pregnant) at 4000 and 7000 ppm . The average days to litter was increased, and the number of live pups per litter, pup body weight, and the proportion of pups born alive was decreased at the mid- and high-dose levels. Live pup weight was also decreased in low-dose F2 pups. F2 pups born to DMF-treated Fl pairs exhibited malformations similar to those observed for Fl litters of F0 pairs. The proportion of litters with one or more externally malformed pups was 0, 27.7, 60, and 75 % in the control, 1000, 4000, and 7000 ppm groups. Fl estrous cycles were monitored with vaginal smears for 12 consecutive days following birth of the F2 litter. Females in the high-dose group had significantly longer cycles and tended to be in either metestrus or diestrus longer than control animals. At necropsy, Fl male and female body weight was reduced at mid- and high-dose DMF (Tables 9 and 10). Absolute and relative liver weight were significantly increased in all DMF-treated groups for both sexes. In addition, female relative kidney plus adrenal weight was increased at the mid- and high-dose levels. Histopathologic evaluation of livers exhibiting gross lesions from animals in the low- and high-dose groups revealed treatment-related centrilobular hepatocellular hypertrophy. These findings indicate a general toxicity at 1000 ppm DMF.

Effect levels (F1)

1

Dose descriptor:

LOAEL

Remarks:

reproductive

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on reduced body weight of pups

2

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F1

Effect level:

< 1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on external malformations or other abnormalities, including domed heads and hematomas along the nose and on the head

3

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: based on reduced body weight of pups, average number of litters per pair, average litter size, proportion of pups born alive at 4000 ppm

Results: F2 generation

General toxicity (F2)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Reduced F2 pup weight was noted at 1000 ppm DMF (219 mg/kg/d).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Craniofacial and sternebral malformations observed at the mid and high doses were characteristic and occurred in offspring of both generations.

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Basis for effect level:

other: not determinable (based on malformations of 27.7 % already at the lowest dose, compared to control of 0 % malformations).

Remarks on result:

not measured/tested

Remarks:

Effect level not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Male and female mice were exposed at doses of 1000, 4000 and 7000 ppm. Average doses in 1000 ppm males ranged from 182 ± 6.9 mg/kg bw/d on week 1 to 187.9 ± 27.7 mg/kg bw/d on week 27. Females consumed 256 ± 38 to 193 ± 11.1 mg/kg bw/d for the same period. Doses for 4000 ppm ranged 545 ± 2 to 845 ± 39 mg/kg bw/d in F0 males and females. At 7000 ppm 1026 ± 42 to 1578 ± 104 mg/kg bw/d were consumed. For F1 animals (week 12 - 16) average doses ranged from 213 ± 16 to 315 ± 13 mg/kg bw/d at 1000 ppm, 1006 ± 30 to 1172 ± 36 mg/kg bw/d at 4000 ppm and from 1684 ± 113 to 2160 ± 72 mg/kg bw/d at 7000 ppm. In general, females consumed more DMF per kg body weight than did males, most likely due to pregnancy. No dose-related clinical signs or increased incidence of mortality were observed for the F0 animals.
At all dose levels in F0 mice, there was increased liver weight for males and increased absolute and relative liver weights and increased relative kidney plus adrenal weights in females. Moreover at necropsy body weight was significantly depressed in the females of the 7000 ppm group. Although liver histopathology was only examined in DMF-treated mice exhibiting gross hepatic lesions (2/10 high dose males and 2/10 mid-dose females), all those examined exhibited centrilobular hepatic hypertrophy.

Reproductive toxicity was observed in the F0 generation, primarily at the mid- and high dose levels. At 4000 and 7000 ppm, fertility and fecundity were reduced; F1 pup postnatal survival at 4000 and 7000 ppm was reduced during the pre- and post- weaning periods, and F1 pup body weight was reduced at the mid and high doses. Surviving F1 pups in the mid- and high-dose groups exhibited craniofacial malformations. The proportion of litters with one ore more pups with an abnormal appearance was 10.5 %, 90.0 % and 77.8 % for the 1000, 4000 and 7000 ppm groups, respectively, compared to 7.9 % for the control group. Because of decreased fertility, increased prenatal death and postnatal cannibalism in the high dose group a slight reduction in the percentage of litters with malformed pups was seen in comparison to the mid-dose group. At F0 necropsy, sperm parameters, and estrous cycle length were not adversely affected, with the exception of a decreased number of females in the high dose group having normal cycles and of a slight decrease in sperm concentration at the low and the high dose. However, microscopic evaluation of the reproductive organs revealed no histopathology due to DMF treatment.

The crossover mating trial was not able to determine the gender responsible for the decrease in fertility observed in the continuous breeding phase of the study. However, females treated with 7000 ppm produced somewhat smaller litters compared to control pairs or the treated males and pup weights were lower from treated females compared to those sired by treated males. These data suggest that the female was the sex affected by DMF exposure. In addition, 7000 ppm females mated to control males produced pups with malformations similar to those observed in Task 2. Further examinations of pups from 7000 ppm task 3 females revealed abnormal ossification of the cranial plates and abnormal or incomplete formation of the sternebrae.

The selected males and females of the F1 generation for inclusion in the reproductive performance evaluation of the F1 generation showed reduced body weights in the mid- and high-dose group (from PND 74 to necropsy). In the F1 mating trial, the mating index was reduced at 7000 ppm, while the pregnancy index, litter size and proportion of pups born alive/litter were reduced at 4000 and 7000 ppm. Live pup weight was reduced at all doses. Malformations of F2 pups were similar to those observed for F1 litters of F0 pairs. The proportion of externally malformed pups was 0, 27.7, 60 and 75 % in the control, low-, mid- and high- dose groups. The F1 animals of all DMF-treated groups had an increase in liver weight in males and females associated with centrilobular hepatocellular hypertrophy. F1 estrous cycles were significantly longer in the 7000 ppm females compared to the control females. Evaluation of F1 reproductive tissues revealed some significant reproductive effects for males but not for females. Relative prostate weight was decreased at all doses as was absolute prostate weight in males of the mid- and high-dose group and epididymidal spermatozoa concentration was decreased at the high dose. At necropsy, F1 animals from each DMF dose group and the control group (5 females/5 males, each) selected for skeletal evaluation exhibited malformations persistent from birth at 4000 ppm and above. In summary, the MTD for generalized toxicity was 1000 ppm for both the F0 and F1 generation.

According to the authors, the NOAEL for generalized toxicity could not be determined for either the F0 or F1 generation. Significant reproductive and developmental toxicity was observed at 4000 ppm for the F0 and F1 generation in the presence of some general toxicity.

Applicant's summary and conclusion

Conclusions:

Chronic exposure to DMF in drinking water at 0, 1000, 4000, and 7000 ppm (200 to 1300 mg/kg/d) reduced fertility by the first litter at 4000 ppm, reduced body weight in F0 females at 7000 ppm, and increased liver weights at all doses in both sexes. A crossover mating at 7000 ppm identified F0 females as the affected sex. F1 postnatal survival was reduced at > 4000 ppm DMF. F1 mating reduced F2 litter size and live pup weight at > 1000 ppm. At necropsy, body weight of F1 males and females was reduced at > 4000 ppm. DMF-treated pups (both Fl and F2) and Fl adults had cranial and sternebral skeletal malformations. Only DMF caused overt developmental toxicity. A No-Observed-Adverse-Effect-Level for DMF was not established.

Executive summary:

Reproductive toxicity was clearly manifested as reduced fertility in F0 animals at the high dose after FORM and for both the mid and high doses of DMF as an immediate and progressive disruption of fertility. In both cases, litter size was reduced. Developmental toxicity was observed after DMF at the mid and high doses as a decrease in pup body weight and an increase in craniofacial malformations. The crossover mating trial clearly indicated that the female was the affected sex after FORM, and some data suggest the female was also the sex affected for DMF. After DMF at 7000 ppm, females produced malformed pups, contributing to increased mortality. Furthermore, the number of live pups was reduced at the high dose during the continuous breeding phase for both compounds. After DMF, a higher mortality occurred at 4000 ppm, and live pup weight was reduced.

Within the study, the reproductive and developmental effects observed in F1 animals were similar in type and degree to those observed in F0 animals for both the FORM and DMF study. However, the RACB study design did not allow for separation of reproductive (i.e., conception and preimplantation) versus developmental (i.e., post-implantation) toxicity. Between studies, the developmental effects appeared to be confined to DMF, but higher doses were not tested for FORM.

For DMF, the developmental toxicity appeared to be mediated primarily through the treated females, since treated males mated to control females did not produce malformed offspring. However, F0 males and females were similarly sensitive to the general toxicity of DMF. Observation of reproductive toxicity in the presence of general toxicity suggests a possible interaction between the systemic and reproductive effects. Maternal toxicity contributed to fetal and neonatal problems.

Pregnant mice exhibited no overt maternal toxicity. Both doses caused a decrease in fetal body weight and an increase in the incidence of retardation and variations. A significant increase in the incidence of malformations was observed at the higher dose.

Changes in estrous cycle characteristics in females (both F0 and F1) were caused by both DMF and FORM. The cycle was lengthened in F1 animals that were cycling in both experiments; 25 % of high-dose females exhibited no estrous cycles after FORM or DMF, compared to 5 % of controls. In the DMF F0 generation, fewer females had “normal” cycles (25 % of mice treated with DMF at 7000 ppm vs. 0 % controls had long or no cycles during the 12 d observed); 42 % vs. 9 % after FORM. It appears for both compounds that the treated females had a higher frequency of diestrus smears than controls (e.g., 63 % vs. 38 % for F0 form treated versus controls). This likely caused fewer regular cycles and contributed to longer cycles. The changes observed in estrous cycle characteristics could be due to depressed body weight gain.

Other indicators of possible endocrine disruption were reduced sperm numbers and prostate weights in F0 males after high dose DMF and altered ovarian weight function in F0 females after high dose FORM and mid- and high-dose DMF. The proportion of pups born alive in these mating studies was reduced during the crossover mating phase at 14 % for FORM-treated females and a slight amount in DMF females. From these two experiments, the MTD (maximal tolerated dose) can be estimated for both DMF and FORM, but the No-Observed-Adverse-Effect-Level (NOAEL) can only be established for FORM.

In the DMF experiment, a NOAEL was not observed. Toxicity was present at all doses tested. On the basis of indicators of mild general toxicity, the MTD for both the F0 and F1 generations was 1000 ppm DMF (average exposure was 219 mg/kg/d). Sensitivity to toxicity seemed to be similar in each generation and between sexes. The liver appeared to be the primary non-reproductive target organ in both males and females and may be the most sensitive indicator of exposure. Because the liver is the site of metabolism for most chemicals (and presumably DMF), the increase in mass and centrilobular hepatocellular hypertrophy may be due, at least in part, to induction of metabolizing enzymes and increase in cell number and/or size (54). Significant reproductive and developmental toxicity was observed in both generations at 4000 ppm DMF (mean exposure 820 mg/kg/day) and at 7000 ppm (mean exposure 1455 mg/kg/day) in the presence of some general toxicity. Reduced F2 pup weight was noted at 1000 ppm DMF (219 mg/kg/d). Craniofacial and sternebral malformations observed at the mid and high doses were characteristic and occurred in offspring of both generations. The more severe malformations were incompatible with life. Those animals less affected did grow to maturity, although examination after necropsy indicated the malformations present at birth had persisted through young adulthood. These studies indicate that FORM and DMF significantly affect reproduction, and that DMF affects development in Swiss mice. A striking decrease in fertility of the F0 animals was noted at both 4000 and 7000 ppm DMF and at 750 ppm for FORM in the continuous breeding portion of the studies.