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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
published 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable well documented publikation, which meets basic scientific principles
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
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

Constituent 1
Reference substance name:
Automatically generated during migration to IUCLID 6, no data available
IUPAC Name:
Automatically generated during migration to IUCLID 6, no data available
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:
(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 6 0.2°F and 58 6 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).

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 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 +/- 10 d of age, males and females in the control or treated groups were cohabited as nonsibling 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 nonsibling 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.
Doses / concentrations
Remarks:
Doses / Concentrations:
1000, 4000, 7000 ppm (ca. 219, 820 and 1455 mg/kg/d)
Basis:
nominal 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 nonsibling pairs (in case of DMF h igh dose group only 15 pairs, in part siblings).
Control animals:
yes, concurrent vehicle

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 C02 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 (23). Homogenizationresistant spermatid heads were counted from the left testis (26,27).
Litter observations:
Upon delivery of each litter, lethality, gestation length, sex, number, weight of the pups, and dam weight were determined. All newborn 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 6 10 (mating), 84 6 10, and 112 6 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 C02 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’ (30) modification of Dunn’s (31) or Shirley’s (32) nonparametric multiple comparisons procedures. Jonckheere’s test (33) 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 (34) was used to test for a dose-related trend, and pairwise comparisons were performed using a Chi-square test (35). 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 (36). 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 (37). Unadjusted weights were analyzed with Shirley’s or Dunn’s test. An arcsine transformation was performed on vaginal cytology data, then a multivariate analysis of variance was conducted (38). Cycle length was analyzed 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
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
no effect of treatment on male bw or feed / water intake, female bw was significantly reduced
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
no effect of treatment on male bw or feed / water intake, female bw was significantly reduced
Other effects:
effects observed, treatment-related

Details on results (P0)

Dose range-finding study: DMF
During the 2-week DMF exposure (2,500 to 15,000 ppm in drinking water), treatment-related deaths occurred at doses of 10,000 ppm and 15,000 ppm. No clinical signs were observed other than the death of seven males and three females at 15,000 ppm and three males at 10,000 ppm. Body weight was decreased in the remaining 15,000 ppm animals. Water consumption was decreased in both sexes at 1 and 2 weeks of DMF exposure. On the basis of these data, doses of 1000, 4000, and 7000 ppm were chosen for the continuous breeding phase.

Cohabitation and lactation studies (continuous
breeding phase): DMF
For F0 animals, no dose-related clinical signs or increased incidence of mortality was observed. There was no effect of treatment on male body weight or feed and water consumption (data not shown; Reference 25). Female body weight was significantly reduced at the high dose on Weeks 8 and 16, reflecting, at least in part, the nonpregnant 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 (data not shown; Reference 25). During the lactational period, DMF effect seemed to be directly related to pup mass (PND 0 through 4). Relative maternal feed consumption (g/kg/d) was significantly depressed only at 7000 ppm on PND 0 thrugh 4, at $4000 ppm midlactation, and at $1000 ppm on PND 14 through 21. Relative maternal water consumption (g/kg/d) exhibited the same, albeit more pronounced effect (data not shown; Reference 25). A small portion of the water and feed were consumed by pups on PND 10 through 21. For DMF-dosed animals, average doses in 1000 ppm males ranged from 182 6 6.9 mg/kg body weight/d on Week 1 to 187.9 6 27.7 mg/kg/d on Week 27, whereas females consumed 256 6 38 to 193 6 11.1 mg/kg body weight/d for the same time frame (Table 1; Reference 25). In general, females consumed more DMF per kg body weight than did males, most likely because they were pregnant. Doses for 4000 ppm animals ranged from 545 6 29 to 845 6 39 mg/kg/d in F0 males and pregnant females. For 7000 ppm animals, 1026 6 42 to 1578 6 104 mg/kg/d DMF was consumed (Table 1). Exposure of F0 mice to DMF altered measures of fertility and fecundity (Table 2). At 7000 ppm DMF, fertility was reduced in the first litter to 90%, compared to 100% in controls. Over time, this treatment-related effect increased. By the final litter, fertility was further reduced to 55% at 7000 ppm. By this time, reduced fertility was also noted at 4000 ppm. For pairs exposed at 4000 ppm or greater, the average number of litters per pair, average litter size, proportion of pups born alive, and average pup weight were reduced compared to control pairs. DMF treatment had no effect on these parameters in the 1000 ppm group (Table 2). Pups born to DMF-treated pairs had external malformations or other abnormalities, including domed heads and hematomas along the nose and on the head. Those pups affected most severely died shortly after birth, and many were partially cannibalized prior to examination. During the continuous breeding phase, 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 dose groups, respectively, compared to 7.9% for the control group. The slight reduction in the proportion of litters with malformed.

Crossover mating trial and F0 necropsy: DMF
A crossover mating trial was conducted with 7000 ppm treated males and females bred to control mates (Table 4). No differences were detected in DMF-treated groups of either sex for comparisons to controls. A lower than usual pregnancy rate in the control group resulted in fewer litters, thus affecting the power of statistical analysis and the strength of conclusions. However, comparisons between treated groups did differ. For the two groups in which control animals of both sexes were mated to dosed animals, groups with the dosed females had fewer live pups per litter (5.5 6 1.0 vs. 10.2 6 1.2). The smaller litter size was a reduction of 32% from control 3 control pair values, with the difference being significant at P 5 0.065. Both the adjusted and unadjusted pup weights were lower in pups of dosed females compared to those sired by treated males mated to control females. Together, these data suggest that the female was the sex affected by DMF exposure. F1 pups born following the crossover mating were examined for whole body skeletal malformations and soft tissue cranial malformations. Pups born to pairs with the DMF-treated female partner exhibited the same spectrum of malformations as observed during the continuous breeding phase. The proportion of litters with one or more externally malformed pups was 12.5%, 0.0%, and 90.9% for the control male 3 control female, 7000 ppm male 3 control female, and control male 3 7000 ppm female pairs, respectively. A thorough examination of the internal skeletal structure of 252 pups indicated an incidence of litters with one or more malformed pups at 83.3%, 81.8%, and 100% for the same three mating groups, respectively. The percent pups (within litters) with skeletal malformations was significantly increased in the control male 3 7000 ppm female group (95%), compared to the control pairs 3 control mating (40%) and for control female 3 7000 ppm males (38%). Incomplete ossification of the cranial bones, a common indicator of developmental delay, accounted for 82% of the malformations obs rved in the control male 3 control female group, and 97% of the malformations observed in the 7000 ppm male 3 control female group, but was not observed in the control male 3 7000 ppm female group. Malformations observed in the latter group included abnormal ossification of the cranial plates, abnormal suture formation in the cranium, and abnormal or incomplete formation of the sternebrae. Further examination of 95 heads, taken from randomly selected pups, revealed that 6/26 (23.1%) of the pups born to DMF-treated mothers had malformations, including agenesis of the cerebrum, agnathia, abnormally shaped cerebrum or cranium, cleft palate, or enlarged cerebral ventricles. Head malformations observed for pups born to the control male 3 control female pairs (2/29) and 7000 ppm male 3 control female pairs (1/40) were accounted for solely by the observation of enlarged nasal passages (data not shown; Reference 25). After pups were born from the crossover mating, estrous cycles were monitored in control and high-dose females for 12 days. For DMF-treated females, there was no effect of treatment on the length of the estrous cycle or stage frequency distribution, but 86% of the controls had 4- to 5-d estrous cycles, compared to 66% after DMF (Table 5). Thus, the number of animals having normal cycles was decreased by DMF. In DMF-treated animals at necropsy, F0 female but not male body weight was significantly depressed at the high dose (Tables 6 and 7). Male liver weights were increased at all doses (Table 6). Female absolute and relative liver weight and kidney plus adrenal weights were increased at all dose levels (Table 7). Histopathologic evaluation of livers exhibiting gross lesions from four animals in the mid- (two females) and high-dose (two males) groups revealed centrilobular hepatocellular hypertrophy (data not shown), which is considered to be treatment related. Thus, at all doses, DMF caused general toxicity, with some evidence of histologic involvement at the mid and high dose. Of the reproductive organs examined, cauda epididymidal weight was significantly increased at all doses of DMF (Table 6). Further evaluation of sperm parameters indicated a slight decrease in testicular spermatid concentration in the DMF-treated groups that was significant at the low and high doses, with a significant trend. However, DMF had no adverse effect on epididymal spermatozoan concentration, motility, or morphology. Microscopic evaluation of the reproductive organs revealed no histopathology due to DMF treatment. Thus, the effect on testicular spermatids was likely a Type II error and not biologically relevant. Growth and survival of F1 juveniles: DMF Growth and survival of Fl pups were retarded after DMF. The proportion of Fl pups born alive in the final litter and postnatal survival on PND 4 were reduced at the mid- and high-dose levels of DMF (Table 3) and continued to decline throughout the lactational period. Pup body weight during lactation was reduced in the mid- and high-dose groups prior to PND 7 and may have contributed to decreased survival rate (data not shown; Reference 25). F1 pups born to DMF-treated pairs in the mid- and high-dose groups also exhibited craniofacial malformations. Those pups that were severely malformed did not survive the preweaning period. The surviving F1 pups were closely examined during the maturation period. Those in the mid- and high-dose groups were small and appeared to have foreshortened, domed heads. The abnormal appearance of the pups in the mid-dose group was more prominent than in the high-dose group, suggesting that in the high-dose group, pups most affected had not survived. After weaning, pups were randomly selected (within dose group) for rearing and inclusion in the reproductive performance evaluation of the F1 generation. Both male and female body weight was reduced in the midand high-dose groups throughout the remainder of the study (PND 74 to necropsy; data not shown). Feed consumption was unaffected by DMF for the F1 generation. Water consumption was increased for males in the mid- and high-dose groups on Day 84 +/- 10 and in the high-dose group on Day 112 6 10. Estimated mean exposure to DMF was 259 mg/kg body weight/d for the 1000 ppm group, 1023 mg/kg body weight/d for the 4000 ppm group, and 1934 mg/kg body weight/d for the 7000 ppm group, with females receiving slightly more than males (Table 1; Reference 25).

Evaluation of Fl reproductive tissues revealed some significant reproductive effects for males but not for females. Relative prostate weight was decreased at all doses (Table 9), as was absolute prostate weight in males at the mid and high doses (data not shown, 25). Epididymidal spermatozoa concentration was decreased at the high dose, but no other significant effects of treatment were noted for andrologic parameters. Relative ovary weight was increased in the mid-dose group females due to the presence of cystic ovaries in two animals (Table 10) but was not likely treatment related. Microscopic examination of the reproductive organs revealed no other pathology. The developmental effects observed at delivery of DMF were confirmed in surviving Fl adults. Examination of the skeletons of five adult Fl males and five adult Fl females from each DMF dose group revealed a malformation rate of 30, 20, 100, and 100% in the control through high-dose groups, respectively (data not shown). Malformations observed in the mid- and highdose groups were characteristic and consisted of abnormal or incomplete ossification of the cranial plates, abnormal cranial suture formation, and abnormally formed sternebrae. Histopathologic evaluation of the cranium of additional adult Fl animals in the mid- and high-dose groups revealed dysplasia of the cranial bones, primarily at the midline.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
< 1 000 ppm
Sex:
female
Basis for effect level:
other: based on significantly female but not male body weight reduction
Dose descriptor:
NOAEL
Remarks:
reproductive / maternal
Effect level:
< 1 000 ppm
Sex:
male/female
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):
reduced
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Spermatozoa concentration: relatively reduced Spermatozoa (percent motile),: relatively higher
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
relative weight of liver is significantly higher in F1, ,Prostate relatively reduced,
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
malformations
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 (Table 8). 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 (Table 10). 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)

open allclose all
Dose descriptor:
LOAEL
Remarks:
reproductive
Generation:
F1
Effect level:
1 000 ppm
Basis for effect level:
other: based on reduced body weight of pups.
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Generation:
F1
Effect level:
< 1 000 ppm
Basis for effect level:
other: based on external malformations or other abnormalities, including domed heads and hematomas along the nose and on the head

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Basis for effect level:
other: not determable (based on malformations of 27,7 % already at the lowest dose, compared to control of 0 % malfromations.
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

RS-Freetext:
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.
Reproductivce 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 signifcant 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 Fl 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., postimplantation) 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 Fl) were caused by both DMF and FORM. The cycle was lengthened in Fl 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 Fl 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 nonreproductive 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 (;195 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.

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