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EC number: 200-679-5 | CAS number: 68-12-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Effects on fertility
Description of key information
Key (Fail et al., 1998): acceptable well-documented GLP-study, which meets basic scientific principles, two-generation reproductive toxicity, oral, premating exposure period up to F2, ca. 219, ca. 820, ca. 1455 mg/k/d in water, NOAEL (fertility) = 219 mg/kg/d in water
Link to relevant study records
- 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.
- Reason / purpose for cross-reference:
- reference to same study
- 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
- 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). - 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. - Dose / conc.:
- 1 000 ppm (nominal)
- Remarks:
- ca. 219 mg/kg/d in water
- Dose / conc.:
- 4 000 ppm (nominal)
- Remarks:
- ca. 820 mg/kg/d in water
- 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. - 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.
- 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
- Description (incidence and severity):
- At 7000 ppm DMF, fertility was reduced in the first litter to 90 %, compared to 100 % in controls.
- 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
- 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
- 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.
- 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
- 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.
- 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
- 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
- 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
- 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.
- 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
- Reproductive effects observed:
- not specified
- 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.
Reference
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.
Effect on fertility: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 219 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- mouse
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Oral route
In a continuous breeding study CD-1 mice were treated orally with DMF in the drinking water at doses of 1000, 4000 and 7000 ppm (about 219, 820 and 1455 mg/kg bw/day) (Fail et al., 1998). The maximal tolerated dose (MTD) for generalized toxicity was 1000 ppm for the F0 and the F1 generation, thus a systemic NOAEL could not be determined. At all dose levels in the F0 generation liver weights were increased. At necropsy body weight was significantly depressed in the females at 7000 ppm. Reproductive toxicity was observed in the mid and high dose groups represented by reduced fertility. Monitoring of the estrous cycle in control and high dose females revealed a decreased number of females in the high dose group having normal cycles. F1 pup postnatal survival was reduced during pre- and postweaning and body weights of F1 pups in the mid and high dose were also reduced, moreover the surviving pups of these dose groups exhibited craniofacial and sternebral malformations. Data generated by a crossover mating trial in the course of the continuous breeding study suggested that the female was the sex affected by DMF treatment because females treated with 7000 ppm DMF produced smaller litters compared to control pairs or the group of control females mated to treated males. In addition, pups born by treated females mated to controls exhibited malformations similar to those observed in the F1 pups of the F0 parental generation. The selected animals for the F1 parental generation showed reduced body weights in the mid and high dose groups. The F1 animals of all DMF treated groups had increased liver weights associated with hepatocellular hypertrophy. F1 estrous cycle length was significantly longer in the high dose females compared to the control animals. Histopathology did not reveal any findings in the reproductive tissues of the females. Male animals showed decreased relative prostate weight at all doses and epididymal spermatozoa concentration was reduced at the high dose. Affected reproductive performance was seen at the high dose by reduced mating index and at the high and mid dose by reduced pregnancy index and reduced litter size. Live F2 pup body weights were reduced at all doses and malformations observed in F2 pups of all DMF treated groups were similar to those observed for F1 litters. Craniofacial and sternebral malformations at the mid and high doses were characteristic and occurred in offspring of both generations.
Effects on developmental toxicity
Description of key information
Key (Hellwig et al., 1991; BASF AG, 1984),
OECD 414, rabbit, dermal, LOAEL = 100 mg/kg bw/d (according to RAC opinion;
2019)
Key (Hellwig et al. 1991; BASF AG, 1989), OECD 414, inhalative, rabbit,
NOAEC = 0.15 mg/L
Key (Hellwig et al. 1991; BASF AG, 1976a), OECD 414, oral, rat, NOAEL = 165 mg/kg bw/d
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1984
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- not specified
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rabbit
- Strain:
- Himalayan
- Remarks:
- Chbb:HM
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Dr K. Thomae GmbH (Biberach, Germany)
- Age at study initiation: 49 - 56 weeks old (at day of insemination)
- Housing: singly in wire cages (UNO HD II)
- Diet (e.g. ad libitum): daily 130 g of standardized pellet feed (SSNIFF)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: at least 2 weeks prior to artificial insemination.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- dermal
- Details on exposure:
- TEST SITE
- Area of exposure: 9 cm2 for the 100 mg/kg body weight/day dose group; 28 cm2 for the 200 mg/kg bw/d dose group; and 66 cm2 for the 400 mg/kg bw/d dose group
- Type of wrap if used: porous dressing in four layers and gauze and a porous bandage
- Time intervals for shavings or clipplings: The skin area was changed daily during the application period to avoid irritation.
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100, 200 and 400 mg/kg bw/d - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of the test material was determined by gas chromatography.
- Details on mating procedure:
- no details given
- Duration of treatment / exposure:
- day 6-18 post insemination
- Frequency of treatment:
- 6 h/d
- Duration of test:
- 29 d
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 200 mg/kg bw/day
- Dose / conc.:
- 400 mg/kg bw/day
- No. of animals per sex per dose:
- 15
- Control animals:
- yes, sham-exposed
- Details on study design:
- - Dose selection rationale: In a range-finding study 400 and 800 mg/kg bw/d had caused maternal toxicity in pregnant rabbits. A level of 400 mg/kg bw/d was therefore chosen as the highest dose for the main study.
- Rationale for animal assignment (if not random): Randomization of the test animals, grouped according to day 0 of gestation, was performed using computer-generated tables of random numbers on the first day of the acclimatization period. - Maternal examinations:
- DETAILED CLINICAL OBSERVATIONS: Yes ((including abortions or premature birth)
BODY WEIGHT: Yes
FOOD CONSUMPTION: Yes
POST-MORTEM EXAMINATIONS: Yes
-macroscopic pathology in dams - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes all per litter
- Skeletal examinations: Yes all per litter - Statistics:
- The Williams' test (analysis of trend), the Mann-Whitney U test and Fisher test (Lienert, 1973; Williams, 1971 and 1972) were used for statistical analyses. In every case the test was carried out at the 95 and 99% confidence levels and the dose groups were always compared with the control groups.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical signs in the does were significant skin irritation and one abortion, with six implantations in the highest dose group.
- Dermal irritation (if dermal study):
- effects observed, treatment-related
- Description (incidence and severity):
- significant skin irritation in the highest dose group
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A 5.5 and 5.6 % decrease in maternal body weights in relation to the control animals was recorded in the highest dose group (400 mg/kg body weight/day) towards the end of treatment period from day 16 to 18 post insemination.
- Food efficiency:
- not specified
- Ophthalmological findings:
- not examined
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- not specified
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- no effects observed
- Number of abortions:
- effects observed, treatment-related
- Description (incidence and severity):
- one abortion, with six implantations in the highest dose group.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- Preimplantation losses were 18.07, 21.00, 20.57 and 17.73 % with increasing dose level. For postimplantation losses no differences of biological relevance were found between the dose groups.
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- effects observed, treatment-related
- Description (incidence and severity):
- One dead foetus was found in the 400 mg/kg bw/d.
- Changes in pregnancy duration:
- not specified
- Changes in number of pregnant:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 200 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical signs
- dermal irritation
- number of abortions
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- Foetal weight was not influenced by the treatment regimen.
- Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- not specified
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- not specified
- External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- In the highest dose group at 400 mg/kg body weight/day several malformations were observed: two foetuses in two litters showed umbilical hernia. Skeletal (sternal) malformations were found in 15 foetuses in seven litters, and five foetuses in two litters had gall bladder agenesis. In animals of the 200 mg/kg body weight/day group and of the untreated control group no malformations occurred. At 100 mg/kg body weight/day one foetus (out of 80 live foetuses) had a sternal anomaly, two foetuses had gall bladder agenesis and one of the latter a hypertrophic-dilatative cardiac-aortic malformation.
- Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- In the highest dose group at 400 mg/kg body weight/day several malformations were observed: two foetuses in two litters showed umbilical hernia. Skeletal (sternal) malformations were found in 15 foetuses in seven litters, and five foetuses in two litters had gall bladder agenesis. In animals of the 200 mg/kg body weight/day group and of the untreated control group no malformations occurred. At 100 mg/kg body weight/day one foetus (out of 80 live foetuses) had a sternal anomaly, two foetuses had gall bladder agenesis and one of the latter a hypertrophic-dilatative cardiac-aortic malformation.
- Visceral malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- In the highest dose group at 400 mg/kg body weight/day several malformations were observed: two foetuses in two litters showed umbilical hernia. Skeletal (sternal) malformations were found in 15 foetuses in seven litters, and five foetuses in two litters had gall bladder agenesis. In animals of the 200 mg/kg body weight/day group and of the untreated control group no malformations occurred. At 100 mg/kg body weight/day one foetus (out of 80 live foetuses) had a sternal anomaly, two foetuses had gall bladder agenesis and one of the latter a hypertrophic-dilatative cardiac-aortic malformation.
- Dose descriptor:
- LOAEL
- Effect level:
- 100 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- external malformations
- skeletal malformations
- visceral malformations
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- external: trunk
- skeletal: sternum
- visceral/soft tissue: hepatobiliary
- visceral/soft tissue: cardiovascular
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 100 mg/kg bw/day
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects in the absence of maternal toxicity effects
- Dose response relationship:
- yes
- Conclusions:
- Under the conditions of the present study and according to the authors, disregarding the skin reactions, the NOEL for maternal toxicity is 200 mg/kg bw/d and the LOAEL for teratogenicity was 100 mg/kg bw/d.
- Executive summary:
Study design
The teratogenic effects of DMF were studied in groups of 15 rabbits. Rabbits were between 49 and 56 weeks old and had a mean weight of 2.572 kg (calculated from the means of the groups) on the day of artificial insemination, which was designated as day 0 of gestation. The test substance was administered directly (i.e. undiluted) on the shaved dorsal skin daily for 6 hours from day 6 to 18 post insemination. Depending on the dose, DMF was applied to an area of about 9, 28 or 66 cm² (for the low, mid and high dose, respectively). The amount of DMF to be administered at each dose level per kg body weight was 0.105 mL, 0.211 mL and 0.421 mL for the low, mid and high dose group, respectively. The skin area was changed daily during the application period to avoid irritation. The test material was applied semi-occlusively using a cover of a porous dressing in four layers and gauze and a porous bandage. After the 6 hours the patches and bandages were removed. Control animals received a volume of 0.421 ml 0.9 % saline solution/kg bw in the same manner. During the application period (6 hours/day) the animals were placed in a hood. On day 29 post insemination the does were sacrificed and macroscopically examined for pathological changes. Uterus, uterine contents and fetuses were investigated. All fetuses were eviscerated, the organs examined macroscopically and the sex determined. For skeletal examination the fetuses were X-rayed, the heads were fixed in Bouin's solution and after fixations processed and evaluated according to the method of Wilson (1965).
Results
All animals survived until termination of the study. Conception rate varied between 93.33 and 100 %. The repeated dermal application caused a dose dependent skin irritation in all DMF-treated groups. At the end of the treatment period, days 16 and 18 of gestation, a slight statistical significant decrease in body weight was observed at 400 mg/kg/d (5.5 and 5.6 % decrease in relation to the control animals). However, according to the authors, this finding was without biological relevance. One dose of the 400 mg/kg group showed abortion on day 21 post insemination. No further signs of maternal toxicity were noted. Three fetuses with gall bladder agenesis and one of the latter with a hypertrophic-dilatative cardiac-aortic malformation were observed at 100 mg/kg bw/d. Although no malformations were observed in the group exposed to 200 mg/kg/day, the lowest dose of 100 mg/kg bw/d is considered as LOAEL for teratogenicity. There were no differences between the groups concerning the variations and retardations. However, one dead fetus was found at 400 mg/kg/d and several malformations were observed, i.e two fetuses in two litters showed umbilical hernia, a distinct increase of skeletal anomalies in the form of sternal malformations was seen in 15 fetuses in seven litters and 5 fetuses in 2 litters had gall bladder agenesis. Thus 21 fetuses out of 9 litters (31 % fetuses/litter versus 0.0 % in the concurrent control) showed anomalies at 400 mg/kg/d. With the exception of the anomalies of the sternum, the other findings mentioned above for the 400 mg/kg group can be seen in the strain of rabbits used in this experiment, thus they were regarded to be independent of the compound administered.
Conclusion
Under the conditions of the present study and according to the authors, disregarding the skin reactions, the NOEL for maternal toxicity is 200 mg/kg bw/d and the LOAEL for teratogenicity was 100 mg/kg bw/d. This LOAEL is in accordance with the RAC opinion on DMF (20 Septermber 2019).
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1989
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- not specified
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rabbit
- Strain:
- Himalayan
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach, Germany
- Age at study initiation: 32 - 41 weeks
- Weight at study initiation: about 2.7 kg
- Housing: single
- Diet: KLIBA 24-341-4, 10 mm pellets, supplied by Klingentalmahle AG, Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 7 days, before the exposure period the animals were sham-exposed for acclimatization on 4 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation
- Vehicle:
- unchanged (no vehicle)
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- gas-chromatography
- Details on mating procedure:
- - Impregnation procedure: artificial insemination
- Duration of treatment / exposure:
- day 7 - 19 post insemination
- Frequency of treatment:
- 6 h/d
- Duration of test:
- 29 days
- Dose / conc.:
- 51 ppm
- Remarks:
- ca. 0.15 mg/L
- Dose / conc.:
- 148 ppm
- Remarks:
- ca. 0.45 mg/L
- Dose / conc.:
- 452 ppm
- Remarks:
- ca. 1.36 mg/L
- No. of animals per sex per dose:
- 15 (females only)
- Control animals:
- yes, concurrent no treatment
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: on the days of exposure, before, during and after exposure. During the remaining study period clinical signs and findings were recorded on each working day .
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: days 3, 7, 10, 13, 16, 19, 21, 24, 27 and 29 p .i .
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: If heads of fetuses revealed severe findings. - Statistics:
- Dunnett's Test was used for statistical evaluation of body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation loss, resorptions and live fetuses. Fisher's Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
- Clinical signs:
- no effects observed
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Two spontaneous deaths were observed in the control group.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No statistically significant difference in body weights occurred between the exposed group and the control group. Does of the highest exposure group, however, showed a retardation of body-weight gain; these animals lost some weight (about 34.4 g) particularly between days seven and 10 post insemination and showed a static weight until day 19 post insemination. At 150 ppm body weights were static during exposure (+ 3.1 g), while the animals at 50 ppm gained weight during exposure (31 - 42.4g). Corrected body-weight gain (day 29 -day 7 post implantation) showed no clear differences.
- Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The maternal toxicity elicited at 450 and 150 ppm was in accordance with maternal toxicity observed at 300 ppm in the range-finding study, which also led to deviations of blood chemistry parameters: an increase in clotting time, a decrease in serum albumin concentration and an increase in cholesterol levels. These effects may be indicative of some liver toxicity at this exposure level.
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- not specified
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- no effects observed
- Number of abortions:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At 150 ppm one animal aborted. No casualties occurred at 450 ppm.
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- not specified
- Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- One non-pregnancy in control group and at 50 ppm. At 150 and 450 ppm all animals were pregnant.
- Other effects:
- no effects observed
- Details on maternal toxic effects:
- Maternal toxic effects: yes
Details on maternal toxic effects:
With respect to survival of the treated animals, one animal of the mid dose group was sacrificed on day 27 p.i. due to abortion. In the control group one animal with rhinitis was sacrificed on day 7 p.i. and one animal died on day 16 p.i. Necropsy findings for these animals were of incidental nature.
Maternal toxicity was observed at 0.45 mg/L (static weight during exposure) and at 1.36 mg/L (body weight loss of about 34.4 g between days 7 and 10 p.i. and static weight until day 19 p.i.). No clinical symptoms or autopsy findings that could be related to treatment were seen. No effects on uterine weights or reproduction data were observed. - Dose descriptor:
- NOAEC
- Effect level:
- ca. 0.15 mg/L air
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Foetal weights were significantly lowered at 450 ppm.
- Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- not specified
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- not specified
- External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- At 450 ppm there was a significant increase in malformations-- mostly hernia umbilicalis (seven in 86 foetuses in four out of 15 litters)--and some soft tissue malformations, such as missing gall bladder (not statistically significant). In addition, anomalies of the sternum, increases in numbers of split vertebrae and a number of variations were also recorded. At 150 ppm one hernia umbilicalis among 75 foetuses and an increase in sternal variations were observed. At 50 ppm the foetuses did not show any response to treatment.
- Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- At 450 ppm there was a significant increase in malformations-- mostly hernia umbilicalis (seven in 86 foetuses in four out of 15 litters)--and some soft tissue malformations, such as missing gall bladder (not statistically significant). In addition, anomalies of the sternum, increases in numbers of split vertebrae and a number of variations were also recorded. At 150 ppm one hernia umbilicalis among 75 foetuses and an increase in sternal variations were observed. At 50 ppm the foetuses did not show any response to treatment.
- Visceral malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- At 450 ppm there was a significant increase in malformations-- mostly hernia umbilicalis (seven in 86 foetuses in four out of 15 litters)--and some soft tissue malformations, such as missing gall bladder (not statistically significant). In addition, anomalies of the sternum, increases in numbers of split vertebrae and a number of variations were also recorded. At 150 ppm one hernia umbilicalis among 75 foetuses and an increase in sternal variations were observed. At 50 ppm the foetuses did not show any response to treatment.
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Embryo-/fetotoxicity (significantly reduced fetal body weights, i.e. mean fetal body weight was 37.7 g in comparison to 43.7 g in the concurrent control group) was observed at the highest concentration which was maternal toxic. In this group, the incidence of malformations (especially hernia umbilicalis in 7 out of 86 fetuses in 4 out of 15 litters) and variations (mainly skeletal, i.e. skull bones and sternebrae) was significantly increased. A slight increase was found for external variations (i.e. pseudoankylosis in 6 out of 86 fetuses in 2 of 15 litters). Total malformations occurred at a fetal incidence of 15 and a litter incidence of 9 at 1.36 mg/L in comparison to a fetal incidence of 3 and a litter incidence of 2 in the concurrent control. Fetal and litter incidences for total variations at 1.36 mg/l were 77 and 15, respectively in comparison to 29 and 11 in the concurrent control. One hernia umbilicalis among 75 fetuses was observed in the 0.45 mg/L group, the number of skeletal variations was also increased in this group but without being statistical significant. - Dose descriptor:
- NOAEC
- Effect level:
- ca. 0.15 mg/L air
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- external malformations
- skeletal malformations
- visceral malformations
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- external: thorax
- skeletal: sternum
- visceral/soft tissue: hepatobiliary
- Developmental effects observed:
- not specified
- Conclusions:
- Maternal toxicity was seen at 0.45 mg/L and 1.36 mg/L and clear signs of embryo-/fetotoxicity including indications of teratogenicity were seen at the highest concentration tested. The possibility that there was also a minimal toxic/teratogenic effect on the 0.45 mg/L fetuses cannot be excluded .
- Executive summary:
Study design
This fully reliable GLP-study was performed according to OECD TG 414 (Prenatal Developmental Toxicity Study). In the study 15 female Himalayan rabbits per group were used. At the start of the study (i.e. day 0 = day of artificial insemination), the animals weighed about 2.7 kg and were about 32-41 weeks old. Animals of the negative concurrent control were exposed to air, for the DMF-treated groups concentrations of 50, 150 and 450 ppm were chosen. The animals were treated with DMF vapor by whole-body exposure on day 7 through day 19 post insemination. During exposure food and water were withdrawn. Post-treatment period lasted from day 20 p.i. until the day the animals were sacrificed (29 p.i.). The analytically determined concentrations were calculated to the mean of the overall concentration and were 51 ppm, 148 ppm and 452 ppm, respectively.
Results and conclusion
Maternal toxicity was seen at 0.45 mg/L and 1.36 mg/L and clear signs of embryo-/fetotoxicity including indications of teratogenicity were seen at the highest concentration tested. The possibility that there was also a minimal toxic/teratogenic effect on the 0.45 mg/L fetuses cannot be excluded.
- Endpoint:
- developmental 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 report which meets basic scientific principles.
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Principles of method if other than guideline:
- - Principle of test: In accordance with the FDA guidelines (Guidelines for reproduction studies for safety evaluation of drugs for human use, Food and Drug Administration, Washington 1966)
- Short description of test conditions: DMF was dissolved in distilled water and administered by gavage (to a volume of 5 mL/kg body weight) to 19-23 pregnant females/group from day 6 to day 15 of gestation. Each treated group had an untreated control of 18-23 pregnant females. The female rats were impregnated overnight by untreated males of the same strain and of proven fertility. When sperm were detected next morning in the vaginal smear, the day was defined as day 0 of gestation. Cesarian section was carried out on day 20 of gestation
- Parameters analysed / observed: Body weight was determined three times a week, clinical signs and mortality were checked each day. At sacrifice all animals were examined for gross pathological changes and uterine contents were investigated. All fetuses were examined for external changes (malformations, variations, retardations) and 2/3 of the fetuses in each litter were examined for skeletal - and 1/3 of the fetuses of each litter were examined for soft tissue malformations, variations and retardations. - GLP compliance:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: WIGA, Sulzfeld, Germany
- Weight at study initiation: females: 221 g (mean)
- Housing: 2 animals per cage
- Diet: Altromin-R, Altrogge, ad libitum
- Water: tap water ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 5 %
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- no details given
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy - Duration of treatment / exposure:
- days 6 - 15 of gestation
- Frequency of treatment:
- daily
- Duration of test:
- All the dams were sacrificed on day 20 of gestation.
- Dose / conc.:
- 165 mg/kg bw/day
- Remarks:
- 176 µL/kg based on density: d= 0.94 g/cm3, actual ingested
- Dose / conc.:
- 500 mg/kg bw/day
- Remarks:
- 533 µL/kg based on density: d= 0.94 g/cm3, actual ingested
- Dose / conc.:
- 1 500 mg/kg bw/day
- Remarks:
- 1600 µL/kg based on density: d= 0.94 g/cm3, actual ingested
- No. of animals per sex per dose:
- 19-23
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- no information given
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each day
BODY WEIGHT: Yes
- Time schedule for examinations: 3 times/week
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: internal organs - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: No data - Statistics:
- not specified
- Clinical signs:
- not specified
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- With the exception of one animal in the high dose group, that died on day 10 p.c., all animals survived until termination of the study.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Maternal toxicity occurred at 1500 and 500 mg DMF/kg shown by a dose-dependent decrease in body weight gain. (At 1500 mg/kg a clear stagnation of body weight gain during the time of test substance application (day 6-15 of gestation) was observed)
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- 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:
- not specified
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- no effects observed
- Pre- and post-implantation loss:
- effects observed, treatment-related
- Description (incidence and severity):
- At the dose of 500 mg/kg embryolethality occured; about 11 % of the implants died mainly in the early part of pregnancy.
- Total litter losses by resorption:
- effects observed, treatment-related
- Description (incidence and severity):
- Embryolethality occured at 1500 mg/kg bw. About 63 % of the implants were resorbed, thus the number of live fetuses (85 in comparison to 265 live fetuses of the concurrent control) was distinctly reduced.
- Early or late resorptions:
- effects observed, treatment-related
- Description (incidence and severity):
- Embryolethality occured at 1500 mg/kg bw. About 63 % of the implants were resorbed, thus the number of live fetuses (85 in comparison to 265 live fetuses of the concurrent control) was distinctly reduced.
- Dead fetuses:
- not specified
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- not specified
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- - At 1500 mg/kg bw mean placental weight was significantly reduced.
- At the dose of 500 mg/kg signs of embryo-/fetotoxicity were seen in the form of reduced mean placental weight.
- At 165 mg/kg dose-related reduction of mean placental weight (0.50 g versus 0.52 g in the concurrent control) was observed (slightly but significantly) - Dose descriptor:
- NOAEL
- Remarks:
- maternal toxicity
- Effect level:
- ca. 165 mg/kg bw/day
- 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):
- - Embryolethality occured at 1500 mg/kg bw: Live fetuses of this group had significantly reduced mean body weight.
- At the dose of 500 mg/kg signs of embryo-/fetotoxicity were seen in the form of significantly reduced mean fetal body weight.
- At 165 mg/kg bw fetal weights of the low dose were comparable to the respective concurrent control or even higher. - Reduction in number of live offspring:
- effects observed, treatment-related
- Description (incidence and severity):
- - At 1500 mg/kg bw the number of live fetuses (85 in comparison to 265 live fetuses of the concurrent control) was distinctly reduced.
- At 165 mg/kg live fetuses ot the low dose were comparable to the respective concurrent control or even higher. - Changes in sex ratio:
- not specified
- Changes in litter size and weights:
- effects observed, treatment-related
- Description (incidence and severity):
- - At the dose of 500 mg/kg signs of embryo-/fetotoxicity were seen in the form of significantly reduced mean fetal body weight.
- At 165 mg/kg bw fetal weights of the low dose were comparable to the respective concurrent control or even higher. - Changes in postnatal survival:
- not specified
- External malformations:
- not specified
- Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- - Embryolethality occured at 1500 mg/kg bw. Mean placental weight was significantly reduced and live fetuses of this group had significantly reduced mean body weight and showed an increased incidence of skeletal retardations.
- Teratogenicity occured at 1500 mg/kg bw: About 12 %, i.e. 10 fetuses of the 85 live fetuses had one or more malformations in the form of anasarca (9 fetuses), aplasia of the tail (2 fetuses) and micrognathia (1 fetus) as well as malformations of the vertebral column (mainly aplasia), ribs and sternum.
- The oral administration of 500 mg DMF/kg also led to teratogenicity in about 9.47 % (25 of 264 fetuses) of the live fetuses in the form of anasarca (1 fetus), aplasia of the tail (2 fetuses) and atresia ani (1 fetus), cleft palate (1 fetus) and open eyelid (1 fetus) as well as malformations of the vertebral column (split or aplastic vertebrae).
- At the dose of 500 mg/kg embryolethality occured; about 11 % of the implants died mainly in the early part of pregnancy. Signs of embryo-/fetotoxicity were seen in the form of increased incidence of skeletal and organ retardations and/or variations. - Visceral malformations:
- not specified
- Other effects:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Embryolethality occured at 1500 mg/kg bw. About 63 % of the implants were resorbed, thus the number of live fetuses (85 in comparison to 265 live fetuses of the concurrent control) was distinctly reduced. Mean placental weight was significantly reduced and live fetuses of this group had significantly reduced mean body weight and showed an increased incidence of skeletal retardations. At this dose teratogenicity occurred. About 12 %, i.e. 10 fetuses of the 85 live fetuses had one or more malformations in the form of anasarca (9 fetuses), aplasia of the tail (2 fetuses) and micrognathia (1 fetus) as well as malformations of the vertebral column (mainly aplasia), ribs and sternum.
At the dose of 500 mg/kg embryolethality occured; about 11 % of the implants died mainly in the early part of pregnancy. Signs of embryo-/fetotoxicity were seen in the form of significantly reduced mean fetal body weight and reduced mean placental weight as well as an increased incidence of skeletal and organ retardations and/or variations. The oral administration of 500 mg DMF/kg also led to teratogenicity in about 9.47 % (25 of 264 fetuses) of the live fetuses in the form of anasarca (1 fetus), aplasia of the tail (2 fetuses) and atresia ani (1 fetus), cleft palate (1 fetus) and open eyelid (1 fetus) as well as malformations of the vertebral column (split or aplastic vertebrae).
At the dose of 165 mg/kg neither maternal toxicity nor clear embryo-/fetotoxicity or teratogenicity occured. The only finding was slightly but significantly and dose-related reduced mean placental weight (0.50 g versus 0.52 g in the concurrent control), however the number of live fetuses and fetal weights of the low dose were comparable to the respective concurrent control or even higher. - Dose descriptor:
- NOAEL
- Effect level:
- ca. 165 mg/kg bw/day
- Basis for effect level:
- other: teratogenicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- In conclusion, a NOAEL for fetal toxicity of 165 mg/kg bw was determined based teratogenic effects (number of total litter losses by resorption and body weight change at 1500 and 500 mg DMF/kg). A NOAEL for maternal toxicity of 165 mg/kg/bw was determined based dose-dependent decrease in body weight gain at 1500 and 500 mg DMF/kg.
- Executive summary:
Study design
The present non-GPL study was conducted in accordance with the FDA guidelines (Guidelines for reproduction studies for safety evaluation of drugs for human use, Food and Drug Administration, Washington 1966). In the study N,N, Dimethylformamide (DMF) was dissolved in distilled water and administered by gavage (to a volume of 5 mL/kg body weight) to 19-23 pregnant females/group from day 6 to day 15 of gestation. Each treated group had an untreated control of 18-23 pregnant females. The female rats were impregnated overnight by untreated males of the same strain and of proven fertility. When sperm were detected next morning in the vaginal smear, the day was defined as day 0 of gestation. Cesarian section was carried out on day 20 of gestation. Body weight was determined three times a week, clinical signs and mortality were checked each day. At sacrifice all animals were examined for gross pathological changes and uterine contents were investigated. All fetuses were examined for external changes (malformations, variations, retardations) and 2/3 of the fetuses in each litter were examined for skeletal - and 1/3 of the fetuses of each litter were examined for soft tissue malformations, variations and retardations.
Results
Embryotoxic / teratogenic effects:yes
- Embryolethality occured at 1500 mg/kg bw. About 63 % of the implants were resorbed, thus the number of live fetuses (85 in comparison to 265 live fetuses of the concurrent control) was distinctly reduced. Mean placental weight was significantly reduced and live fetuses of this group had significantly reduced mean body weight and showed an increased incidence of skeletal retardations. At this dose teratogenicity occurred. About 12 %, i.e. 10 fetuses of the 85 live fetuses had one or more malformations in the form of anasarca (9 fetuses), aplasia of the tail (2 fetuses) and micrognathia (1 fetus) as well as malformations of the vertebral column (mainly aplasia), ribs and sternum.
- At the dose of 500 mg/kg embryolethality occured; about 11 % of the implants died mainly in the early part of pregnancy. Signs of embryo-/fetotoxicity were seen in the form of significantly reduced mean fetal body weight and reduced mean placental weight as well as an increased incidence of skeletal and organ retardations and/or variations. The oral administration of 500 mg DMF/kg also led to teratogenicity in about 9.47 % (25 of 264 fetuses) of the live fetuses in the form of anasarca (1 fetus), aplasia of the tail (2 fetuses) and atresia ani (1 fetus), cleft palate (1 fetus) and open eyelid (1 fetus) as well as malformations of the vertebral column (split or aplastic vertebrae).
- At the dose of 165 mg/kg neither maternal toxicity nor clear embryo-/fetotoxicity or teratogenicity occured. The only finding was slightly but significantly and dose-related reduced mean placental weight (0.50 g versus 0.52 g in the concurrent control), however the number of live fetuses and fetal weights of the low dose were comparable to the respective concurrent control or even higher.
Maternal toxic effects: yes
- With the exception of one animal in the high dose group, that died on day 10 p.c., all animals survived until termination of the study.
- Maternal toxicity occurred at 1500 and 500 mg DMF/kg shown by a dose-dependent decrease in body weight gain. At 1500 mg/kg a clear stagnation of body weight gain during the time of test substance application (day 6-15 of gestation) was observed.
Conclusion
In conclusion, a NOAEL for fetal toxicity of 165 mg/kg bw was determined based teratogenic effects (number of total litter losses by resorption and body weight change at 1500 and 500 mg DMF/kg).
A NOAEL for maternal toxicity of 165 mg/kg/bw was determined based on a dose-dependent decrease in body weight gain at 1500 and 500 mg DMF/kg.
Referenceopen allclose all
All animals survived until termination of the study. Conception rate varied between 93.33 and 100 %. The repeated dermal application caused a dose dependent skin irritation in all DMF-treated groups. At the end of the treatment period, days 16 and 18 of gestation, a slight statistical significant decrease in body weight was observed at 400 mg/kg/d (5.5 and 5.6 % decrease in relation to the control animals). However, according to the authors, this finding was without biological relevance. One dose of the 400 mg/kg group showed abortion on day 21 post insemination. No further signs of maternal toxicity were noted. Three fetuses with gall bladder agenesis and one of the latter with a hypertrophic-dilatative cardiac-aortic malformation were observed at 100 mg/kg bw/d. There were no differences between the groups concerning the variations and retardations. However, one dead fetus was found at 400 mg/kg/d and several malformations were observed, i.e two fetuses in two litters showed umbilical hernia, a distinct increase of skeletal anomalies in the form of sternal malformations was seen in 15 fetuses in seven litters and 5 fetuses in 2 litters had gall bladder agenesis. Thus 21 fetuses out of 9 litters (31 % fetuses/litter versus 0.0 % in the concurrent control) showed anomalies at 400 mg/kg/d. With the exception of the anomalies of the sternum, the other findings mentioned above for the 400 mg/kg group can be seen in the strain of rabbits used in this experiment, thus they were regarded to be independent of the compound administered. Although no malformations were observed in the group exposed to 200 mg/kg/day, the lowest dose of 100 mg/kg bw/d is considered as LOAEL forteratogenicity. This is in accordance with th RAC opinion on DMF (20 September 2019). They draw the folowing conclusion: DMF seems to affect the skeletal system in all three species, with the rabbit as the most sensitive species. Relevance to humans must be assumed. The first signs of malformations in rabbits are seen at dermal doses of 100 mg/kg/day (sternal malformations and gallbladder agenesis) and following inhalation exposure to 150 ppm (umbilical hernia and sternal malformations). Although low incidences, and not always supported by clear dose response, the malformations are rare and the incidences exceed the only available (improper) HCD for Himalayan rabbits. Sternal malformations, umbilical hernia and gallbladder agenesis are serious effects supporting using 100 mg/kg/day as LOAEL for dermal developmental toxicity and 150 ppm as LOAEC for inhalation developmental toxicity (NOAEC 50 ppm = 150 mg/m³).
Thus, under the conditions of the present study and according to the authors, disregarding the skin reactions, the NOEL for maternal toxicity is 200 mg/kg bw/d and the LOAEL for teratogenicity was 100 mg/kg bw/d.
Maternal toxicity was seen at 0.45 mg/L and 1.36 mg/L and clear signs of embryo-/fetotoxicity including indications of teratogenicity were seen at the highest concentration tested. The possibility that there was also a minimal toxic/teratogenic effect on the 0.45 mg/L fetuses cannot be excluded .
Weight (g):
| Dose (mg/kg) | day 0 | day 6 | day 11 | day 15 | day 20 | |
| 165 | 203 | 226 | 238 | 256 | 328 | |
| 500 | 230 | 254 | 268 | 274 | 338 | |
| 1500 | 227 | 250 | 245 | 246 | 285 | |
| control | 223 | 245 | 266 | 266 | 353 |
Clinical signs:
| Dose (mg/kg) | 1500 | control | 500 | control | 165 | control | ||
| Total animals | 22 | 24 | 26 | 25 | 20 | 20 | ||
| Pregnant animals | 20 | 23 | 23 | 22 | 19 | 18 | ||
| Dead animals | 1 | 0 | 0 | 0 | 0 | 0 | ||
| Total implantations | 235 | 291 | 296 | 296 | 252 | 230 | ||
| Implantations per dam | 11.6 | 12.65 | 12.87 | 13.45 | 13.26 | 12.78 | ||
| Live fetuses | 85 | 265 | 264 | 279 | 235 | 223 | ||
| Live fetuses per dam | 4.25 | 11.52 | 11.48 | 12.68 | 12.37 | 12.39 | ||
| Male fetuses | 52 | 141 | 146 | 135 | 132 | 111 | ||
| Female fetuses | 33 | 124 | 118 | 144 | 103 | 112 | ||
| Dead fetuses | 0 | 0 | 0 | 0 | 0 | 0 | ||
| Early resorptions | 22 | 25 | 21 | 16 | 15 | 6 | ||
| Medium-term resorptions | 116 | 1 | 1 | 1 | 1 | 0 | ||
| Late resorptions | 9 | 0 | 10 | 0 | 1 | 1 | ||
| Dead implants | 147## | 26 | 32## | 17 | 17 | 7 | ||
| Weight of males | 2.77xx | 3.96 | 3.34xx | 3.93 | 3.91xx | 3.79 | ||
| Weight of females | 2.66xx | 3.77 | 3.09xx | 3.76 | 3.64 | 3.62 | ||
| Overall weight | 2.73xx | 3.87 | 3.23xx | 3.84 | 3.79xx | 3.71 | ||
| Lenght of males | 3.19xx | 3.68 | 3.52xx | 3.66 | 3.66xx | 3.64 | ||
| Length of females | 3.09xx | 3.61 | 3.41xx | 3.62 | 3.59 | 3.56 | ||
| Overall length | 3.15xx | 3.65 | 3.47xx | 3.64 | 3.63xx | 3.60 | ||
| Placentae of males | 0.34xx | 0.53 | 0.44xx | 0.58 | 0.51 | 0.52 | ||
| Placentae of females | 0.34xx | 0.53 | 0.45xx | 0.57 | 0.50 | 0.53 | ||
| Overall placentae | 0.34xx | 0.53 | 0.44xx | 0.57 | 0.50xx | 0.52 | ||
| Runts overall | 55 | 0 | 28 | 1 | 2 | 1 | ||
| Malformation | 10# | 13 | 25## | 2 | 0 | 0 | ||
| % (live fetuses) | 11.76 | 9.91 | 9.47 | 0.72 | 0 | 0 |
Significance: t-test > 99%: xx; chi-squared-test >99%: ##; chi-squared-test >95%: #.
Indications of developmental toxic/teratogenic effects were seen in animal studies.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 165 mg/kg bw/day
- Study duration:
- subacute
- Experimental exposure time per week (hours/week):
- 9
- Species:
- rat
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 150 mg/m³
- Study duration:
- subacute
- Experimental exposure time per week (hours/week):
- 42
- Species:
- rabbit
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEL
- 100 mg/kg bw/day
- Study duration:
- subacute
- Experimental exposure time per week (hours/week):
- 42
- Species:
- rabbit
Additional information
Dermal route
In a dermal developmental toxicity study (OECD Guideline 414, (1981)) with rabbits, doses of 100, 200 and 400 mg/kg bw/day were administered on the shaved dorsal skin for 6 hours/day from day 6 to 18 post insemination (Hellwig et al., 1991; BASF, 1984). At the high dose signs of maternal toxicity and embryo-/fetotoxicity were observed. One dead fetus and several malformations (e.g. hernia umbilicalis, skeletal malformations) were found at this dose level. No embryo-/fetotoxic effects were found at the low and mid dose. 3 fetuses with malformations were seen in the low dose. Although no malformations were observed in the group exposed to 200 mg/kg/day, one sternal malformation and two cases of gallbladder agenesis were observed at 100 mg/kg/day. Therefore, 100 mg/kg bw/d is considered as LOAEL for teratogenicity (according to RAC opinion; 2019).
Inhalation route
In an inhalation developmental toxicity study NOAEC of 150 mg/m³ was established for rabbits for maternal as well as for embryo-/fetotoxicity including teratogenicity (BASF AG 87/586, 1989). Female Himalayan rabbits per concentration group were used. Animales were exposed to DMF vapour by whole-body exposure on day 7 through day 19 post insemination (p.i.). During exposure food and water were withdrawn. Post-treatment period lasted from day 20 p.i. until the day the animals were sacrificed (29 p.i.). The analytically determined concentrations were calculated to the mean of the overall concentration and were 51 ppm, 148 ppm and 452 ppm respectively (ca. 0.15, 0.45 and 1.36 mg/L). Maternal toxicity was seen at 0.45 mg/L and 1.36 mg/L and clear signs of embryo-/fetotoxicity including indications of teratogenicity were seen at the highest concentration tested. Embryo-/fetotoxicity resulted in significantly reduced fetal body weights (i.e. mean fetal body weight was 37.7 g in comparison to 43.7 g in the concurrent control group). In this group, the incidence of malformations (especially hernia umbilicalis in 7 out of 86 fetuses in 4 out of 15 litters) and variations (mainly skeletal, i.e. skull bones and sternebrae) was significantly increased. A slight increase was found for external variations (i.e. pseudoankylosis in 6 out of 86 fetuses in 2 of 15 litters). Total malformations occurred at a fetal incidence of 15 and a litter incidence of 9 at 1.36 mg/L in comparison to a fetal incidence of 3 and a litter incidence of 2 in the concurrent control. Fetal and litter incidences for total variations at 1.36 mg/L were 77 and 15, respectively in comparison to 29 and 11 in the concurrent control. One hernia umbilicalis among 75 fetuses was observed in the 0.45 mg/L group, the number of skeletal variations was also increased in this group but without being statistical significant.
In two inhalation supporting studies Long-Evans rats (Kimmerle and Machemer, 1975) and Sprague-Dawley rats (TSCATS: OTS 0516779, 1978) were exposed from day 6 to day 15 of gestation, 6 hours/day to exposure levels of 18 and 172 ppm (about 55 and 520 mg/m3) and to 30 and 300 ppm (about 90 and 910 mg/m3), respectively. In both studies teratogenicity was not observed, however fetotoxicity occurred at 172 ppm in the Long-Evans fetuses without signs of maternal toxicity whereas maternal toxicity and fetotoxicity were observed in the Sprague-Dawley rats at the exposure level of 300 ppm. In the Long-Evans fetuses fetotoxicity was represented by significantly reduced body weights in comparison to the control fetuses and in the Sprague-Dawley fetuses by significantly reduced fetal weights and a significant higher incidence of fetuses with ossification variations in comparison to the control fetuses.
Oral route
A present non-GPL study was conducted in accordance with the FDA guidelines (Guidelines for reproduction studies for safety evaluation of drugs for human use, Food and Drug Administration, Washington 1966). In the study N,N, Dimethylformamide (DMF) was dissolved in distilled water and administered by gavage (to a volume of 5 mL/kg body weight) to 19-23 pregnant females/group from day 6 to day 15 of gestation. Each treated group had an untreated control of 18-23 pregnant females. The female rats were impregnated overnight by untreated males of the same strain and of proven fertility. When sperm were detected next morning in the vaginal smear, the day was defined as day 0 of gestation. Cesarian section was carried out on day 20 of gestation. Body weight was determined three times a week, clinical signs and mortality were checked each day. At sacrifice all animals were examined for gross pathological changes and uterine contents were investigated. All fetuses were examined for external changes (malformations, variations, retardations) and 2/3 of the fetuses in each litter were examined for skeletal - and 1/3 of the fetuses of each litter were examined for soft tissue malformations, variations and retardations.
Embryotoxic / teratogenic effects:yes
Embryolethality occured at 1500 mg/kg bw. About 63 % of the implants were resorbed, thus the number of live fetuses (85 in comparison to 265 live fetuses of the concurrent control) was distinctly reduced. Mean placental weight was significantly reduced and live fetuses of this group had significantly reduced mean body weight and showed an increased incidence of skeletal retardations. At this dose teratogenicity occurred. About 12 %, i.e. 10 fetuses of the 85 live fetuses had one or more malformations in the form of anasarca (9 fetuses), aplasia of the tail (2 fetuses) and micrognathia (1 fetus) as well as malformations of the vertebral column (mainly aplasia), ribs and sternum. At the dose of 500 mg/kg embryolethality occured; about 11 % of the implants died mainly in the early part of pregnancy. Signs of embryo-/fetotoxicity were seen in the form of significantly reduced mean fetal body weight and reduced mean placental weight as well as an increased incidence of skeletal and organ retardations and/or variations. The oral administration of 500 mg DMF/kg also led to teratogenicity in about 9.47 % (25 of 264 fetuses) of the live fetuses in the form of anasarca (1 fetus), aplasia of the tail (2 fetuses) and atresia ani (1 fetus), cleft palate (1 fetus) and open eyelid (1 fetus) as well as malformations of the vertebral column (split or aplastic vertebrae). At the dose of 165 mg/kg neither maternal toxicity nor clear embryo-/fetotoxicity or teratogenicity occured. The only finding was slightly but significantly and dose-related reduced mean placental weight (0.50 g versus 0.52 g in the concurrent control), however the number of live fetuses and fetal weights of the low dose were comparable to the respective concurrent control or even higher.
Maternal toxic effects: yes
- With the exception of one animal in the high dose group, that died on day 10 p.c., all animals survived until termination of the study.
- Maternal toxicity occurred at 1500 and 500 mg DMF/kg shown by a dose-dependent decrease in body weight gain. At 1500 mg/kg a clear stagnation of body weight gain during the time of test substance application (day 6-15 of gestation) was observed.
In conclusion, a NOAEL for fetal toxicity of 165 mg/kg bw was determined based teratogenic effects (number of total litter losses by resorption and body weight change at 1500 and 500 mg DMF/kg).
A NOAEL for maternal toxicity of 165 mg/kg/bw was determined based dose-dependent decrease in body weight gain at 1500 and 500 mg DMF/kg.
Justification for classification or non-classification
The classification as Repr.1B; H360D "May damage the unborn child" is warranted according to the criteria of Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
