Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reproductive toxicity data are available for the registered substance. A reproductive/developmental screening study in rats with 2,2’-azobis(isobutyronitrile) was used as a read-across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances are similar in physicochemical properties, have common effects seen at acute exposures, and are similar in metabolic pathway.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Based on the results, the NOEL for reproductive and developmental toxicity was 50 mg/kg/day in males and 10 mg/kg in females and in pups.The abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. GLP guideline study with acceptable restrictions. Restrictions : no english data on study design, no details on functional observational battery tests.
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across: supporting information
Qualifier:
according to
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 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
Gross pathological findings:
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 50 mg/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 50 mg/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 10 mg/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 50 mg/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 2 mg/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, NOEL was considered to be 2 mg/kg/day for rats of 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:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
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.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
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

No reproductive toxicity data are available for the registered substance. A reproductive/developmental screening study in rats with 2,2’-azobis(isobutyronitrile) was used as a read-across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances are similar in physicochemical properties, have common effects seen at acute exposures, and are similar in metabolic pathway.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

An OECD 422 repeated dose, reproductive/developmental toxicity screening study with 2,2’-azobis(isobutyronitrile) was evaluated in male and female rats after gavage administration at doses of 0, 2, 10 and 50 mg/kg/day until day 43 for males and day 4 of post-partum for females. Reduced body weight gain and feed consumption was observed in females at ≥10 mg/kg. At 50 mg/kg, reduced weight gain and feed consumption were also observed during gestation, and one animal died 3 days after parturition. Increased liver and kidney weights were observed, and histopathological examinations suggested centrilobular hypertrophy of hepatocytes at ≥10 mg/kg.

No effects were observed on copulation index, fertility index, length of gestation, delivery index, parturition index, live pups delivered, overall delivery index, or sex ratio and body weight of pup on Day 0. Abnormal nursing behaviour was noted in 3 animals from the 50 mg/kg group.

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. This was attributed to maternal toxicity at the 50 mg/kg/day dose level. No offspring from the treated 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. The abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.


Short description of key information:
NOEL for reproduction/developmental toxicity is 50 mg/kg/day in males and 10 mg/kg/day in females (abnormal lactation behavior) and offspring (viability index and body weight decrease on day 4 of lactation). The abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.

Justification for selection of Effect on fertility via oral route:
NOAEL is based on OECD Guideline, GLP study with read-across chemical, 2,2'azobis(isobutyronitrile).

Effects on developmental toxicity

Description of key information

No developmental toxicity data are available for the registered substance. A reproductive/developmental screening and OECD 414 study in rats with 2,2’-azobis(isobutyronitrile) was used as a read-across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances are similar in physicochemical properties, have common effects seen at acute exposures, and are similar in metabolic pathway.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Administration of the read-across substance 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.

In an OECD 422 study conducted on the read-across substance the NOEL for reproduction/developmental toxicity is 50 mg/kg/day in males and 10 mg/kg/day in females (abnormal lactation behavior) and offspring (viability index and body weight decrease on day 4 of lactation). The abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. GLP guideline study with acceptable restrictions. Restrictions : no english data on study design, no details on functional observational battery tests.
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Not available in English language
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Not available in English language
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
Duration of test:
Day four of lactation
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Details on study design:
See below
Maternal examinations:
See chapter 7.8.1
Ovaries and uterine content:
See chapter 7.8.1
Fetal examinations:
See chapter 7.8.1
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Abnormal nursing behavior was noted in 3 animals from the 50 mg/kg group.
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: At a maternally toxic dose level

Details on embryotoxic / teratogenic effects:
The findings in offspring showed that ABMPN 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 ABMPN groups showed any morphological anomaly.
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

CLINICAL SIGNS AND MORTALITY

Males : Temporary salivation after each administration was observed in the 10 mg/kg or more groups.

Females : One female died on post-partum day 3 in the 50 mg/kg group. Abnormal nursing behavior was noted in 3 animals from the 50 mg/kg group.

BODY WEIGHT AND WEIGHT GAIN

Males : Suppression of body weight gain during the early administration period was noted in the 50 mg/kg group.

Females : Slight decreases in body weight gain and food consumption during the early administration period were observed in the 10 mg/kg or moregroups. In addition, body weight gain and food consumption were decreased during pregnancy in the 50 mg/kg group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

Males : Suppression of food consumption during the early administration period was noted in the 50 mg/kg group.

Females : No effects.

HAEMATOLOGY Males : Elevated platelet and white blood cell counts were apparent after the administration of the compound at the dose level of 50 mg/kg.

Females : No effects.

CLINICAL CHEMISTRY

Males : Increases in total protein and concentrations of albumin, total cholesterol, Ca and inorganic phosphorus and decreases in the A/G ratio an Cl concentration were apparent after the administration of the compound at the dose level of 50 mg/kg.

Females : No effects.

ORGAN WEIGHTS

Males : Increases in the kidney weights in all the treated groups and in the liver weights in the 10 mg/kg or more groups were observed at autopsy. Females : Liver and kidney weights showed a tendency for increase in the 50 mg/kg group (observed at autopsy on post-partum day 4).

GROSS PATHOLOGY No effects.

HISTOPATHOLOGY: NON-NEOPLASTIC

Males : Increased eosinophilic bodies and basophilic changes of the renal tubular epithelial cells in the kidneys of males treated with the compound atany dose levels tested were noted, as well as granular casts in the lower nephrons. Centrilobular hypertrophy of hepatocytes was also detected in the 10 mg/kg or more groups.

Females : Centrilobular hypertrophy of hepatocytes was observed in the 10 mg/kg or more groups.

No effects on the copulation index or fertility index at 50 mg/kg or lower doses. ABMPN 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 abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.

Conclusions:
NOEL for reproduction/developmental toxicity is 50 mg/kg/day in males and 10 mg/kg/day in females (abnormal lactation behavior) and offspring (viability index and body weight decrease on day 4 of lactation). The abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.
Executive summary:

No reproductive toxicity data are available for the registered substance. A reproductive/developmental screening study in rats with 2,2’-azobis(isobutyronitrile) was used as a read-across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances are similar in physicochemical properties, have common effects seen at acute exposures, and are similar in metabolic pathway.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

An OECD 422 repeated dose, reproductive/developmental toxicity screening study with 2,2’-azobis(isobutyronitrile) was evaluated in male and female rats after gavage administration at doses of 0, 2, 10 and 50 mg/kg/day until day 43 for males and day 4 of post-partum for females. Reduced body weight gain and feed consumption was observed in females at ≥10 mg/kg. At 50 mg/kg, reduced weight gain and feed consumption were also observed during gestation, and one animal died 3 days after parturition. Increased liver and kidney weights were observed, and histopathological examinations suggested centrilobular hypertrophy of hepatocytes at ≥10 mg/kg.

No effects were observed on copulation index, fertility index, length of gestation, delivery index, parturition index, live pups delivered, overall delivery index, or sex ratio and body weight of pup on Day 0. Abnormal nursing behaviour was noted in 3 animals from the 50 mg/kg group.

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. This was attributed to maternal toxicity at the 50 mg/kg/day dose level.No offspring from the treated 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.The abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
Qualifier:
according to
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 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
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
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).
Dose descriptor:
NOAEL
Effect level:
> 20 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effect
Abnormalities:
not specified
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
20 mg/kg bw/day
Species:
rat
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

No developmental toxicity data are available for the registered substance. A reproductive/developmental screening study in rats with 2,2’-azobis(isobutyronitrile) and OECD 414 study was used as a read-across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances are similar in physicochemical properties, have common effects seen at acute exposures, and are similar in metabolic pathway.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Administration of the read-across substance 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.

An OECD 422 repeated dose, reproductive/developmental toxicity screening study with 2,2’-azobis(isobutyronitrile) was evaluated in male and female rats after gavage administration at doses of 0, 2, 10 and 50 mg/kg/day until day 43 for males and day 4 of post-partum for females. Reduced body weight gain and feed consumption was observed in females at ≥10 mg/kg. At 50 mg/kg, reduced weight gain and feed consumption were also observed during gestation, and one animal died 3 days after parturition. Increased liver and kidney weights were observed, and histopathological examinations suggested centrilobular hypertrophy of hepatocytes at ≥10 mg/kg.

No effects were observed on copulation index, fertility index, length of gestation, delivery index, parturition index, live pups delivered, overall delivery index, or sex ratio and body weight of pup on Day 0. Abnormal nursing behaviour was noted in 3 animals from the 50 mg/kg group.

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. This was attributed to maternal toxicity at the 50 mg/kg/day dose level.No offspring from the treated 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.The abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.


Justification for selection of Effect on developmental toxicity: via oral route:
NOEL is based on OECD Guideline, GLP study with read-across chemical, 2,2'azobis(isobutyronitrile).

Justification for classification or non-classification

Based on the lack of reproductive effects in a rat OECD 422 guideline study and OECD 414 developmental study, the substance does not need to be classified for reproductive/developmental toxicity according the EU Directive 67/548/EEC.