C,C'-azodi(formamide)

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

C,C'-azodi(formamide)

EC Number:

204-650-8

EC Name:

C,C'-azodi(formamide)

Cas Number:

123-77-3

Molecular formula:

C2H4N4O2

IUPAC Name:

diazene-1,2-dicarboxamide

Details on test material:

- Name of the test material: Azodicarboxamide (hereafter referred to as ADC; CAS No. 123-77-3)
- Physical state: orange-yellow powder
- Molecular formula, C2H4N4O2
- Structural formula, H2NCON=NCONH2
- Molecular weight, 116.08
- Decomposition point, 195-202ºC
- Purity, 99.5 wt%
- Storage: at room temperature
- Stability: The analytical results showed that its purity remained unchanged, suggesting that it remained stable throughout the study period.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc., Tsukuba Rearing Center
- Age at study initiation: Males at 3 weeks, Females at 6 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: Each animal was individually housed in a metallic cage with a metallic mesh floor (220w x 270d x 190h mm) in an animal room (Room 15).
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Animals were given free access to pellet food (CE-2, Japan Clea Inc.)
- Water (e.g. ad libitum): Animals were given free access to drinking water (tap water supplied by Hatano Waterworks Department).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24+/-1ºC
- Humidity (%):50-65%
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Nakalai Tesque, Inc.; V8N6540 and V9F1299

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The stability of dosing suspensions was evaluated by comparing the absorption spectrum obtained at 300-600 nm to that obtained with ADC suspensions prepared at the same concentrations as dosing suspensions. The results obtained showed that 20 and 200 mg/mL dosing suspensions were stabile for at least 8 days in refrigerator when protected from light. It was therefore decided to prepare dosing suspensions at least once a week and to store them in refrigerator protected from light and consume them within 7 days after preparation. In addition, the homogeneity and content of ADC were evaluated once (homogeneity) or twice (content) during the study period by determining absorption at 424 nm. The results obtained indicated that thoroughly stirred dosing suspensions evenly contained the designated amount of ADC.

Daily treatment was conducted in the designated time zone (between 9:00 and 13:00). The dosing volume (5 mL/kg) for each animal was calculated based on body weight determined once a week in males. In females, the dosing volume was calculated based on body weight determined once a week before and during the mating period, on days 0, 7, 14 and 20 of gestation (day 0 of gestation = day on which vaginal plugs or sperms were detected) after copulation was confirmed, and on days 0, 4, 7 and 14 of lactation after parturition.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle, corn oil was added to ADC, weighed to attain each concentration, and the mixture was stirred to suspend.
- Concentration in vehicle: The ADC content was adjusted so that the dosing volume was 5 mL/kg of body weight regardless of the dose.
- Purity: 99.5 wt%

Details on mating procedure:

Animals were stratified based on body weight determined the day before starting administration (males) or the day before starting observation of the estrous cycle (females) and randomly assigned to groups of 25 males and 25 females.
- M/F ratio per cage: 1:1
- Length of cohabitation: 3 weeks
- Proof of pregnancy: vaginal plug referred to as day 0 of gestation
- Further matings after two unsuccessful attempts: not required
- After successful mating, females were separated from males and housed individually starting from the day copulation was confirmed (gestation day 0).

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

In males, 98 consecutive days until the day before necropsy: maximum of 3 week-mating period starting 10 weeks before starting mating.
In females, 2 weeks before starting mating, during the mating period (until copulation was confirmed), during the gestation period, and during a 20-day lactation period (day 0 of lactation=day of parturition).

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

The doses used in the present study were selected based on the results of an oral one-generation reproductive toxicity study of ADC in rats (preliminary dose-finding study; protocol No.: R-98-012).
Briefly, ADC, purchased from Tokyo Chemical Industry Co., Ltd. (lot No. GF01) and suspended in corn oil, was administered daily to groups of 5 males and 5 females of the same strain as that used in the present study at 0, 250, 500 or 1,000 mg/kg for 2 weeks before mating.
Females were necropsied on day 14 of gestation to determine the numbers of corpus lutea and implantation scars and the embryonic viability.
Males were necropsied the day after day 28 of treatment, and the testes and prostate ventral lobe were weighed.

No male or female showed any toxic signs or symptoms and their reproductive performance was not affected regardless of the dose.
The oral LD50 of ADC in rats was high (6400 mg/kg), indicating that ADC shows no marked toxic effects in rats.
Based on these findings, the highest dose was set at 1,000 mg/kg, followed by 300 mg/kg (intermediate dose) and 100 mg/kg (low dose) with a common ratio of about 3.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the treatment period, animals were observed before and after administration
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS:No data

BODY WEIGHT: Yes
- Time schedule for examinations: All males were weighed once a week on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 92 of treatment and the day of necropsy.
All females were weighed once a week until copulation was confirmed (days 1, 8, 15 and 22 of treatment). After copulation was confirmed, they were weighed on days 0, 7, 14 and 20 of gestation and on days 0, 4, 7, 14 and 21 of lactation following parturition. Body weight data obtained on day 22 of treatment were excluded from evaluation since they were obtained only from animals in which copulation had not been confirmed.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: In males, on days 2-3, 9-10, 16-17, 23-24, 30-31, 37-38, 44-45, 51-52, 58-59, 65-66, 71-72, 79-80 and 86-87 of treatment. However, data obtained on days 79-80 and days 86-87 were excluded from evaluation since animals were in the mating period and feeding condition differed from one animal to another. In females, on days 2-3 and 9-10 before mating, days 0-7, 7-14 and 14-20 of gestation after confirmation of copulation and days 0-4, 4-7, 7-14 and 14-21 of lactation after parturition.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

Oestrous cyclicity (parental animals):

Vaginal smear specimens were prepared and examined in all females from 2 weeks before starting treatment through the mating period until the day copulation was confirmed.
The estrous cycle was divided into three staged, estrus, early estrus and diestrus, based on the cellular composition of vaginal smears.
Females showing estrus only once during each of the two 2-week periods, before and after starting treatment, were regarded as monestrous.
In each of the other animals, the number of days between one estrus (the final day, if estrus was constant) and the next estrus was counted to calculate the mean number of days needed for the return of estrus.
The estrous cycle was classified into the “4-day cycle” if estrus returned in 4 days, the “5-day cycle” if estrus returned in 5 days, and the “4/5-day cycle” if both 4-day and 5-day intervals were found.
The estrous cycle was classified as irregular in all other cases.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.The size was not adjusted if it was less than 8.

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

GROSS EXAMINATION OF DEAD PUPS: Dead newborns were subjected to necropsy after determining the presence or absence of external abnormalities and fixed in 10% formalin solution for storage. The lungs were excised from newborns found dead on day 0 after birth and placed in saline. If the lungs floated in the saline, the newborn was considered as stillborn. Dead newborns with major injury or maceration were fixed for storage without any further processing.

Postmortem examinations (parental animals):

GROSS NECROPSY
- Gross necropsy: external and internal examinations including the cervical, thoracic, and abdominal viscera.
In dead males, major thoracic and abdominal organs were macroscopically examined to determine the presence or absence of abnormalities, and major thoracic and abdominal organs were excised including the pituitary glands, thyroids, testes, epididymides, coagulating glands, seminal vesicle, and prostate.

In females which died or were sacrificed in extremis, major thoracic and abdominal organs were macroscopically examined to determine the presence or absence of abnormalities, and major thoracic and abdominal organs including the pituitary glands, thyroids, ovaries, uterus, cervix and vagina were excised.

HISTOPATHOLOGY / ORGAN WEIGHTS
MALES: The excised testes and epididymides were fixed in Bouin's solution (0.1M phosphate-buffered 10% formalin solution for long-term storage). All other excised organs and tissues were fixed in 0.1M phosphate-buffered 10% formalin solution for storage.
Organs and tissues with abnormalities, as well as the pituitary glands, thyroids, testes, epididymides, coagulating glands, seminal vesicle and prostate ventral lobe, were processed according to the conventional method to prepare paraffin sections, which were then stained with hematoxylin-eosin to prepare specimens for histopathological examinations.
These sections were prepared and examined for all animals.

All animals which serviced until day 98 of treatment were exsanguinated to death the following day under anesthesia with sodium pentobarbital and subjected to necropsy (routine anatomy). During necropsy, major thoracic and abdominal organs were macroscopically examined to determine the presence or absence of abnormalities. Tissues with abnormalities were fixed in 0.1M phosphate-buffered 10% formalin solution for storage. The pituitary glands, thyroids, testes, epididymides, coagulating glands, seminal vesicle and prostate, as well as the kidneys, stomach, spleen, thymus and adrenals, which showed abnormalities in females sacrificed in extremis before males were necropsied, were macroscopically examined to determine the presence or absence of abnormalities. The testes and epididymides were fixed in Bouin’s solution (0.1M phosphate-buffered 10% formalin solution for long-term storage). All other organs and tissues were fixed in 0.1M phosphate-buffered 10% formalin solution for storage. Tissues with abnormalities were processed to prepare paraffin sections according to the conventional method and stained with hematoxylin-eosin to prepare specimens for histopathological examinations. Specimens for histopathological examinations were prepared for the pituitary glands, thyroids, testes, epididymides, seminal vesicle, prostate ventral lobe and coagulating glands from all animals. All males from the control and high-dose groups, as well as males whose mates copulated but did not become pregnant in the mid and low-dose groups, were examined. Specimens for histopathological examinations were also prepared for the kidneys, which showed enlargement in necropsy in the high-dose group, in all males from the control and high-dose groups.

FEMALES: Excised ovaries were fixed in Bouin's solution (0.1M phosphate-buffered 10% formalin solution for long-term storage), while the uterus was fixed in 0.1M phosphate-buffered 10% formalin solution together with other tissues after counting the number of implantation scars. Organs and tissues with abnormalities, as well as the pituitary glands, thyroids, ovaries, uterus, cervix and vagina, were processed according to the conventional method to prepare paraffin sections and stained with hematoxylin-eosin to prepare specimens for histopathological examinations of all females.

Surviving animals were exsanguinated to death under anesthesia with an overdose of sodium pentobarbital and necropsied, those which showed parturition on day 21 of lactation and those which copulated but did not show parturition on presumed day 25 of gestation. Major thoracic and abdominal organs were macroscopically examined to determine the presence of absence of abnormalities. Tissues with abnormalities were fixed in 10% phosphate-buffered formalin solution for storage. The uterus was excised and fixed in 0.1M phosphate-buffered 10% formalin solution for storage after macroscopically counting the number of implantation scars. In addition to these organs, the pituitary glands, thyroids, ovaries, uterus, cervix and vagina, as well as the kidneys, heart, stomach, spleen, thymus and adrenals, which showed abnormalities during necropsy of dead animals and those sacrificed in extremis, were excised and macroscopically examined to determine the presence or absence of abnormalities. Ovaries were fixed in Bouin's solution (0.1M phosphate-buffered 10% formalin solution for long-term storage), while other organs and tissues were fixed in 0.1M phosphate-buffered 10% formalin solution for storage. Tissues with abnormalities were processed to prepare paraffin sections according to the conventional method and stained with hematoxylin-eosin to prepare specimens for histopathological examinations. The pituitary glands, thyroids, ovaries, uterus, cervix and vagina from all females in the control group and the high dose group, as well as females found to be infertile in other groups, were also processed to prepare specimens for histopathological examinations. Specimens for histopathological examinations were also prepared for the kidneys from all females because abnormalities were observed during necropsy in animals sacrificed in extremis in the mid-dose group.

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All live pups were sacrificed by ether inhalation and necropsied on day 21 after birth. Organs with abnormalities were fixed in 0.1M phosphate-buffered 10% formalin solution for storage.

Statistics:

The incidence of estrous cycles by type, copulation index, fertility index and incidence of morphological abnormalities in newborns were analyzed by Fischer’s exact test.
Findings of histopathological examinations were compared between the control group and each of the ADC groups: graded data by Mann-Whitney’s U-test and sums of positive grades by Fisher’s one-sided exact test.
All other data were first analyzed by Bartlett’s method with respect to the uniformity of variance in each group using data obtained from each animal and means determined for each litter as one sample.
If the variance was uniform, one-way analysis of variance was conducted.
If significant inter-group differences were found, multiple comparison was conducted by Dunnett’s method.
Kruskal-Wallis’ rank test was conducted if the variance was 0 in any group or if the variance was not uniform.
If significant inter-group differences were found, multiple comparison was made by the Dunnett-type test.

Reproductive indices:

Fertility index
Number of pairing days until copulation
Number of vaginal estruses during the mating period

Offspring viability indices:

Delivery index [(number of newborns/number of implantations) x 100],
Birth index [(number of live newborns/number of implantations) x 100]
Live birth index [(number of live newborns/number of newborns) x 100].
Sex ratio on day 0 after birth [(number of male live newborns/number of live newborns) x 100]
Viability index on day 4 after birth [(number of live pups on day 4 before adjustment of the litter size/number of live newborns on day 0 after birth) x 100]
Sex ratio on day 4 after birth [(number of male live pups on day 4 before adjustment of the litter size/number of live pups on day 4 before adjustment of the litter size) x 100]
Weaning index [(number of live pups on day 21 of lactation/number of live pups on day 4 after adjustment of the litter size) x 100]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For details, see attached information below.

Mortality:

mortality observed, treatment-related

Description (incidence):

The following animals died: one from the control group (death on day 47 of treatment) and 5 from the 1,000 mg/kg group (on days 6, 12, 12, 41 and 69). None of these animals showed any abnormality until death except one from the 1,000 mg/kg group which showed crust formation. These deaths were thought to be attributable to errors in administration procedures because necropsy revealed the findings described below. Findings which were considered to be unrelated to errors in administration procedures were included in evaluation.

Animal No. MX1006: esophageal perforation and retention of oily pleural effusion
Animal No. MX04001: yellow soilage of perioral fur, test material-like yellow area mainly on the right lung lobes and yellow pigmentation on the tracheal lumen
Animal No. MX04003: esophageal perforation and retention of test material-like yellow, semi-transparent effusion in the thoracic cavity
Animal No. MX04004: hemorrhage-like dark red area in the esophagus around the hilar region and retention of a large volume of plural effusion with scattered yellow masses in the thoracic cavity
Animal No. MX04007: retention of a large volume of oily plural effusion and adhesion of yellow substances to the anterior diaphragm
Animal No. MX04023: dark red material in the esophagus and test material-like yellow material in the posterior lobe of the right lung

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: Body weight gain between days 92 and 99 was significantly greater (p<0.01) in the 300 mg/kg group than in the control group. However, no other significant differences were observed in body weight or body weight gain between the control group and each of the ADC groups.
Females: There were no significant differences in body weight or body weight gain between the control group and each of the ADC groups during the pre-mating, gestation or lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences between the control group and each of the ADC groups for males and females.

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Findings in males which died during the dosing period: No abnormalities were found in one animal each in the control and 1,000 mg/kg groups (MX01006 and MX04023). Of the other four males which died in the 1,000 mg/kg group, one (MX04001) showed atrophy of the seminiferous tubules associated with diffuse hyperplasia of Leydig cells in one testis. This animal also showed spermatic granuloma and multinucleated giant cells in the seminiferous tubules. The lumen of the epididymis on the affected side contained almost no sperm and was filled with cell debris. The ventral lobe of the prostate of this animal showed lymphocyte infiltration in the interstitium and neutrophil infiltration in the epithelium and lumen. Reproductive organs of the other three males which died before sexual maturation at an early stage of treatment (MX04003, MX04004 and MX04007) showed findings consistent with the age (5-6 weeks after birth). No abnormality was found. These animals were excluded from the histopathological evaluation of reproductive organs.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

In the observation of the estrous cycle during the 2-week pre-treatment period, one animal each in all groups, including the control group, showed monoestrus. After starting treatment, however, these animals showed normal estrous cycle. Before starting treatment, animals in the 100 mg/kg group and the 1,000 mg/kg group showed 5-day cycles, which were not seen in the control group. In the 1,000 mg/kg group, the mean number of days needed for return of estrus was slightly extended compared to the control group.
After starting treatment, the percentage of animals which showed changes in the estrous cycle showed no significant differences between the control group and any of the ADC groups. The mean number of days needed for return of estrus showed no significant changes in any ADC group, but significant differences (p<0.05) were noted between the 1000 mg/kg group and the control group.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

All animals copulated, but fertility was not confirmed due to the failure of females to become pregnant in the following animals: one male (MX01019) and one female (FB01019) in the control group, two males (MX02010 and MX02021) and two females (FB02010 and FB02021) in the 100 mg/kg group, three males (MX03003, MX03006 and MX03010) and three females (FB03003, FB03006 and FB03010) in the 300 mg/kg group, and one male (MX04009) and three females (FB04003, FB04004 and FB0009) in the 1000 mg/kg group. However, the fertility index, the number of pairing days until copulation, and the number of vaginal estruses during the mating period were comparable between the control group and each of the ADC groups.

Details on results (P0)

Details are included in the attached document below.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects observed up to and including highest dose level tested (1000 mg/kg bw/day)

2

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Treatment with 1000 mg/kg to females caused death during the lactation period due to pelvic nephritis-like lesions.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the control group, all newborns showed decreases in temperature of the body surface on day 0 after birth, and all pups from one litter (dam No.: FB01017) which did not show any milk spot died on day 1 after birth. Pups from the ADC groups showed abnormalities due to poor lactation activities associated with worsening of general signs and symptoms in F0 females. In the 100 mg/kg group, all pups from one litter (dam No.: FB02005) showed no milk spot on day 1 after birth and died following decreases in temperature of the body surface the following day. Pups from another litter (dam No.: FB02019) showed no milk in the stomach on day 3 after birth. Temperature of the body surface decreased and milk spot disappeared the following day. All these pups showed emaciation thereafter and died by day 7 after birth. In the 300 mg/kg group, one litter (dam No.: FB03004) showed no milk spot on day 1 after birth. All pups from this litter died the following day following decreases in temperature of the body surface. In the 1,000 mg/kg group, pups from two litters (dam Nos.: FB04014 and FB04001) showed emaciation when the F0 females died on days 3 and 14 after birth. The former litter showed no milk spot. Another litter from the 1,000 mg/kg group (dam No.: FB04013) showed no milk spot on day 4 after birth. Milk spots became unclearly visible the following day and reappeared one day later. However, pups showed emaciation on day 12 after birth, followed by decreases in motor activity on day 14 after birth, and all died the following day. In addition, external malformations of the tail were suspected in some pups from two litters (dam Nos.: FB02002 and FB02025) in the 100 mg/kg group and one litter (dam No.: FB03009) from the 300 mg/kg group. However, this disappeared in the 300 mg/kg group as pups grew up. Other pups showed no abnormality.

Mortality / viability:

no mortality observed

Description (incidence and severity):

All of the four dead newborns found on day 0 after birth in the control group were stillbirths, while in the 100, 300, and 1,000 mg/kg groups, 12/17, 13/15, and 3/6 newborns found dead were judged to be stillbirths, respectively. The incidence of stillbirths was not significantly different between the control group and each of the ADC groups.
As mentioned above, all pups from some litters died in all groups including the control group. However, the number of live newborns, delivery index, number of live pups on days 0, 4 and 21 after birth, birth index, live birth index, viability of pups on day 4 after birth showed no significant differences between the control group and each of the ADC groups. The weaning index showed significant decreases only in the 100 mg/kg group (p<0.05).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight of male and female pups showed no significant differences between the control group and each of the ADC groups at any time point.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy of dead pups, excess pups culled in the adjustment of the litter size, and live pups sacrificed on day 21 after birth showed various morphological changes including variations and malformations in pups from one to 4 litters per group in all groups including the control group. External malformations observed included a kinked tail in one pup (excess pup on day 4 after birth) from one litter in the control group (dam No.: FB01018). In the 100 mg/kg group, one (dead) pup from one litter (dam No.: FB02002) showed short trunk, club foot and brachyury. Excess pups from the same litter culled on day 4 after birth showed kinked tail (one pup), rudimentary tail (one pup) and brachyury/kinked tail (two pups). One pup from this litter sacrificed on day 21 after birth showed brachyury/kinked tail. One excess pup from another litter (dam No.: FB02025) culled on day 4 after birth showed kinked tail. No external malformation was observed in the 300 mg/kg group. In the 1,000 mg/kg group, excess pups from one litter (dam No.: FB04002) culled on day 4 after birth showed anal atresia/anury (one pup) and kinked tail (another pup). Two excess pups from another litter (dam No.: FB04019) culled on day 4 after birth showed kinked tail. Visceral malformations observed in the 100 mg/kg group included a horseshoe kidney and bifid apex observed in the dead pup from dam No. FB02002 which showed external malformations such as short trunk, as well as hydronephrosis observed in one pup from another litter (dam No.: FB02008) sacrificed on day 21 after birth. In addition, dilatation of the renal pelvis, a visceral variation, was observed in all groups including the control group. The total number of pups with morphological changes was counted, and the percentage of pups with these changes was compared between the control group and each of the ADC groups. The percentage of pups with external changes increased significantly in the 100 mg/kg group compared to the control group (p<0.05), but no significant inter-group differences were noted with respect to the percentage of pups with visceral changes.

Morphological examinations of live pups showed one pup with external malformations (short trunk, rudimentary tail, anal atresia and club foot) from one litter (dam No.: FB02002) in the 100 mg/kg group. This pup showed no milk spot. Six other pups from this litter had short tails. One pup from another litter (dam No.: FB02004) showed a bite in the rostral area. In the 300 mg/kg group, one pup from one litter (dam No.: FB03013) showed cyanosis. In the 1,000 mg/kg group, one pup from one litter (dam No.: FB04002) showed external malformations (anury and anal atresia). One pup from another litter (dam No.: FB04013) showed a loss of the tail tip due to trauma. One litter from the control group (dam No.: FB01017) showed no abnormality, but all pups from this litter showed decreases in temperature of the body surface without milk spot.

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

The sex ratio was comparable between the control group and each of the ADC groups at all time points.

Details on results (F1)

VIABILITY (OFFSPRING)
All of the four dead newborns found on day 0 after birth in the control group were stillbirths, while in the 100, 300, and 1,000 mg/kg groups, 12/17, 13/15, and 3/6 newborns found dead were judged to be stillbirths, respectively. The incidence of stillbirths was not significantly different between the control group and each of the ADC groups.
As mentioned above, all pups from some litters died in all groups including the control group. However, the number of live newborns, delivery index, number of live pups on days 0, 4 and 21 after birth, birth index, live birth index, viability of pups on day 4 after birth showed no significant differences between the control group and each of the ADC groups. The weaning index showed significant decreases only in the 100 mg/kg group (p<0.05). The sex ratio was comparable between the control group and each of the ADC groups at all time points.

CLINICAL SIGNS (OFFSPRING)
In the control group, all newborns showed decreases in temperature of the body surface on day 0 after birth, and all pups from one litter (dam No.: FB01017) which did not show any milk spot died on day 1 after birth. Pups from the ADC groups showed abnormalities due to poor lactation activities associated with worsening of general signs and symptoms in F0 females. In the 100 mg/kg group, all pups from one litter (dam No.: FB02005) showed no milk spot on day 1 after birth and died following decreases in temperature of the body surface the following day. Pups from another litter (dam No.: FB02019) showed no milk in the stomach on day 3 after birth. Temperature of the body surface decreased and milk spot disappeared the following day. All these pups showed emaciation thereafter and died by day 7 after birth. In the 300 mg/kg group, one litter (dam No.: FB03004) showed no milk spot on day 1 after birth. All pups from this litter died the following day following decreases in temperature of the body surface. In the 1,000 mg/kg group, pups from two litters (dam Nos.: FB04014 and FB04001) showed emaciation when the F0 females died on days 3 and 14 after birth. The former litter showed no milk spot. Another litter from the 1,000 mg/kg group (dam No.: FB04013) showed no milk spot on day 4 after birth. Milk spots became unclearly visible the following day and reappeared one day later. However, pups showed emaciation on day 12 after birth, followed by decreases in motor activity on day 14 after birth, and all died the following day. In addition, external malformations of the tail were suspected in some pups from two litters (dam Nos.: FB02002 and FB02025) in the 100 mg/kg group and one litter (dam No.: FB03009) from the 300 mg/kg group. However, malformations disappeared in the 300 mg/kg group as pups grew up. Other pups showed no abnormality.

BODY WEIGHT (OFFSPRING): Body weight of male and female pups showed no significant differences between the control group and each of the ADC groups at any time point.

GROSS PATHOLOGY (OFFSPRING)
Necropsy of dead pups, excess pups culled in the adjustment of the litter size, and live pups sacrificed on day 21 after birth showed various morphological changes including variations and malformations in pups from one to 4 litters per group in all groups including the control group. External malformations observed included a kinked tail in one pup (excess pup on day 4 after birth) from one litter in the control group (dam No.: FB01018). In the 100 mg/kg group, one (dead) pup from one litter (dam No.: FB02002) showed short trunk, club foot and brachyury. Excess pups from the same litter culled on day 4 after birth showed kinked tail (one pup), rudimentary tail (one pup) and brachyury/kinked tail (two pups). One pup from this litter sacrificed on day 21 after birth showed brachyury/kinked tail. One excess pup from another litter (dam No.: FB02025) culled on day 4 after birth showed kinked tail. No external malformation was observed in the 300 mg/kg group. In the 1,000 mg/kg group, excess pups from one litter (dam No.: FB04002) culled on day 4 after birth showed anal atresia/anury (one pup) and kinked tail (another pup). Two excess pups from another litter (dam No.: FB04019) culled on day 4 after birth showed kinked tail. Visceral malformations observed in the 100 mg/kg group included a horseshoe kidney and bifid apex observed in the dead pup from dam No. FB02002 which showed external malformations such as short trunk, as well as hydronephrosis observed in one pup from another litter (dam No.: FB02008) sacrificed on day 21 after birth. In addition, dilatation of the renal pelvis, a visceral variation, was observed in all groups including the control group. The total number of pups with morphological changes was counted, and the percentage of pups with these changes was compared between the control group and each of the ADC groups. The percentage of pups with external changes increased significantly in the 100 mg/kg group compared to the control group (p<0.05), but no significant inter-group differences were noted with respect to the percentage of pups with visceral changes.

OTHER FINDINGS (OFFSPRING)
Morphological examinations of live pups showed one pup with external malformations (short trunk, rudimentary tail, anal atresia and club foot) from one litter (dam No.: FB02002) in the 100 mg/kg group. This pup showed no milk spot. Six other pups from this litter had short tails. One pup from another litter (dam No.: FB02004) showed a bite in the rostral area. In the 300 mg/kg group, one pup from one litter (dam No.: FB03013) showed cyanosis. In the 1,000 mg/kg group, one pup from one litter (dam No.: FB04002) showed external malformations (anury and anal atresia). One pup from another litter (dam No.: FB04013) showed a loss of the tail tip due to trauma. One litter from the control group (dam No.: FB01017) showed no abnormality, but all pups from this litter showed decreases in temperature of the body surface without milk spot.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed on F1 up to and including highest dose level tested (1000 mg/kg bw/day)

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the present conditions, NOELs for reproductive toxicity of 1,1'azobisformamide are considered to be 1000 mg/kg/day for males and females, and those for toxicity other than reproduction are considered to be 1000 mg/kg/day for males and 300 mg/kg/day for females.

Executive summary:

One-generation reproduction toxicity was investigated for 1,1 '-azobisformamide by daily oral administration of 0, 100, 300 and 1000 mg/kg to 5 week-old male and to 10 week-old female Sprague-Dawley strain (Crj:CD) rats (25 animals each sex/group). The test substance was given to the males for 10 weeks before mating and thereafter in total for 14 weeks, and to the females for 2 weeks before mating, and throughout the mating, gestation and lactation periods, thus in total 8 weeks.

The treatment did not affect their reproductive ability, including the estrous cycle, mating, parturition of the animals and viability, growth and morphology of offspring. While no adverse effects of 1,1 '-azobisformamide were found in males, treatment with 1000 mg/kg to females caused death during the lactation period due to pelvic pyelonephritic kidney lesions.

Under the present conditions, NOELs for reproductive toxicity of 1,1'azobisformamide are considered to be 1000 mg/kg/day for males and females, and those for toxicity other than reproduction are considered to be 1000 mg/kg/day for males and 300 mg/kg/day for females.