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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comprehensive NTP Guideline study. Comparable to guideline study.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Formamide and diemethylformamide: reproductive assessment by continous breeding in mice
Author:
Fail, PA, George JD, Grizzle TB and Heindel J
Year:
1998
Bibliographic source:
Reprod. Toxicol. 12, 317-332.
Reference Type:
publication
Title:
Potential reproductive toxicity of formamide (FORM) as evaluated by the continous breeding protocol.
Author:
Fail PA, George JD, Grizzle TB and Heindel J
Year:
1993
Bibliographic source:
Toxicologist 13, 76; abstract no. 197.
Reference Type:
publication
Title:
Formamide.
Author:
Heindel J, George J, Fail P, Grizzle T and Feldman D
Year:
1997
Bibliographic source:
Environ. Health Perspect. 105 (suppl. 1), 311-312.
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
Principles of method if other than guideline:
Continuous breeding; F0 animals were allowed to produce 5 litters.
Crossover mating trial
Two generation study; mating of F1 animals
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Formamide
EC Number:
200-842-0
EC Name:
Formamide
Cas Number:
75-12-7
Molecular formula:
CH3NO
IUPAC Name:
formamide
Details on test material:
- substance name: formamide
- analytical purity: >99%

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh. NC, USA
- Age: 6 weeks old upon arrival
- Age at study initiation: (P) 8-11 wks; (F1) 10-11 wks
- Fasting period before study: no
- Housing: singly during exposure week 1; in breeding pairs during weeks 2 through 15; singly starting at week 16
- Diet: pelleted feed (NIH-07 Rodent Chow, Zeigler Brothers, Gardners, PA) ad libitum.
- Water: deionized/filtered waterad libitum.
- Acclimation period: 2- 5 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 (72± 0.2°F)
- Humidity (%): 58 ± 0.1 %
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
mixing formamide with water.

Details on mating procedure:
Fo cohabitation:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 weeks
- Proof of pregnancy: vaginal plug

Crossover 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 Fo animals were created : (1) control male X control female, (2) high-dose male X control female, and (3) control male X high-dose female.
- M/F ratio per cage: 1:1
- Length of cohabitation: 1 week
- Proof of pregnancy: vaginal plug
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged: singly
- Any other deviations from standard protocol: no dosing during cohabitationin order to avoid dosing of control animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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:
Exposure period: depending on the sub-part of the study: approx. 24 (F0, Task 2), and up to 30 wk (Task 3, F0)
Premating exposure period (males): 1 wk (F0); approx. 11 wk post-lactation exposure (F1)
Premating exposure period (females): 1 wk (F0); approx. 11 wk post-lactation exposure (F1)
Frequency of treatment:
continuously in the drinking water
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 10-11 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
ca. 16-32, 48-110, 144-226 mg/kg bw/d (100, 350, 750 ppm in the drinking water); for details see free text
Basis:
actual ingested
No. of animals per sex per dose:
Fo generation: control group: 40 /sex; treated groups: 20/dose/sex
F1 generation: 20/dose/sex
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on results of a range finding study
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations:
Fo generation: weeks 1, 8, 16
F1 generation: at birth, weeks 12, 16


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Fo generation: weeks 1, 8, 16
F1 generation: at days 74, 84, and 119 of age (all ± 10 days)
Oestrous cyclicity (parental animals):
Yes, control and high-dose Fo and F1 females (publication Fail (1998, tables 5 and 10, resp.)
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations: testis weight, epididymis weight, 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:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes; and on day 0


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain


GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
SACRIFICE
F0 generation:
- Male animals: All surviving animals in week 29 , i.e. as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals in week 29 , i.e. as soon as possible after the last litters in each generation were produced.

F01generation:
- Male animals: All surviving animals at day 119 of age, i.e. as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals at day 119 of age, i.e. as soon as possible after the last litters in each generation were produced.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively, as indicated below.

Liver and paired kidney (with attached adrenal) weights were collected for both sexes. For males, right testis, right epididymis, prostate,
and seminal vesicles with coagulating glands (glandular secretions not removed) were weighed at necropsy. 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 µm 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 .
Postmortem examinations (offspring):
As described for the F0 animals.
Statistics:
Statistical methods were extensively used; cf. publication Fail (1998), page 320.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no dose-related clinical signs of toxicity or mortalities.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The body weights of the male mice at 750 ppm was reduced by 7% after 8 weeks and remained decreased until week 27 (14% at
week 16, 7% at termination). High-dose females had also significantly decreased body weights on weeks 16 and 23. The reduced
body weights occurred in the presence of increased feed consumption, especially in males (about 20 % more than controls at 750 ppm).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

---------------------------------------------------------
Dose groups
(ppm in drinking water) Actually ingested doses
(mg/kg bw/day)
F0 males F0 females
(week 1 / week 27) (week 1 / week 27)
---------------------------------------------------------
100 23 / 16 30 / 31
350 81 / 48 110 / 95
750 220 / 144 227 / 217
---------------------------------------------------------

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
After the crossover mating, the status of reproductive cycles was monitored in the control and high-dose Fo females (cf. Fail (1998),
Table 5, p. 326) . Evaluation of vaginal smears for 12 consecutive days indicated significant differences in the relative frequency
of time spent in the individual estrous stages. Females in the 750 ppm group spent more time in diestrus and less time in proestrus,
metestrus, and estrus than did the control females. Furthermore, only 58% of the treated females had 4- to 5-d estrous cycles,
compared to 91% of controls .

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No dose-related changes were noted for sperm parameters.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The number of litters per pair was slightly reduced in the group at 750 ppm compared to controls (4.6 vs. 5.0; p < 0.05) as was
the number of live pups per litter (8.8 vs. 12.3; p < 0.05), whereas the number of the days to first litter was increased (26.2 vs.
21.9 days; p < 0.05). Other reproductive parameters (i.e. fertility, percent of live pups, live pup weight of the first litter) were not
affected at any dose. In the final 5th litter, the fertility was decreased at 750 ppm to 65% compared 95% in controls (p < 0.05);
fertility was not affected in the groups at 100 and 350 ppm (cf. Fail (1998), Table 2, p. 322).

The cross-over experiment revealed that the reduced fertility was mainly due to impairment of reproduction in females. Mating
of control males with 750 ppm females gave a fertility of 37% (p < 0.05), whereas mating of 750 ppm with control females reduced
fertility to 70% which was not statistically significantly different from the control male and female mating (94%) (cf. Fail (1998), Table 4, p. 325).

ORGAN WEIGHTS (PARENTAL ANIMALS)
Most organ weights were unaffected in FORM-treated animals (cf. data in NTIS, 1992), except the weight of the seminal vesicles of
high-dose males (421 .9 ± 27.7 vs . 551 .6 ± 29.9 mg in controls) was decreased.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No dose-related changes were noted.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No dose-related changes were noted for the histopathology of somatic or reproductive organs of either sex

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
144 - 220 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: body weight reduction of less than 10% at termination at 750 ppm
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
217 - 227 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: body weight reduction of less than 10% at termination at 750 ppm
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
144 - 220 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: no adverse effects noted at highest tested dose
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
95 - 110 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No effect at 350 ppm. At 750 ppm: prolonged time to litter; reduced litter size; reduced number of litters per pair; reduced fertility in final (5th) litter; prolonged time in diestrus. Reduced female fertility in the crossover trial.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction, F1 pups
Generation:
F1
Effect level:
217 - 227 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: F1 pup viability, pup weight not affected in dams at 750 ppm (217-227 mg/kg bw/day)
Dose descriptor:
NOAEL
Remarks:
general toxicity, F1 parental
Generation:
F1
Effect level:
144 - 227 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No relevant adverse effect at teh highest tested dose in F1 parental animals. Doses received: males 144-220 mg/kg bw/day; females: 217-227 mg/kg bw/day.
Dose descriptor:
NOAEL
Remarks:
reproduction, F1 parental
Generation:
F1
Effect level:
152 - 183 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: no effect on reproductive parameters, organs at highest tested dose of 750 ppm
Dose descriptor:
NOAEL
Remarks:
reproduction, F1 parental
Generation:
F1
Effect level:
85 - 101 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

As to the F1 generation, the doses of formamide were comparable to that of the F0 animals. Again, females had higher doses than

males, and doses increased during lactation. Feed and water consumption was also increased in the male and female high dose

groups, and the terminal body weights were reduced at necropsy.

 

 

Dose groups

(ppm in drinking water)

Actually ingested doses

(mg/kg bw/day)

 

F1 males

(week 12 / week 16)

F1 females

(week 12 / week 16)

100

23  /  27

25  /  31

350

77  /  73

101  /  85

750

183  /  152

219  /  201

 

 

At 750 ppm, the fertility rate was significantly decreased (60% vs. 89% in controls; p<0.05), the number of live pups was reduced

(8.0 vs. 10.9; p < 0.05), and the average days to delivery of litters was increased (24.3 days vs. 20.9; p < 0.05). The live pup weight

was not affected (cf. Fail (1998), Table 8, p. 328).

The estrous cycle of F1 mice at 750 ppm was extended (6.5 vs. 4.8 days in controls), and the high-dose group tended to be in estrous

for a shorter time than controls (17.4 vs. 30.3%), and to be longer in diestrous (43.8 vs. 30.7% in controls) (cf. Fail (1998), Table 10, p. 330).  

At necropsy, histopathological examinations revealed no treatment related effect on the non-reproductive tissues.

In the reproductive tissues a significant increase in relative right corpus and caput epididymis and right testis weight was noted,

and a decrease in relative seminal vesicle weight at the high dose level. The evaluation of sperm parameters (sperm concentration,

motility, morphology, spermatid head count) revealed no treatment-related changes. In females, the absolute and relative ovarian

weight was reduced (p < 0.05) at the mid- and high-dose level.

Applicant's summary and conclusion

Conclusions:
Formamide was a reproduction toxicant in female mice, as evidenced by reduced fertility, litter size, and number of viable pups. The reproduction NOAEL was 85-110 mg/kg bw/day in females. The NOAEL for generalized toxicity was 144 to 226 mg/kg bw/day for the male and female F0 generation.
Executive summary:

The reproduction toxicity of formamide was thoroughly investigated in a Reproductive Assessment by Continuous Breed (RACB) protocol which includes several tasks (continuous breed; crossover breed trial, 2 -generation study). The study was conducted according to EPA guidelines and under GLP conditions. Formamide was examined In a continuous breeding study in mice with the substance in drinking water at concentrations of 0, 100, 350, and 750 ppm reproductive toxicity was observed at 750 ppm in drinking water (144-226 mg/kg bw/day) in the parental and offspring generation, mainly decrease in fertility rate and reduction in live litter size. In a crossover experiment, this was shown to be mainly due to impairment of reproduction in females. At 750 ppm (approx. 210 mg/kg/day) a prolongation of the time to litter from 22 to 26 days was seen. The estrous cycle of F0 and F1 mice at 750 ppm was extended (F1: 6.5 vs. 4.8 days in controls), and the high-dose group tended to be in estrous for a shorter time than controls, and to be longer in diestrous. At necropsy, histopathological examinations revealed no treatment related effect on the non-reproductive tissues. In the reproductive tissues a significant increase in relative corpus and caput epididymis and testis weight was noted, and a decrease in relative seminal vesicle weight at the high dose level. The evaluation of sperm parameters (sperm concentration, motility, morphology, spermatid head count) revealed no treatment-related changes. The absolute and relative ovarian weight was reduced at the mid- and high-dose level.

The NOAEL for generalized toxicity was 144 to 226 mg/kg bw/day for the F0 generation. The NOAEL for reproductive toxicity was 152 - 220 mg/kg bw/day for males and 85 - 110 mg/kg bw/day for females of both generations (NTIS, 1992; Fail, 1998).

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