Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

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
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
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 cageenrichment, 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
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
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 (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
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.
Positive control:
None
Observations and examinations performed and frequency:
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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY & CLINICAL CHEMISTRY & HORMONES: Yes
- collected on the day of scheduled necropsy. Samples will be collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility.
- Animals fasted: Males: yes; Females: No
- How many animals: selected 5/sex/group
- Parameters checked in table were examined:
HEMATOLOGY:
White blood cells (WBC)
Neutrophils (absolute)
Lymphocytes (absolute)
Monocytes (absolute)
Eosinophils (absolute)
Basophils (absolute)
Large unstained cells (LUC) (absolute)
Red blood cells
Reticulocyte (absolute)
Red Blood Cell Distribution Width (RDW)
Haemoglobin
Haematocrit
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Platelets
COAGULATION
Prothrombin Time (PT)
Activated Partial Thromboplastin Time (APTT)
CLINICAL CHEMISTRY
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Bile Acids
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate (Inorg. Phos)
THYROIUD HORMONE
Thyroxine (T4)
Thyroid-Stimulating Hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Once during the treatment period. The selected 5 males will be tested once during Week 4 of treatment and the selected 5 females will be tested once during the last week of lactation (i.e. PND 6-13). These tests will be performed after clinical observations (including arena observation, if applicable).
• hearing ability, pupillary reflex and static righting reflex
• fore- and hind-limb grip strength
• locomotor activity

ESTROUS CYCLE EVALUATIONS:
Daily vaginal lavage will be performed beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed.

IMMUNOLOGY: No.

OTHER
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: Yes
Brian
Cervix a
Epididymis b
Gland, adrenal b
Gland, coagulation b, c
Gland, parathyroid d
Gland, prostate
Gland, seminal vesicle b
Gland, thyroid b
Heart
Kidney b
Liver
Ovaries b
Spleen
Testes b
Thymus
Uterus
a Weighed together with the uterus.
b Paired organ weight.
c Weighed together with the seminal vesicles.
d Weighed together with the thyroid.

HISTOPATHOLOGY: Yes
Animal identification
Artery, aorta
Body cavity, nasopharynx
Bone marrow
Bone, femur
Bone, sternum
Brain (eight levels)
Cervix
Epididymides a
Esophagus
Eye a
Gland, adrenal
Gland, coagulation
Gland, Harderian a, b
Gland, lacrimal
Gland, mammary
Gland, parathyroid c
Gland, pituitary
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Larynx
Liver
Lung
Lymph node (mandibular and mesenteric site)
Muscle, skeletal
Nerve, optic a, b
Nerve, sciatic
Ovaries
Pancreas
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testes a
Thymus
Tongue
Trachea
Urinary bladder
Uterus
Vagina
a Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours.
b Only collected if present in the routine section of the eye.
c Only collected if present in the routine section of the thyroid.
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.
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
Description (incidence and severity):
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.

% increase weight compared to control:
- Liver, males
absolute 15mg: -2; 50 mg: 14* ; 150 mg: 26**
rel. to BW 15mg: 8; 50 mg: 20**; 150 mg: 42**
Liver, females
absolute 15mg: 5 ; 50 mg: 6 ; 150 mg: 16**
rel. to BW 15mg: 15*; 50 mg: 15*; 150 mg: 33**

- Adrenal, females
absolute 15mg: 3; 50 mg: -5; 150 mg: 1%*
rel. to BW 15mg: 17; 50 mg: 4; 150 mg: 33**
(*: P<0.05, **: P<0.01)
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: 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.
Details on results:
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 (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
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.
Executive summary:

The objectives of this study were to determine the potential toxic effects of 2,2’-Azodi(2-methylbutyronitrile) 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 this 10-day range finder, 3 females group received 150 or 300 mg/kg bw/day in 5 mL/kg bw corn oil for 10 days.
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.


 


Results:


10-day range finder:


300 mg: 2/3 dead on day 2. The other animal was sacrificed in extremis on day 2. Macroscopic evaluations revealed no abnormalities.


150 mg: no mortality was observed. Clinical signs observed were hunched posture (3/3), piloerection (2/3), salivation (3/3), lethargy slight (3/3), reduced breathing frequency (2/3). Recovery from these signs was observed between Days 5-10. Food consumption was very low during days 1-5, but normal again thereafter. BW showed a loss of 3% before day 5 in 2/3 animal, whereas the other animal showed no gain during this period. All animals showed normal gain again between days 5-10.


Organ weight evaluation revealed an increase of liver weight (up to 1.5x of the historical mean).


 


Main study:


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 statistically 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 (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.


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.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Remarks:
oral Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 22 April 2020 to 18 November 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:
other: 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
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following
guidelines:
• OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
• EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
• EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
• OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
• EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: 1708360421
- Purity, including information on contaminants, isomers, etc.: 99% of purity. Other components below 1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature. Maximum storage temperature: 25°C.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: in corn oil stability for at least 24 hours at room temperature under normal laboratory light conditions, stability of at least 8 days in the refrigerator and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) has been confirmed over the concentration range 1 to 200 mg/mL (suspensions).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during the dose administration procedure.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.
Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10-11 weeks old and females were 13-14 weeks old
- Weight at study initiation: males weighed between 261 and 312 g and females weighed between 185 and 247 g
- Fasting period before study: no
- 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.
- Diet: 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.
- Water: ad libitum, municipal water. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 7 days prior to the start of the pretest period (females) or 7 days before the commencement of dosing (males). Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. There were no known contaminants that would interfere with the objectives of the study.

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. There were no contaminants that could have interfered with the objectives of the study.
Analysis of water did not reveal the presence of contaminants interfering with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures of 18 to 24 °C. Actual daily mean temperature during the study period was 20 to 21 °C.
- Humidity (%): Target humidity of 40 to 70%. Actual daily mean relative humidity of 48 to 80% was achieved during the study period.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 of June 2020 To: 20 of August 2020
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical method
Ultra performance liquid chromatographic method with spectrophotometric detection (UPLC-UV).

Concentration analysis
For all groups, during week 1, concentration analyses were performed. The results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.

Homogeneity analysis
In week 1, results obtained from the top, middle and bottom for the Group 2 (low dose) and 4 (high dose) preparations were averaged and used as the concentration results. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Stability analysis
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males were treated for 28 days, up to and including the day before scheduled necropsy. This included a minimum of 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.

The first day of dosing was designated as Day 1.

The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during the dose administration procedure.

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:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week.
Duration of test:
Males which sired and failed to sire were euthanized following completion of the mating period (a minimum of 28 days of administration).

Females which delivered were euthanized on PND 14-16.

Females which failed to deliver having evidence of mating were euthanized on post-coitum Day 27. Females without evidence of mating were euthanized 27 days after the last day of the mating period.

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.
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Group 4 - high dose
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Group 3 - intermediate dose
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Group 2 - low dose
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 - Control group
No. of animals per sex per dose:
5 animals per sex per dose group (10 animals per dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels in this study were selected to be 0, 15, 50, 150 mg/kg bw/day, based on the results of the Dose Range Finder (DRF).

The dose levels for the DRF study were selected based on the results of an acute oral toxicity study in rats with 2,2'-azobis(2-methylbutyronitrile) although in another vehicle (0.5% methylcellulose) at dose levels of 202, 254, 320 and 402 mg/kg bw in 5 male and 5 female CD rats (combined LD50: 337 mg/kg bw, obtained from the ECHA registration dossier of 2,2'-azobis(2-methylbutyronitrile)). In that study, mortality was observed for 4 males and 1 female at 320 mg/kg bw and 3 males and 5 females at 402 mg/kg bw. Moreover, severe clinical signs of toxicity were noted (ante mortem signs comprised of lethargy, reduced mobility, staggering gait, reddening, muscle tremor, piloerection, salivation and hunched posture). Clinical signs in surviving animals were similar to those of the decedents with the addition of irritability, breathing irregularities, ungroomed appearance, diarrhea, pigmented orbital secretion, pigmented staining of the snout and closed eyes). All the surviving animals were overtly normal by the sixth day.

As the dose range finder study was a repeated dose study (at least 10 days), given the abovementioned observed mortality and clinical signs, a staggered start approach was chosen for this dose range finder study, starting with a dose level of 150 mg/kg bw, and due to limited toxicity observed, followed by a dose level of 300 mg/kg bw.

The DRF study was performed on females of Crl: WI(Han) rats (Charles River Deutschland, Sulzfeld, Germany). The rats of Group 1 (150 mg/kg bw/day) were 12-13 weeks old and weighed between 222 and 234 g at initiation of dosing. The rats of Group 2 (300 mg/kg bw/day) were 14-15 weeks old and weighed between 206 and 223 g at initiation of dosing.

On arrival and following assignment to groups at random, animals were group housed (3 animals of the same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
The actual daily mean temperature during the study period was 21°C with an actual daily mean relative humidity of 43 to 52%.

The test item and vehicle (corn oil) were administered to the appropriate animals by once
daily oral gavage for 10 consecutive days.

All animals were inspected twice daily for signs of moribundity and mortality. Clinical observations were performed daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body weights were recorded on Day 1 prior to dosing and on Days 5 and 10. Food consumption was measured over Days 1-5 and 5-10.

Mortality, clinical observations, body weights and food consumptions were monitored. All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy) or sooner (decedents). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy and all gross lesions were recorded.

No mortality occurred in Group 1. At 300 mg/kg bw/day 2/3 were found dead on day 2 of treatment. The remaining animal was sacrificed in extremis on Day 2 of treatment (post-dose). On the day of death, 3/3 animals presented with hunched posture, piloerection, lethargy (up to severe), muscle
twitching, shallow respiration, and hypothermia. In the two animals that died spontaneously, additionally, salivation, gasping and ptosis (both eyes, up to moderate) were noted (2/2) as well as squeaking and rales (1/2). In the animal sacrificed in extremis, additionally, decreased locomotor activity was noted 3h post-dose.

At 150 mg/kg bw/day, clinical signs of toxicity were noted starting 3h after treatment on Day 1 and continued to Day 5 of treatment, followed by an apparent recovery between Days 5-10. Hunched posture was noted in 3/3 animals up to 3h post-dose from Days 1-5, as well as piloerection in 2/3 animals from Days 1-3 and in 1/3 animals from Days 1-4 and Days 6-7. Moreover, lethargy (slight) was noted in 3/3 animals on Days 2-3 of treatment as well as reduced breathing frequency in 2/3 animals on Day 3 of treatment. Salivation (up to moderate) was noted at least directly after dosing in 3/3 animals on multiple days throughout the treatment period.
At 300 mg/kg bw/day, clinical signs of toxicity were noted starting 1 or 3h post-dose on Day 1 and included Hunched posture (3/3 animals), piloerection (3/3 animals), slight lethargy and hypersensitivity to touch (1/3 animals).

In Group 1, body weight loss up to 3% was noted in 2/3 animals between Days 0-5 of treatment. No body weight gain was noted in the remaining 1/3 animals between Days 0-5 of treatment. Between Days 5-10 all animals showed normal weight gain (between 3-8% compared to Day 5).
At 150 mg/kg bw/day, food consumption between Days 0-5 of treatment was very low compared to available historical control data (approximately 50% reduction). Between Days 5-10, food consumption appeared to recover, although it was still at the low end of the available historical control range. In the same animals' group, absolute and relative liver weights were significantly increased compared to the available historical control data in all animals (up to 1.5x the historical mean for 10-day dose range finders). Kidney weights were considered within normal range.

For Group 2 food consumption, body weight and organ weights were not recorded due to mortality occurred in all animals prior to Day 5.

At gross pathology, no abnormalities were noted for all the treatment groups.
Maternal examinations:
F0 GENERATION

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy. During the dosing period, these observations were performed directly after dosing. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of each day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first administration of the test item and at weekly intervals during the treatment period. Animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was 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 was monitored on regular basis throughout the study by visual inspection of the water bottles.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters checked are presented in section "Other examinations".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters checked are presented in section "Other examinations".

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5/sex/group for clinical chemistry and coagulation determinations, 10/sex/group for thyroid hormones measurement

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13). These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable).
- Dose groups that were examined: all groups
- Battery of functions tested: hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R), fore- and hind-limb grip strength, locomotor activity

OTHER:
Estrous cycle determination: Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.

General reproduction data: From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No*
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

*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.
Blood sampling:
Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a. m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility (order of sample collection: Groups 1, 4, 2 and 3). Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room.

F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was
available. F0-females were not fasted overnight.

Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room.

On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a. m., and samples were pooled to one sample per litter (samples collected in no specific order). If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. As for Litters No. 44, 47, 54, 55 and 59 the target volume of 0.4 mL was not reached by pooling blood of two pups, blood of a third surplus pup of the same litter was added.

On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female, if possible). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure (samples collected in no specific order).
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: yes

CLINICAL OBSERVATIONS
Clinical observations were performed at least once daily for all pups. Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

BODY WEIGHT
Live pups were weighed individually on PND 1, 4, 7 and 13.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioral 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, and sexed (both internally and externally); If possible, defects or cause of death were evaluated.

TERMINAL PROCEDURES: 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 (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

POST-MORTEM EXAMINATIONS
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, 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.

More details on blood sampling procedure is reported in "Any other information on materials and methods incl. tables" section.
Statistics:
Refer to "Any other information on materials and methods incl. tables"
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 bw/day dose groups. Hunched posture was noted incidentally on the second day of dosing in females of the 50 and 150 mg/kg bw/day dose groups and in an individual female of the 15 mg/kg bw/day dose group during week 6 of treatment.

Red discharge from the vulva was noted on a single occasion, i.e. on Day 17 of treatment (Day 0 post-coitum), for one female of the mid-dose group (Group 3). At the incidence observed, this finding was considered not toxicologically relevant.
Salivation seen after dosing among animals of the 50 and 150 mg/kg bw/day dose groups from the
second week of treatment onwards and in animals of the 15 mg/kg bw/day dose group from the third week of the treatment period onwards was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
This sign was considered to be a physiological response and a sign of local toxicity.

Incidental findings that were noted included wounds, scabs, alopecia and chromodacryorrhoea. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidences observed, these were considered not to be signs of toxicological relevance.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In females treated at 150 mg/kg bw/day, mean body weight gain was lower compared to concurrent controls during the post-coitum phase, from Day 11 post-coitum onwards, resulting in lower body weights at the end of the post-coitum period and at the start of the lactation phase (respectively, 9% and 9% lower compared to concurrent controls). During the lactation phase, the same rate for body weight gain was observed for all groups. The apparent lower body weight observed at 150 mg/kg bw/day during the lactation phase was not a true test item-related effect but secondary to the reduction in body weight gain during the post-coitum phase.
The statistically lower body weight in females at 15 mg/kg bw/day, from Day 8 of treatment onwards was considered the result of the lower body weight of these females at the start of the treatment period, as body weight gain was unaffected by treatment.
Any other statistically significant changes in body weights and body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.
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 animals (up to 32% of control in females at 150 mg/kg bw/day), with recovery to control levels during the second and third week of treatment.

The statistically significant lower absolute food consumption at 150 mg/kg bw/day between Days 4-7, 7-11 and 17-20 post-coitum and Days 7-13 of the lactation phase was in line with the lower body
weights; however, relative food consumption during these intervals was unaffected by treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters were considered not to have been affected by treatment with the test item.
Decreased platelets in females at 50 and 150 mg/kg bw/day (0.84 and 0.86x of control, respectively) were considered unrelated to treatment with the test item in the absence of a dose-related trend.
While the decrease in mean hemoglobin in females at 150 mg/kg bw/day was statistically significant, this alteration was considered not toxicologically relevant due to the minimal magnitude of the change.

Any other statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Coagulation parameters of treated rats were considered not to have been affected by treatment with
the test item.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were found (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
- Higher mean calcium concentration in females at 150 mg/kg bw/day and females at 50
mg/kg bw/day (1.05x of control)
- Lower mean total bilirubin level in females at 50 and 150 mg/kg bw/day (0.77x and 0.78x of control)
- Higher mean total protein concentration in females at 150 mg/kg bw/day (1.09x of control), resulting from the higher albumin value in females at 150 mg/kg bw/day (1.10x of control)
- Lower mean urea concentration in females at 150 mg/kg bw/day (0.83x of control)

The statistically significant higher calcium and lower total bilirubin levels in females at 50 and 150
mg/kg bw/day were considered not toxicologically relevant due to the minimal magnitude of the effect (calcium) and/or in the absence of a dose related trend (calcium and bilirubin).

The apparent higher mean bile acid value in females of all treatment groups (without statistical
significance) when compared to controls, was considered to have arisen as a result of a slightly low
control value and therefore no toxicological significance was attached to this finding.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone analyses:
Dose-dependent lower total T4 values were noted in F0-females treated at 50 and 150 mg/kg bw/day (0.79x and 0.64x of control, respectively).
Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment. The apparent higher TSH value in females at 150 mg/kg bw/day (1.50x of control), although not statistically
significant, was due to a high individual value of one female and was considered not toxicologically relevant.
Please refer to table in "Any other information on results incl. tables" section.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment with the test item up to 150 mg/kg bw/day.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined
animals up to and including 150 mg/kg bw/day.
Motor activity was considered not to be affected by treatment with the test item. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher mean liver and adrenal gland weights (absolute and/or relative to body weights) were noted
in males and/or females (refer to table Mean Percent LIVER and ADRENAL GLAND Weight differences from Control Groups in "Any other information on results incl. tables" section.

Statistically significant higher mean relative liver weights were noted in the 15, 50 and 150 mg/kg bw/day group females. Mean absolute weights were higher in females at 150 mg/kg bw/day.
Statistically significant higher mean absolute and relative adrenal gland weights were noted in 150 mg/kg bw/day group females.

Any other differences, including those that reached statistical significance (females: mean relative brain weight at 15 and 150 mg/kg bw/day and mean relative heart weight at 150 mg/kg bw/day) were considered not to be test item-related due to the direction of the change and/or lack of a dose-related pattern.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic findings were noted in the kidneys of females.

An increased severity of tubular basophilia (slight) passing gradually into areas with tubular degeneration/necrosis (minimal) was recorded in a single female at 150 mg/kg bw/day.

These findings were considered test item-related histopathologic changes. The remainder of the recorded microscopic findings were considered within the range of background pathology encountered in rats of this age and strain.

There were two couples of the control group and one couple of the 50 mg/kg bw/day group with no offspring. Microscopic examination did not reveal lesions which could explain their lack of offspring.
Based on the lack of a microscopic correlate and in the absence of a dose-related response, the failure to produce offspring was considered unrelated to treatment with the test item.

Table summarizing the results of relevant histopathology findings is attached as image named "Histopathology table_CRL20237946".
Details on results:
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%).
Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantation sites was considered not to be affected by treatment with the test
item.

The total number of offspring born compared to the total number of uterine implantations was
considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 99, 89, 93 and 88% for the control, 15, 50 and 150 mg/kg bw/day groups, respectively.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment with the test item.
The gestation indices were 100% for all groups.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 89, 100, 100 and 100% for the control, 15, 50 and 150 mg/kg bw/day groups, respectively.
One control female was not pregnant.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Abnormalities:
no effects observed
Fetal body 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 bw/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 bw/day), reaching statistical significance on Day 13 for male, female and combined pup weights at 150 mg/kg bw/day and female pup weights at 50 mg/kg bw/day.

A table summarizing the pups weight data is attached as image.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the test item. The live birth indices were 100, 94, 97 and 100% for the control, 15, 50 and 150 mg/kg bw/day groups, respectively.

Noteworthy, seven out of eight pups of one litter at 15 mg/kg bw/day were found dead at first
litter check. Only one pup of this litter survived until scheduled necropsy. As this was observed at the low dose level and a poor litter is also occasionally seen in the control group in this type of studies, this finding was considered unrelated to treatment.

Three pups at 50 mg/kg bw/day (from two litters) were found dead at first litter check. 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.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment with the test item.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size was considered not affected by treatment with the test item. Live litter sizes were 12.5, 10.6, 11.6 and 11.2 living pups/litter for the control, 15, 50 and 150 mg/kg bw/day groups, respectively.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
Changes in postnatal survival:
no effects observed
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 bw/day groups, respectively.

Two pups of the control group, two pups at 50 mg/kg bw/day and three pups at 150 mg/kg bw/day were found missing on PND 2, 3 or 4. Pups missing were most likely cannibalized. One pup of one female of the high-dose group (150 mg/kg bw/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.

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups considered to be related to treatment with
the test item.
Visceral malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups considered to be related to treatment with
the test item.

Left testis and left epididymis were smaller in size in one male pup of a low-dose group female (15 mg/kg bw/day) and in one male pup of high-dose group (150 mg/kg bw/day). At the incidences observed and in the absence of a dose-related trend these findings were considered not toxicologically relevant.

No milk in the stomach was noted for one pup and two pups of litters belonging to the mid-dose group (50 mg/kg bw/day), and one pup of a high.-dose group litter (150 mg/kg bw/day) that were found dead at first litter check or were sacrificed in extremis on PND 1. Lean appearance was noted for one pup of a high-dose group litter (150 mg/kg bw/day). The nature and incidences of these and any other macroscopic findings remained within the range considered normal for pups of this strain and age and were therefore considered not to be related to treatment with the test item.
Other effects:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Table 5 - Mean Percent LIVER and ADRENAL GLAND Weight Differences from Control Groups


























 



Males



Females



Dose level (mg/kg bw/day):



15          50          150



15          50          150



LIVER


              Absolute


              Relative to body weight



 


-2           14*        26**


8            20**      42**



 


5            6            16**


15*        15*        33**



ADRENAL GLAND


              Absolute


              Relative to body weight



 


-5           3            3


6            11          17



 


3            -5           15*


17          4            33**



*: P<0.05, **: P<0.01


 


Table 6 - Thyroid hormones measurements
































































Male



T4


(ng/mL)



TSH


(mU/L)



0 mg/kg bw/d



Mean


SD


N



50.96


12.16


10



0.0577


0.0253


10



15 mg/kg bw/d



Mean


SD


N


tCtrl



41.08*


8.10


10


0.81



0.2489


0.2916


10


4.31



50 mg/kg bw/d



Mean


SD


N


tCtrl



36.19**


7.89


10


0.71



0.1856


0.2040


10


3.22



150 mg/kg bw/d



Mean


SD


N


tCtrl



27.28**


6.66


10


0.54



0.0956


0.0511


10


1.66



Female



T4


(ng/mL)



TSH


(mU/L)



0 mg/kg bw/d



Mean


SD


N



26.75


6.59


10



0.1891


0.0894


10



15 mg/kg bw/d



Mean


SD


N


tCtrl



24.85


4.47


10


0.93



0.2137


0.2258


10


1.13



50 mg/kg bw/d



Mean


SD


N


tCtrl



21.06*


4.53


10


0.79



0.2021


0.1029


10


1.07



150 mg/kg bw/d



Mean


SD


N


tCtrl



17.02**


3.03


10


0.64



0.2833


0.2975


10


1.50



Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

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

Wistar Han rats were treated with 2,2’-Azodi(2-methylbutyronitrile) by daily oral gavage at dose levels of 15, 50 and 150 mg/kg bw/day. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 28 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (for 50-57 days).
Females that failed to deliver pups were treated for 42-54 days.


Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.


Short-term toxicity was observed in females adult rats at kidney level. Microscopic findings were noted in a single female at 150 mg/kg bw/day in which a higher 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.


Clinical signs were noted but not considered toxicologically relevant at the incidences observed.


In females treated at 150 mg/kg bw/day, mean body weight gain was lower compared to controls during the post-coitum phase (from Day 11 post-coitum onwards), 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. Food consumption in females was lower during the first week of treatment in all treated groups and recovered to control levels afterwards. Since recovery of food consumption and body weight gain were noted, and due to the small magnitude of the effects on body weights (<10%), the effects were not considered adverse.


At 150 mg/kg bw/day, changes in clinical chemistry were noted and consisted of higher albumin in females and subsequent higher total protein, and lower urea values in females. In the absence of microscopic correlates, these findings were considered not adverse.


Statistically significant higher adrenal gland weights (absolute and relative) were noted in 150 mg/kg bw/day group females. In the absence of any microscopic correlate, this finding was considered not adverse.


In addition, statistically significant higher liver weights were noted in females at 15, 50 and 150 mg/kg bw/day that were considered test item-related.
This correlated in the 150 mg/kg bw/day males with the microscopic finding of hepatocellular hypertrophy. However, because of the recorded minimal severity and in the absence of other test item-related degenerative findings or related changes in liver enzymes, this finding was considered non-adverse.


A dose-dependent lower total T4 was noted in females treated at 50 and 150 mg/kg bw/day. This finding 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 bw/day). However, under the conditions of this 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.


No treatment-related toxicologically significant findings were noted in any of the remaining parameters investigated in this study (i.e. viability/mortality, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), clotting parameters and macroscopic examination).


Focusing on developmental effects, a dose-related lower body weight gain of pups (both sexes) was noted at 50 and 150 mg/kg bw/day from Day 7 of treatment onwards. Due to the magnitude of the response at 150 mg/kg bw/day (>10% at PND 13), this was considered adverse.
No treatment-related toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size, and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).


The NOAEL for maternal effects is set at 50 mg/kg bw/day (based on adverse microscopic findings in the kidneys (up to a slight degree) of a single female treated at 150 mg/kg bw/day).


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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-azobis[2-methylbutyronitrile]
EC Number:
236-740-8
EC Name:
2,2'-azobis[2-methylbutyronitrile]
Cas Number:
13472-08-7
Molecular formula:
C10H16N4
IUPAC Name:
2,2'-diazene-1,2-diylbis(2-methylbutanenitrile)
Test material form:
solid: granular
Details on test material:
Product name : 2,2'-Azo-bis (2-methyl-butyronitrile)
Trade name : Perkadox AMBN
CoA analysis: Jan 5, 2020
Batch no. : 1708360421
Appearance: White Granules
- Purity: 99.0%
- Impurities: below 1%
Storage temperature: 20 °C
Expiration date: 08/01/2021 (month/day/year)

Test animals

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)

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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%).

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

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

Overall reproductive toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.