Registration Dossier
Registration Dossier
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EC number: 201-132-3 | CAS number: 78-67-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data

Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Link to relevant study records
- 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
- 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
- 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
- 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.
Reference
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
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.
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 (%)
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
- 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
- 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.
Reference
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
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.
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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