2,2'-azobis[2-methylbutyronitrile]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A reproductive/developmental screening study in rats did not indicate a specific concern for reproduction toxicity.

The overall NOAEL for parental toxicity was 15 mg/kg/day for males and 50 mg/kg/day for females based on adverse microscopic findings in the kidneys of a single male at 50 mg, and in a single male and female at 150 mg/kg, as well as lower BW gain and increased liver weight in both males and females at this level. The reproduction NOAEL was considered 50 mg/kg bw, based on 3 females at 150 mg/kg displaying irregular or acyclic estrous cycle (although all three became normally pregnant).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 Apr 2020 to 19 Nov 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

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)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) rats

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent
species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and
reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: At initiation of dosing, males were 10-11 weeks old and weighed between 261 and 312 g and females were 13-14 weeks old and weighed between 185 and 247 g.
- Housing:
On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
In order to avoid hypothermia of pups, pups were not left without their mother or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment, bedding material, food and water.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The animals were allowed to acclimate to the Test Facility accommodation for 7 days prior to the start of the pretest period (females) or 7 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY:
Food: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
Based on this analysis there were no known contaminants in the feed that would interfere with the objectives of the study.
Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
Based on this analysis there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
The actual daily mean temperature during the study period was 20 to 21°C with an actual daily mean relative humidity of 48 to 80%. The relative humidity values that were outside the targeted range occurred on 30 days and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

IN-LIFE DATES: Start dosing 24 Jun 2020; Completion of In-life: 28 Aug 2020 (Last date of necropsy)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw data of these trials will be retained by the Test Facility.
- Concentration in vehicle: Corn oil
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group. Sibling mating was avoided by mating males and females with a different age range (also see Section 4.7.1). Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 13 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was based on the analytical method validated for the test item in corn oil: An ultra performance liquid chromatographic method with spectrophotometric detection (UPLC-UV)
Accuracy: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
No test item was detected in the Group 1 formulation.
Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Dose formulation samples were collected for analysis: Week 1 (24 Jun 2020), all groups; Homogeneity: Groups 2 and 4 (The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.)

Duration of treatment / exposure:

minimum of 28 days.
Males were treated for 28 days, up to and including the day before scheduled necropsy. (including 14 days prior to mating and during the mating period.)
Females that delivered were treated for 50-57 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 42-54 days.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

Daily, 7 days a week

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of 2,2’-Azodi(2-methylbutyronitrile) in rats (Test Facility Reference No. 20237944), and in an attempt to produce graded responses to the test.
- Rationale for animal assignment (if not random): Random: First 5 animals in group; Females only with live pups
Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified.
- Fasting period before blood sampling for clinical biochemistry:
F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- Dose range finding studies: 10-day Dose Range Finder: 3 females per subsequent dose group. Two dose groups with staggered start: 150 mg/kg bw respectively 300 mg/kg (dosing in corn oil, 5 mL/kg bw). The dose levels were selected based on the results of an acute oral toxicity study in rats.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals will be observed for general health/mortality and moribundity at least twice daily throughout the study.
Clinical Observations – F0: at least once daily, up to the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
Arena Observations – F0: Once before the first administration of the test item and at weekly intervals during the treatment period.

BODY WEIGHT: Yes
Males and females will be weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated females will be weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
Weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females will be measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION:
Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

OTHER:
HAEMATOLOGY & CLINICAL CHEMISTRY & HORMONES: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes

Oestrous cyclicity (parental animals):

Daily vaginal lavage were performed beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups,

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

On PND 4, the surplus pups were euthanized by decapitation. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Following completion of the mating period (a minimum of 28 days of administration). Order of necropsy: Group 1, 4, 2 and 3.
- Maternal animals:
Females which delivered: PND 14-16. Order of necropsy: Group 1, 4, 2 and 3, if possible.
Females which failed to deliver: With evidence of mating: Post-coitum Day 27 (No. 43). Without evidence of mating: 27 days after the last day of the mating period (Nos. 50 and 61).

All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition. This was performed for female No. 43 (not pregnant) and Nos. 50 and 61 (not mated).

HISTOPATHOLOGY / ORGAN WEIGHTS
Yes - See same study reported under repeated dose toxicity.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
Pups found dead during the weekend were fixed in labelled containers containing 70% ethanol as they were not necropsied on that day.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

On PND 4, the surplus pups were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

In addition, blood was collected from two pups per litter (see also section 4.11.1), and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

Statistics:

Statistics for data collected in ToxData (For data collected in Provantis see 'other information')

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with 4 groups (the designated control group and 3 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (4 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-Parametric
Datasets with 4 groups were compared using a Steel-test (many-to-one rank test).
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Mating index (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Offspring viability indices:

Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was noted on multiple days during the treatment period in individual animals of the 15, 50 and 150 mg/kg/day dose groups (both males and females). Hunched posture was noted incidentally on the second day of dosing in females of the 50 and 150 mg/kg/day dose groups and in an individual female of the 15 mg/kg/day dose group during week 6 of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first week of treatment with the test item, body weight gains were decreased in all dosed groups. After that BW-gains were similar for all groups.
This resulted to a statistical significant decrease at 150 mg group for the males only (-6.6% lower BW than controls), whereas in the females all three dose groups showed about 8.5% lower BW compared to control at end of gestation and day 13 of lactation (end of study), with statistical significance for low and high dose groups (15 mg and 150 mg). (See attached graphs)
The effect seems not related to toxicity, but to lower food consumption in all dose groups during the first week only.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (absolute and relative to body weight) was reduced during the first week of treatment in all treated male and female groups, with recovery to control levels during the second and third week of treatment.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Dose-dependent lower total T4 values were noted in F0-males treated at 15, 50 and 150
mg/kg/day (0.81, 0.71 and 0.54x of control, respectively) and in F0-females treated at 50 and
150 mg/kg/day (0.79x and 0.64x of control, respectively).
Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment.
(See attached graphs) Overall the T4 change may be secondary to the increased T4 turnover resulting from metabolic enzyme induction in the liver (dose dependent higher liver weights in males and females and hepatocellular hypertrophy, present in males at 150 mg/kg/day).

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The liver of all males at 150 mg/kg showed minimal hepatocellular hypertrophy.

Kidneys: An increased incidence and severity of hyaline droplet accumulation (up to moderate) was noted in all 15, 50 and 150 mg/kg/day males. This was considered alpha 2u-globulin related nephropathy, a male rat specific finding, not seen in female and was therefore considered not adverse.
In a single male at 50 and a single male at 150 mg/kg/day, alpha 2u-globulin
nephropathy was observed (i.e. the combination of hyaline droplet accumulation (moderate), tubular basophilia (slight) in similar areas and the presence of granular casts (slight; representing intratubular degenerated cells)). Based on the degenerative nature of the granular casts, the combination of findings at 50 and 150 mg/kg/day in these two males was considered adverse within the context of this study.
In a single female treated at 150 mg/kg/day, a higher severity of tubular basophilia (slight) with adjacent tubules with degeneration/necrosis (minimal) was recorded.

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

During the pre-test period, all females had regular cycles of 4 days.


During the pre-mating period, i.e. after start treatment with the test item, an irregular cycle (out of two) was noted for Female No. 71 at 150 mg/kg/day (with normal litter). Additionally, Female Nos. 75 and 77 at 150 mg/kg/day (with normal litters) were acyclic. At the incidence observed this finding was considered adverse.
Considering that the three involved females became normally pregnant with viable litters following normal precoital times, this finding is of no toxicological relevance.

Reproductive performance:

no effects observed

Dose Formulation Analyses
Accuracy
The concentrations analyzed in the formulations of Groups 2, 3 and 4 ranged from 94% to 107%, achieving accuracy means of 96%, 100% and 104% for Groups 2, 3 and 4, respectively. The results of these analyses were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.

Homogeneity
The formulations of Groups 2 and 4 achieved coefficient variation values of 2.6% and 2.4% for the low-dose and the high-dose groups, respectively. The results confirmed that the formulations were homogeneous (i.e. coefficient of variation ≤ 10%).

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item.
Viability indices were 98, 100, 98 and 96% for the control, 15, 50 and 150 mg/kg/day groups, respectively.
Two pups of the control group, two pups at 50 mg/kg/day and three pups at 150 mg/kg/day were found missing on PND 2, 3 or 4. Pups missing were most likely cannibalized. One pup of Female No. 72 at 150 mg/kg/day was killed in extremis on PND 1 as it was cold to touch and had no milk in the stomach. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this strain and age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain of pups was lower compared to controls at 50 and 150 mg/kg/day from Day 7 of treatment onwards (up to 10 and 14% lower for combined pup weight compared to concurrent controls at respectively 50 and 150 mg/kg/day), reaching statistical significance on Day 13 for male, female and combined pup weights at 150 mg/kg/day and female pup weights at 50 mg/kg/day.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day

Treatment related:

not specified

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

The report indicates a Reproduction NOAEL of 50 mg/kg/day (based on adverse effects on estrous cycle in
three females at 150 mg/kg/day)

Observations: During the pre-test period, all females had regular cycles of 4 days.
During the pre-mating period, i.e. after start treatment with the test item, an irregular cycle (out of two) was noted for Female No. 71 at 150 mg/kg/day (with normal litter). Additionally, Female Nos. 75 and 77 at 150 mg/kg/day (with normal litters) were acyclic.
Considering that the three involved females became normally pregnant follwing normal precoital time, this finding is of ofcertain toxicological relevance.

Conclusions:

The overall NOAEL for parental toxicity was 15 mg/kg/day for males and 50 mg/kg/day for females based on adverse microscopic findings in the kidneys consisting of tubular basophilia and granular cast, both of slight degree) of a single male treated at 50 mg/kg/day and a single male at 150 mg/kg/day, and slight tubular basophilia with minimal tubular degeneration/necrosis in a single female at 150 mg/kg/day.
Reproduction NOAEL was considered 50 mg/kg bw, based on 3 females at 150 mg/kg displaying irregular or acyclic estrous cycle (although all three became normally pregnant)
Developmental NOAEL was considered 50 mg/kg bw, based reduced body weight gain of pups at 150 mg/kg/day; this finding was observed in the presence of reduced maternal body weight gain.

Executive summary:

The objectives of this study were to determine the potential toxic effects of 2,2’-Azodi(2-methylbutyronitrile) (AMBN) when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. The dose levels in this study were selected to be 0, 15, 50, 150 mg/kg/day, based on the results of a Dose Range Finder.
In the main study, 4 groups of 10 animals/sex/group received test substance at 0, 15, 50 or 150 mg/kg bw/day in 5 mL corn oil/kg bw by daily oral gavage. Males were treated for 28 days. Females that delivered were treated for 50-57 days (i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Females which failed to deliver were treated for 42-54 days.

Chemical analyses of formulations were conducted once during the main study to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormone T4 and TSH, gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

 

Results:

In females treated at 150 mg/kg/day, mean body weight gain was lower compared to controls during the post-coitum phase, resulting in lower body weights at the end of the post-coitum period and at the start of the lactation phase. During the lactation phase, the same rate for body weight gain was observed for females of all groups.

Higher mean liver weights (absolute and/or relative to body weights) were noted in males and females. Additionally, females at 150 mg/kg showed a significant higher adrenal gland weight compared to controls.

Microscopic findings were noted in a single male each at 50 and 150 mg/kg/day and in a single female at 150 mg/kg/day. In these males, alpha 2u-globulin nephropathy was observed (i.e. hyaline droplet accumulation accompanied by an increased incidence and severity of tubular basophilia (up to slight) in similar areas and the presence of granular casts (slight) representing intratubular degenerated cells). Based on the degenerative nature of the granular casts, the combination of hyaline droplet accumulation, increased incidence and severity of tubular basophilia and the presence of granular casts at 50 and 150 mg/kg/day was considered adverse. In addition, in a single female treated at 150 mg/kg/day, an increased severity of tubular basophilia (slight) with adjacent tubules with degeneration/necrosis (minimal) was recorded. Based on their degenerative nature, the combination of these findings was regarded adverse.

A marked reduction of total T4 was observed in all male dose groups and in the mid and high dose groups in females. Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment. (See attached graphs) Overall the T4 change may be secondary to the increased T4 turnover resulting from metabolic enzyme induction in the liver. Under the conditions of this screening study no adverse effect was observed that could be linked to the reduction of total T4 and therefore this reduction was not taken into account when determining the parental NOAEL.

 

Reproductive results:

During the pre-test period, all females had regular cycles of 4 days.

During the pre-mating period, i.e. after start treatment with the test item, an irregular cycle (out of two) was noted for Female No. 71 at 150 mg/kg/day (with normal litter). Additionally, Female Nos. 75 and 77 at 150 mg/kg/day (with normal litters) were acyclic. At the incidence observed this finding was considered adverse.

Considering that the three involved females became normally pregnant with viable litters following normal precoital times, this finding is of no toxicological relevance.

 

Developmental results:

A dose-related reduced body weight gain of pups (both sexes) was noted at 50 and 150 mg/kg/day from Day 7 of treatment onwards. Due to the magnitude of the response (>10% at PND 13), the finding at 150 mg/kg/day was considered adverse. 

 

Conclusion:

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of 2,2’-Azodi(2-methylbutyronitrile) were established: 

Parental NOAEL (males): 15 mg/kg/day

Based on adverse microscopic findings in the kidneys (up to a slight degree) of a single male treated at 50 mg/kg/day and a single male at 150 mg/kg/day). The kidney findings at 50 and 150 mg/kg/day were considered related to alpha 2u-globulin nephropathy and adverse within the context of this study. However, as alpha 2u-globulin nephropathy is a rat specific finding it is considered to have no relevance to man.

Parental NOAEL (females): 50 mg/kg/day

Based on adverse microscopic findings in the kidneys (up to a slight degree) of a single female treated at 150 mg/kg/day)

Reproduction NOAEL: 50 mg/kg/day

based on adverse effects on estrous cycle in three females at 150 mg/kg/day

Developmental NOAEL: 50 mg/kg/day

based on reduced body weight gain of pups at 150 mg/kg/day; this finding was observed in the presence of reduced maternal body weight gain.

Effect on fertility: via oral route

Endpoint conclusion:

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

The potential of 2,2’-Azodi(2-methylbutyronitrile) to affect male and female reproductive performance by was evaluated in a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422). The following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

The dose levels in this study were selected to be 0, 15, 50, 150 mg/kg/day. Males were treated for 28 days. Females that delivered were treated for 50-57 days (i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery).

Effects observed in the parent animals involved lower BW gain and reduced and feed consumption compared to control, and higher liver weights in males and females. Additionally, females at 150 mg/kg showed a significant higher adrenal gland weight compared to controls.

The overall NOAEL for parental toxicity was 15 mg/kg/day for males and 50 mg/kg/day for females based on adverse microscopic findings in the kidneys consisting of tubular basophilia and granular cast, both of slight degree) of a single male treated at 50 mg/kg/day and a single male at 150 mg/kg/day, and slight tubular basophilia with minimal tubular degeneration/necrosis in a single female at 150 mg/kg/day.

No effects were observed on copulation index, number of implantation sites and fertility index.

Reproduction NOAEL was considered 50 mg/kg bw, based on 3 females at 150 mg/kg displaying irregular or acyclic estrous cycle (although all three became normally pregnant)

No effects were observed in length of gestation, post-implantation survival, litter size, viability index, lactation index sex ratio, AGD, areola/nipple retention and clinical signs.

Developmental NOAEL was considered 50 mg/kg bw, based reduced body weight gain of pups at 150 mg/kg/day; this finding was observed in the presence of reduced maternal body weight gain.

 

 

An additional OECD 422 repeated dose, reproductive/developmental toxicity screening study with the read-across substance 2,2’-azobis(isobutyronitrile) (AIBN) is available. In this study male and female rats were administered 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. 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.

Effects on developmental toxicity

Description of key information

A reproductive/developmental screening and OECD 414 study in rats with 2,2’-azobis(isobutyronitrile) (AIBN) was used as a read-across to fulfill the data gap for the test substance.

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

 

On both 2,2’-Azodi(2-methylbutyronitrile) (AMBN) itself and read-across substance (AIBN) are results available from an OECD 422 study. Results between the two studies are very comparable.

In the study with AMBN, no effects were observed in length of gestation, post-implantation survival, litter size, viability index, lactation index sex ratio, AGD, areola/nipple retention and clinical signs.

The developmental NOAEL was considered 50 mg/kg bw, based reduced body weight gain of pups at 150 mg/kg/day; this finding was observed in the presence of reduced maternal body weight gain.

For AIBN the NOAEL for reproduction/developmental toxicity was 10 mg/kg/day in females (abnormal lactation behavior at 50 mg) and offspring (viability index and body weight decrease on day 4 of lactation at 50 mg). The abnormal lactation behavior and subsequent effects on offspring on day 4, are considered secondary to maternal toxicity.

Link to relevant study records

Reference

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:

guideline study

Justification for type of information:

see 13.2 for attached read across rationale

Reason / purpose for cross-reference:

read-across source

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

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

Study duration:

subacute

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

A reproductive/developmental screening study in rats with 2,2’-azobis(isobutyronitrile) (AIBN) and OECD 414 study was used as a read-across to fulfill the data gap for the test substance 2,2’-Azodi(2-methylbutyronitrile) (AMBN). The underlying hypothesis for the read-across between the test substance AMBN and AIBN 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.

 

 

2,2’-Azodi(2-methylbutyronitrile) (AMBN) was evaluated in a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), at dose levels of 0, 15, 50, 150 mg/kg/day. Males were treated for 28 days. Females that delivered were treated for 50-57 days (i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Effects observed in the parent animals involved lower BW gain and reduced and feed consumption compared to control, and higher liver weights in males and females. Additionally, females at 150 mg/kg showed a significant higher adrenal gland weight compared to controls. The overall NOAEL for parental toxicity was 15 mg/kg/day for males and 50 mg/kg/day for females based on adverse microscopic findings in the kidneys consisting of tubular basophilia and granular cast, both of slight degree) of a single male treated at 50 mg/kg/day and a single male at 150 mg/kg/day, and slight tubular basophilia with minimal tubular degeneration/necrosis in a single female at 150 mg/kg/day.

No effects were observed on copulation index, number of implantation sites and fertility index.

Reproduction NOAEL was considered 50 mg/kg bw, based on 3 females at 150 mg/kg displaying irregular or acyclic estrous cycle (although all three became normally pregnant).

Developmental NOAEL was considered 50 mg/kg bw, based reduced body weight gain of pups at 150 mg/kg/day; this finding was observed in the presence of reduced maternal body weight gain. No effects were observed in length of gestation, post-implantation survival, litter size, viability index, lactation index sex ratio, AGD, areola/nipple retention and clinical signs.

 

In an OECD 422 repeated dose, reproductive/developmental toxicity screening study, 2,2’-azobis(isobutyronitrile) (AIBN) was administration to male and female rats 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.

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.

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.