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Developmental toxicity / teratogenicity

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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
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
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

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 .

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.

Reason / purpose for cross-reference:
reference to other study
Reference
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

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    
Malformation  10#  13  25##  2  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.
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.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1984
Report date:
1984
Reference Type:
publication
Title:
Studies on the prenatal toxicity of N,N-dimethylformamide in mice, rats and rabbits.
Author:
Hellwig, J. et al.
Year:
1991
Bibliographic source:
Fd. Chem. Tox. 29, 193-201, (1991)

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethylformamide
EC Number:
200-679-5
EC Name:
N,N-dimethylformamide
Cas Number:
68-12-2
Molecular formula:
C3H7NO
IUPAC Name:
N,N-dimethylformamide
Test material form:
liquid
Details on test material:
N,N-Dimethylformamide, purity: 99.99 %

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
external: trunk
skeletal: sternum
visceral/soft tissue: hepatobiliary
visceral/soft tissue: cardiovascular

Overall developmental toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).