2,2'-dimethyl-2,2'-azodipropiononitrile

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions. Restrictions : no english data on study design, no details on functional observational battery tests.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not available in English language.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

No english data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males were exposed for 42 days.
Females were exposed from 14 days prior to mating to day 3 of lactation.
Terminal killing at day 43 for males and on day 4 of lactation for females.

Frequency of treatment:

No English data

Details on study schedule:

No English data

Remarks:

Doses / Concentrations:
2 mg/kg bw (low dose)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
10 mg/kg bw (intermediate dose)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
50 mg/kg bw (high dose)
Basis:
actual ingested

No. of animals per sex per dose:

13

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week the first week ; weekly afterwards, including during the pregnancy of females. Additionally for females, days 0 & 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption determined in four day blocks during the first week and weekly afterwards.
- Compound intake calculated in mean diet as g food/rat/average over the period of calcul.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Sperm parameters (parental animals):

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

Litter observations:

STANDARDISATION OF LITTERS
no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live births, postnatal mortality (at day 4), body weight recorded at day 0 and day 4.


GROSS EXAMINATION OF DEAD PUPS:
yes, morphological (no more data - this information comes from the result in the publication indicating "no morphological abnormalities")

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals at day 43.
- Maternal animals: All surviving animals day 4 post partum.


GROSS NECROPSY
no data.


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination :
Heart, thymus, spleen
AND Liver, kidney, adrenal gland, testis, epididymis. These ones were also weighed.

Postmortem examinations (offspring):

SACRIFICE
- Supposed at day 4. Not accurately indicated.

GROSS NECROPSY
- Gross necropsy consisted of external examinations as supposed by the result in the publication indicating "no morphological abnormalities".

HISTOPATHOLOGY / ORGAN WEIGHTS
No

Statistics:

No data

Reproductive indices:

Copulation index, Fertility index, Gestation index, Duration of pregnancy.

Offspring viability indices:

Viability index

Clinical signs:

effects observed, treatment-related

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Males: Temporary salivation after each administration was observed in the animals exposed at 10 mg/kg and more.
Females: One female in the 50mg/kg group died on post-partum day 3.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: Suppression of body weight gain and food consumption during the early administration period were noted in the 50 mg/kg group.
Females: Slight decrease in body weight gain and food consumption during the early administration period were observed in the animals exposed at 10 mg/kg and more.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The compound showed no adverse effects on copulation and fertility, duration of pregnancy, gestation index and parturition at any of the dose levels tested. Three dams in the 50mg/kg group showed abnormal lactation.
(In the Japan HPV document, it was more accurately reported that 3 dams of 12 showed difficulty of nursling and two of them let all their offsprings die within the first 4 days after birth).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males: At autopsy, increases in the kidney weights in all of the compound-treated groups and in the liver weights in the animals exposed at 10mg/kg and more were observed.
Females: At autopsy on post-partum day 4, liver and kidney showed a tendency for increase after the administration of the compound at the dose level of 50mg/kg.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males: Increased eosinophilic bodies and basophilic changes of the renal tubular epithelial cells in the kidneys of all the animals exposed were noted, as well as granular casts in the lower nephrons. Centrilobular hypertrophy of hepatocytes was also detected in the groups of animals exposed at 10 mg/kg and more.
Females: Centrilobular hypertrophy of hepatocytes was observed in the groups of animals exposed at 10 mg/kg and more.

The NOEL for toxicity was suggested by the authors to be less than 2mg/kg/day in males and 2 mg/kg in females. In the Japan HPV document, as renal pathological changes were observed only in males, accumulation of alpha-2-macroglobulin was suspected as a cause of male specific renal toxicity. Therefore, based on pathological changes in liver of both sexes, NOAEL was considered to be 2 mg/kg/day for both sexes, based on liver toxicity.

For reproductive and developemental toxicity the NOEL was 50 mg/kg/day in males and 10 mg/kg in females

Dose descriptor:

NOEL

Effect level:

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

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Effect level:

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

Sex:

female

Basis for effect level:

other: General Toxicity body weight; food consumption ; histopathology (centrilobular hypertrophy of hepatocytes).

Dose descriptor:

NOEL

Effect level:

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

Sex:

male

Basis for effect level:

other: No effect on reproductive toxicity in male

Dose descriptor:

NOEL

Effect level:

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

Sex:

female

Basis for effect level:

other: At 50 mg/kg bw/d, abnormal lactation in 3 dams

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

The compound did not demonstrate any adverse effects on viability (statistically), sex ratio and body weight gain of pups.
However, viability of newborns at birth and body weight of nurslings on postnatal day 4 in the 50mg/kg bw group were lower than controls.

For reproductive and developemental toxicity the NOEL was 10 mg/kg in pups according to the authors.

In Japan HPV document, it was considered that the effects on pups were caused by maternal toxicity since a difficulty in nursling (lactation) was reported. Then the NOAEL for reproduction in offspring was considered to be 50 mg/kg.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

other: decreased viability index and body weight on day 4 at 50 mg/kg/day

Reproductive effects observed:

not specified

Summary of developement of pups

Dose (mg/kg)	0	2	10	50
Number of pregnant females	12	12	11	12
Number of pregnant females with pup alive	12	12	11	12
Gestation Index	100	100	100	100
Gestation length in days	22.3 +/-0.5 (12)	22.1 +/-0.3 (12)	22.2 +/-0.4 (11)	22.4 +/-0.5 (12)
Number of corpora lutea	18.8 +/-2.5 (12)	17.8 +/-1.5 (12)	18.1 +/-2.0 (11)	16.9 +/-2.0 (12)
Number of implantation sites	17.3 +/-25 (12)	16.8 +/-1.5 (12)	17.1 +/-2.1 (11)	15.6 +/-1.4 (12)
Implantation index	92.9 +/-10.6 (12)	95.0 +/-5.7 (12)	94.7 +/-7.7 (11)	92.8 +/-9.3 (12)
Day 0 of lactation
Number of pups born	15.4 +/-2.5 (12)	15.8 +/-1.9 (12)	15.5 +/- 1.8 (11)	14.8 +/-1.5 (12)
Delivery index	88.8 +/-6.2 (12)	94.0 +/-6.1 (12)	91.6 +/-10.3 (11)	94.6 +/-4.4 (12)
Number of pups alive	15.0 +/-2.5 (12)	15.8 +/-1.9 (12)	15.5 +/-1.8 (11)	14.8 +/-1.5 (12)
Birth index	86.6 +/- 8.3 (12)	93.5 +/- 5.8 (12)	91.6 +/-10.3 (11)	94.6 +/-4.4 (12)
Live birth index	97.4 +/-4.9 (12)	99.4 +/- 1.9 (12)	100.0 +/-0.0 (11)	100.0 +/- 0.0 (12)
Pup weight in grams
Male	6.2 +/-0.6 (12)	6.3 +/-0.5 (12)	6.3 +/-0.4 (11)	6.1 +/-0.2 (12)
Female	5.9 +/-0.5 (12)	6.0 +/-0.5 (12)	6.0 +/-0.3 (11)	5.9 +/-0.2 (12)
Sex ratio	50.5 +/-9.9 (12)	43.9 +/-12.8(12)	54.4 +/-10.9 (11)	53.6 +/-11.7 (12)
Day 4 of lactation
Number of pups alive	14.6 +/-2.3 (12)	15.6 +/-1.9 (12)	15.5 +/-1.8 (11)	11.6 +/-5.9 (12)
Viability index	97.5 +/-4.1 (12)	99.0 +/-2.4 (12)	100.0 +/-0.0 (11)	77.9 +/-38 (12)
Pup weight in grams
Male	9.8 +/-1.4 (12)	9.8 +/-1.1 (12)	9.8 +/-1.0 (11)	9.0 +/-1.2 (12)
Female	9.5 +/-1.2 (12)	9.5 +/-1.1 (12)	9.4 +/-1.0 (11)	8.5 +/-1.1 (12)

Gestation Index: Number of pregnant female with pups alive/Number of pregnant females

Implantation Index: Number of implantation sites/Number of corpora lutea *100 (%)

Delivery Index: Number of pups born/Number of implantation sites *100 (%)

Birth Index: Number of pups alive on day 0/ Number of implantation sites *100 (%)

Live Birth Index: Number of pups alive on day 0/ Number of pups born *100 (%)

Sex ratio: Number of male pups alive on day 0/ Number of pups alive on day 0 *100 (%)

Viability index: Number of pups alive on day 4/ Number of pups alive on day 0 *100 (%)

Conclusions:

In conclusion, the NOEL for reproductive and developemental toxicity was 50 mg/kg/day in males and 10 mg/kg in females and in pups.
According to CLP criteria, the test substance is not classified.

Executive summary:

The reproductive / developmental toxicity of 2,2'-Azobis(2-methylpropionitrile) was evaluated in male and female rats after oral administration (gavage) at doses of 0, 2, 10 and 50 mg/kg/day until day 43 for males and day 4 of post partum for females.

AZDN had no effects on the copulation index or fertility index at 50 mg/kg or lower doses. AZDN did not affect the length of the gestation period or delivery index in maternal animals, either. No abnormal parturition was observed. Abnormal nursing behaviour was noted in 3 animals from the 50 mg/kg group.

The findings in offspring showed no effects on the parturition index, live pup delivery index, overall delivery index, or the sex ratio and body weight on Day 0 at all dose-levels. In the 50 mg/kg group, the offspring viability index and body weight on Day 4 of lactation showed a tendency to decrease. No offspring from the AZDN groups showed any morphological anomaly.

Based on these results, the NOEL for reproductive and developmental toxicity was 50 mg/kg/day in males and 10 mg/kg in females and in pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The reproductive / developmental toxicity of 2,2'-Azobis(2-methylpropionitrile) was evaluated in male and female rats after oral administration (gavage) at doses of 0, 2, 10 and 50 mg/kg/day until day 43 for males and day 4 of post partum for females (Hatano, 1999).

In the females, AZDN inhibited weight gain and feed consumption at an early stage of treatment at 10 mg/kg or greater doses. 

At 50 mg/kg, it also inhibited weight gain and feed consumption during gestation. One animal died 3 days after parturition. Necropsy conducted 4 days after parturition revealed a tendency toward increases in weight of the liver and kidneys. Findings obtained in histopathological examinations suggested centrilobular hypertrophy of hepatocytes at 10 mg/kg or greater doses.

Findings revealed that AZDN had no effects on the copulation index or fertility index at 50 mg/kg or lower doses. AZDN did not affect the length of the gestation period or delivery index in maternal animals, either. No abnormal parturition was observed. Abnormal nursing behavior was noted in 3 animals from the 50 mg/kg group. 

The findings in offspring showed that AZDN had no effects on the parturition index, live pup delivery index, overall delivery index, or the sex ratio and body weight on Day 0. In the 50 mg/kg group, the offspring viability index and body weight on Day 4 of lactation showed a tendency to decrease. No offspring from the AZDN treated groups showed any morphological anomaly.

It was concluded that under the conditions of the present study, the NOEL for parental toxicity of AZDN is slightly lower than 2 mg/kg/day in males and 2 mg/kg/day in females and that the NOEL for reproduction/developmental toxicity is 50 mg/kg/day in males and 10 mg/kg/day in females and offspring.

Short description of key information:
The reproductive/developmental toxicity of AZDN was evaluated in male and female rats after oral administration (gavage) at doses of 2, 10 and
50 mg/kg/day for 2 weeks before mating and for 2 weeks after mating followed by additional 2 weeks after completion of mating (key study) for males and day 4 of post partum for females following the OECD Guideline 422.

Effects on developmental toxicity

Description of key information

There is a rat developmental study by oral route performed according to regulatory guideline OECD 414 (J. Rhodes, 2014).

The NOAEL for maternal toxicity in this study was set at 1 mg/kg/day based on decrease in maternal food consumption and body weight changes at 5 mg/kg/day. The NOAEL for fetal development was set at 20 mg/kg/day. No effects were seen on fetal development at any dose-level.

There is a second developmental toxicity study performed in a second species according to OECD 414 in rabbits. Dose-levels of 3, 9 and 24 mg/kg/day were selected in the study.

Severe maternal toxicity occured at 24 mg/kg/day . the No Observed Adverse Effect Levels (NOAELs) for maternal parameters and for embryodevelopment were considered to be 9 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 OCTOBER 2012 to 24 JANUARY 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to current test guidelines and GLP compliant

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Age at study initiation: 9-10 weeks old at time of mating
- Weight at study initiation: 200.1 to 271.8 g at time of mating
- Fasting period before study: No
- Housing: Animals housed singly in cages that conform with the 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989). Suitable wood bedding provided weekly to each cage. Wooden Aspen chew blocks and sizzle nest provided as environmental enrichment.
- Diet (e.g. ad libitum): SQC Rat and Mouse Breeder Diet No 3, Expanded (Special Diets Services Ltd, Witham, UK) ad libitum
- Water (e.g. ad libitum): Mains water ad libitum
- Acclimation period: No. Female animals delivered to the testing laboratory on Day 3 of gestation and examined on receipt. All animals found to be in good health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24C
- Humidity (%): 45 to 65%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: 19 October 2012 to 08 November 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared on three occasions. The test substance was formulated as a suspension in corn oil following dispensary SOPs and the formulation method 8266543_O_01D.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil selected due to stability of test substance in the vehicle, and use in previous study R-95-007.
- Concentration in vehicle: Not specified
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity previously assessed at 0.1 and 15 mg/mL in study 8266542.
CONCENTRATION VERIFICATION: Samples (three random aliquots from test substance formulations; two random aliquots from control formulations) prepared for use on the first and last day of dosing were taken for analysis of achieved concentration.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
Mating was performed overnight at the supplier's laboratory and confirmed by the presence of a vaginal plug in situ.

Duration of treatment / exposure:

The test and control substances were administered orally, by gavage, to mated female rats daily from Day 6 to Day 19 of gestation.

Frequency of treatment:

Daily

Duration of test:

The females were maintained to Day 20 of gestation when they were killed and their uterine contents examined.

No. of animals per sex per dose:

22 females per dose concentration

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose level of 20 mg/kg/day was selected as this was expected to elicit mild maternal toxicity and was based on the range-finding study 8266558, where a dose level of 25 mg/kg/day elicited maternal toxicity in the form of slight body weight loss and reduced food consumption. The intermediate dose level of 5 mg/kg/day was selected as this is the geometric mean of the high and low dose levels. The low dose level of 1 mg/kg/day was selected as this was expected to be a no observed effect level (NOEL).

- Rationale for animal assignment (if not random): On Day 3 of gestation the animals were assigned to treatment groups using a randomisation procedure based on body weight and day of mating.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Beginning and end of the working day for signs of ill health and overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination on the days of body weight recording.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each female was recorded on Days 3, 6, 7, 8, 9, 12, 15, 17, 19 and 20 of gestation.

FOOD CONSUMPTION):
Individual food consumption was recorded for Days 3 to 5, 6, 7, 8, 9 to 11, 12 to 14, 15 to 16 and 17 to 18 and 19 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: Ovaries and uteri

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
Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue. Dead foetuses were classified as those which appeared to have died shortly before necropsy.

Fetal examinations:

Live foetuses were killed by a subcutaneous injection of sodium pentobarbitone.
Individual foetal and placental weights were recorded and foetuses were examined externally and sexed. Approximately one half of the foetuses in each litter, selected by systematic sampling, were examined for visceral abnormalities by microdissection. They were then eviscerated and the carcasses processed to stain the ossified skeleton by the Alizarin technique and cartilage processed to stain using Alcian Blue. The skeletons were examined, preserved and stored in glycerol/propylene glycol.
The remaining foetuses were placed in Bouin's solution for at least two weeks to allow fixation and partial decalcification. At examination, the head was removed by a cut through the mouth, pharynx and back of the head and coronal sections of the head were examined. The remaining portion of the foetus was examined by dissection and was preserved, with the head sections, in 10% neutral buffered formalin and stored in plastic vials.
The dissection of the foetuses and the examination of the stained skeletons were performed using low power binocular magnification.
Foetal abnormalities were classified as malformations (rare and/or potentially lethal defects) and variations (commonly occurring non lethal abnormalities).

Statistics:

The control group (Group 1) was taken as the baseline group with which the treated groups (Groups 2, 3 and 4) were compared.
Statistical methods included one-way analysis of variance (ANOVA), Levene's test, Dunnett's test, Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test, the Wilcoxon rank sum test, analysis of covariance (ANCOVA), Cochran-Armitage test and Fisher's exact test.

Indices:

Not applicable

Historical control data:

Not applicable

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal receiving 1 mg/kg/day (number 23) died on Day 7 of gestation and one animal receiving 5 mg/kg/day (number 62) died on Day 20 of gestation. Findings for these animals were consistent with dosing accidents including thoracic cavity containing abnormal liquid contents.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight body weight loss was seen in animals receiving 5 and 20 mg/kg/day from Days 6 to 8 of gestation inclusive. Statistically significant reduced body weight gain from Days 7 to 8 of gestation at 5 and 20 mg/kg/day (79% and 173% less, or P<0.05 and P<0.001 respectively). Percentage body weight change (corrected) on Day 20 of gestation was lower than control at 5 and 20 mg/kg/day (12% and 29% lower respectively). These were considered to be effects of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In animals receiving 5 and 20 mg/kg/day there was a statistically significant treatment-related reduction in food intake from Days 6 to 8 of gestation inclusive (between 5% and 47% less, or P<0.05 for both), with recovery seen afterwards. This was an effect of treatment.

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:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There were 22, 22, 22 and 22 pregnancies leading to 22, 21, 21 and 22 litters in animals receiving 0, 1, 5 and 20 mg/kg/day respectively. The mean number of corpora lutea, the mean incidence of pre- and post-implantation loss and mean litter size were all unaffected by treatment.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Sex ratio, mean litter weight, mean placental weight and mean foetal weight all showed no effect of treatment.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Morbidity and mortality:
One animal receiving 1 mg/kg/day (number 23) died on Day 7 of gestation and one animal receiving 5 mg/kg/day (number 62) died on Day 20 of gestation. Findings for these animals were consistent with dosing accidents including thoracic cavity containing abnormal liquid contents.
There were no unscheduled treatment-related deaths.

Clinical signs:
There were no significant, treatment-related clinical signs recorded.

Post-dosing observations:
On the first dosing the post-dosing observation mouth rubbing was seen in two animals receiving 1 mg/kg/day and three animals receiving 5 mg/kg/day. During the rest of the dose period mouth rubbing was seen on most days in up to six animals receiving 20 mg/kg/day. This commonly recorded observation is considered to be a result of taste-aversion rather than systemic toxicity.

Body weight:
Slight body weight loss was seen in animals receiving 5 and 20 mg/kg/day from Days 6 to 8 of gestation inclusive. Statistically significant reduced body weight gain from Days 7 to 8 of gestation at 5 and 20 mg/kg/day (79% and 173% less, or P<0.05 and P<0.001 respectively). Percentage body weight change (corrected) on Day 20 of gestation was lower than control at 5 and 20 mg/kg/day (12% and 29% lower respectively). These were considered to be effects of treatment.

Food consumption:
In animals receiving 5 and 20 mg/kg/day there was a statistically significant treatment-related reduction in food intake from Days 6 to 8 of gestation inclusive (between 5% and 47% less, or P<0.05 for both), with recovery seen afterwards. This was an effect of treatment.

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations were noted in three foetuses from three litters in the control group (one external/visceral, two skeletal), four foetuses from three litters at 1 mg/kg/day (skeletal), five foetuses from two litters at 5 mg/kg/day (skeletal), and nine foetuses from eight litters at 20 mg/kg/day (three external/visceral, six skeletal). The incidence and intergroup distribution of these foetal malformations and variations do not indicate an adverse effect of treatment. Findings which on occasion achieved statistical significance generally fell within the background range (abnormal shaped head, thymus cervical remnant, metacarpal 5 unossified), or were commonly seen findings which did not show a clear treatment-related increase across the groups and/or were not considered to be biologically significant (kidney cavitation increased, ureter distended – fluid contents).

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Uterine/implantation data:
There were 22, 22, 22 and 22 pregnant females leading to 22, 21, 21 and 22 litters in animals receiving 0, 1, 5 and 20 mg/kg/day respectively. The mean number of corpora lutea, the mean incidence of pre- and post-implantation loss and mean litter size were all unaffected by treatment.

Foetal data:
Sex ratio, mean litter weight, mean placental weight and mean foetal weight all showed no effect of treatment.

Foetal defect data:
Malformations were noted in three foetuses from three litters in the control group (one external/visceral, two skeletal), four foetuses from three litters at 1 mg/kg/day (skeletal), five foetuses from two litters at 5 mg/kg/day (skeletal), and nine foetuses from eight litters at 20 mg/kg/day (three external/visceral, six skeletal). The incidence and intergroup distribution of these foetal malformations and variations do not indicate an adverse effect of treatment. Findings which on occasion achieved statistical significance generally fell within the background range (abnormal shaped head, thymus cervical remnant, metacarpal 5 unossified), or were commonly seen findings which did not show a clear treatment-related increase across the groups and/or were not considered to be biologically significant (kidney cavitation increased, ureter distended – fluid contents) (see attached tables).

Dose descriptor:

NOAEL

Effect level:

> 20 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no effect

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

not specified

Summary of Foetal Defects
Defect	Mean incidence – number of foetuses (mean percentage of foetuses)				Background Incidence minimum %, maximum %
	Group
	1	2	3	4
External/visceral findings
Malformations
Aortic arch, retro-oesophageal	-	-	-	1 (0.3)
Eye, anterior chamber, internal haemorrhage	-	-	-	1 (0.3)
Eyes, one absent	-	-	-	1 (0.5)
Eyes, severely reduced in size	1 (0.4)	-	-	1 (0.5)
Variations
Head, abnormal shape, domed	2 (0.9)	-	2 (0.7)	9 (3.4)^DR*	0.7, 4.4
Kidney, cavitation increased	6 (3.0)	5 (1.7)	4 (1.7)	18 (10.7)*	0.8, 4.9
Thymus, cervical remnant	1 (0.4)	6 (2.1)	7 (2.6)	11 (4.0)*	0.0, 11.2
Ureter, distended – fluid contents	10 (4.6)	6 (2.1)	8 (3.5)	18 (10.8)	1.9, 6.6
Skeletal findings
Malformations
Forelimbs, severely shortened and bent with bent scapulae	-	-	-	1 (0.9)
Ribs, ossification interrupted	-	1 (0.8)	-	-
Vertebral thoracic centrum, cleft cartilaginous centres	2 (1.3)	1 (0.7)	2 (1.4)	2 (1.6)
Vertebral cervical arches, additional cartilaginous ventral plate	-	1 (0.6)	-	-
Variations
Metacarpal 5 , unossified	26 (21.7)	14 (11.6)	38 (30.2)	38 (26.7)*	17.1, 45.2

* P<0.05

DR = significant dose response test

Conclusions:

In conclusion, administration of AZDN to pregnant rats from Days 6 to 19 of gestation at dose levels of 1 ,5 and 20 mg/kg/day resulted in maternal toxicity from 5 mg/kg/day upwards based on transient effects of reduced body weight gain and reduced mean food intake while no effects on foetal development were observed.
At 1 mg/kg/day, no adverse effects were observed in both parental and foetal development.
There was no evidence of embryotoxicity at any dose level tested.
The maternal no observed adverse-effect level (NOAEL) was 1 mg/kg/day based on a significant decrease in maternal body weight gain and food consumption.
The embryo-foetal NOAEL was 20 mg/kg/day.

Executive summary:

The objective of the study was to determine the effects of the test substance, AZDN(2,2’-dimethyl-2,2’azodipropionitrile), on the embryonic and foetal development of the rat.

The test item and control substance (corn oil) were administered orally, by oral gavage, to mated female rats daily from Day 6 to Day 19 of gestation, inclusive. The animals were dosed in ascending group order. The females were maintained to Day 20 of gestation when they were killed and their uterine contents examined.

Groups of rats of the Crl:CD(SD) strain were dosed as follows:

Group Number	Description	Dose level (mg/kg/day)	Number of animals in group
1	Control	0	22
2	Low	1	22
3	Intermediate	5	22
4	High	20	22

 

Two females one from each of the low and intermediate dose groups died on Days 7 and 20 of gestation respectively due to dosing accidents. Findings for these animals were consistent with dosing accidents including thoracic cavity containing abnormal liquid contents. There were no treatment-related deaths, clinical signs, or macroscopic necropsy findings.

The minor post-dosing observation mouth rubbing was seen in all treated groups with frequency of observation increasing with increasing dose.

Treatment-related reduced food intake (between 5% and 47% less, or P<0.05) for both 5 and 20 mg/kg/day and a consequent slight body weight loss (79% and 173% less, or P<0.05 and P<0.001 respectively) were seen from Days 6 to 8 of gestation inclusive.

Mean uterine/implantation and mean foetal data showed no adverse effect of treatment. There was no overall increase in the mean incidence or inter-group distribution of external, visceral or skeletal foetal variations or malformations.

In conclusion, administration of AZDN to pregnant rats from Days 6 to 19 of gestation at dose levels of 1 ,5 and 20 mg/kg/day resulted in maternal toxicity from 5 mg/kg/day upwards based on transient effects of reduced body weight gain and reduced mean food intake while no effects on foetal development were observed.

At 1 mg/kg/day, no adverse effects were observed in both parental and foetal development.

There was no evidence of embryotoxicity at any dose level tested.

The maternal no-observed-adverse-effect-level (NOAEL) was 1 mg/kg/day based on a significant decrease in maternal body weight gain and food consumption from 5 mg/kg/day upwards.

The embryo-foetal NOAEL was 20 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was performed according to regulatory guideline OECD 414 and is quoted Klimish 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Administration of AZDN to pregnant rats from Days 6 to 19 of gestation at dose levels of 1 ,5 and 20 mg/kg/day resulted in maternal toxicity from 5 mg/kg/day upwards based on transient effects of reduced body weight gain and reduced mean food intake while no effects on foetal development were observed.

At 1 mg/kg/day, no adverse effects were observed in both parental and foetal development.

There was no evidence of embryotoxicity at any dose level tested.

The maternal no-observed-adverse-effect-level (NOAEL) was 1 mg/kg/day.

The embryo-foetal NOAEL was 20 mg/kg/day.

Justification for classification or non-classification

According to the criteria laid down in Council Directive 67/548/EEC and EU CLP 1272/2008, the test item 2,2'-AZOBIS(2-methylpropionitrile) (ABMPN),is not categorised as toxic to reproduction.

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