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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

There are one combined reproduction toxicity study (OECD 422) performed in rat in 1999 and one new reproduction study (OECD 421)- performed in 2021 as a preliminary study to the OECD 443 and an OECD 443 study performed in 2022 both performed according to GLP.

1-Combined screening Reproduction toxicity study (Hatano, 1999-OECD 422):

The reproductive / developmental toxicity of 2,2'-Azobis(2-methylpropionitrile) was evaluated in male and female rats after oral administration (gavage) at doses of 0, 2, 10 and 50 mg/kg/day until day 43 for males and day 4 of post partum for females (Hatano, 1999). In the females, AZDN inhibited weight gain and feed consumption at an early stage of treatment at 10 mg/kg or greater doses. 

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

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

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

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

2- Reproduction toxicity study (OECD 421, CRL,2021)

The potential toxic effects of the test item was evaluated following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 21 post-partum (p.p.). The study was conducted in accordance with the OECD TG 421 and GLP principles.

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item daily by the oral route (gavage) at dose levels of 5, 20 or 50 mg/kg bw/day. Males were treated for 2 weeks before mating, throughout mating and then until the day before euthanasia (i.e. after an 8-week treatment period). Females were treated for an overall period of 8 to 10 weeks: 2 weeks before pairing, then throughout the mating and gestation periods until Day 21 p.p. inclusive.

Another group of 10 males and 10 females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg bw/day was used.

The dams were allowed to deliver and rear their progeny until Day 21 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible four males and four females per litter. The pups were observed daily for clinical signs and abnormal behavior and were weighed at designated intervals. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p.

Thyroid hormone (TSH and T4) levels were determined in all F0 males at final euthanasia and in pups euthanized on Day 21 p.p.

F0 males were euthanized after completion of the gestation period (i.e. study Day 56). Dams were euthanized on Day 22 p.p.

A full macroscopic post-mortem examination was performed, and particular attention was accorded to the reproductive organs. Designated organs were weighed, and selected tissue specimens were preserved. A microscopic examination was performed on epididymides, oviducts, ovaries, testes, uterus and vagina from the control and high-dose groups on liver from all parent animals and on all macroscopic lesions.

A detailed external examination was performed after euthanasia of pups found dead or prematurely euthanized, or euthanized on Day 21 p.p., and particular attention was paid to the external genital organs.

Under the experimental conditions of the study:
- The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg bw/day in female parents based on unscheduled mortality, signs of poor clinical condition and hypertrophic liver changes at 50 mg/kg bw/day and no NOAEL could be determined in male parents based on the tubular degenerative changes in the kidneys from 5 mg/kg bw/day,
- The No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 50 mg/kg bw/day, based on the absence of adverse findings at this dose level,
- The No Observed Adverse Effect Level (NOAEL) for F1 development was considered to be 20 mg/kg bw/day, based on the decreased pup body weight after culling at 50 mg/kg bw/day.

3-Extended one generation study without F2 extension (CRL, 2022)

The objective of this study was to provide general information concerning reproductive and developmental effects of the test item, AZDN, that may occur as a result of pre- and post-natal exposure. Systemic toxicity in pregnant and lactating females and young and adult offspring were also evaluated.

In the assay, sexually mature male and female rats [parental (P) generation] were exposed to the test item from 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were selected and assigned to cohorts of animals for reproductive/developmental toxicity testing (cohorts 1A, 1B and 1C, without extension to mate the Cohort 1B animals to produce an F2 generation).

The test item was also administered to the F1 offspring from weaning to adulthood or euthanasia. Clinical observations and pathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development of the offspring.

The main effects are summarized below:

P generation: No test item-related mortality was observed. Test item-related increased kidney weights were noted in males treated at ≥ 1 mg/kg/day. This correlated with enlargement, granular aspect and tan discoloration at gross observation and microscopic degenerative/inflammatory lesions in males (considered as adverse at ≥ 3 mg/kg/day).  Increased liver weights were noted in males treated at ≥ 3 mg/kg/day and in females treated at 15 mg/kg/day. This correlated with increased incidence of accentuated lobular pattern in males at ≥ 3 mg/kg/day and microscopic non-adverse hypertrophy and vacuolation. Hepatocellular degeneration/necrosis was recorded at 15 mg/kg/day and was considered to be adverse, although seen in only 2/20 males and 1/24 females at minimal severity. In addition, non-adverse minimal follicular hypertrophy was seen in the thyroid glands from males treated at 15 mg/kg/day and correlated with increased weights. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.

Cohort 1A: No test item-related mortality was observed. Increased kidney weights were noted in males treated at ≥ 1 mg/kg/day. This correlated with enlargement, irregular color and tan discoloration at gross observation and microscopic degenerative/inflammatory lesions in these males (considered as adverse at ≥ 1 mg/kg/day). Increased liver weights were noted in males and females treated at ≥ 3 mg/kg/day. This correlated with microscopic non-adverse hypertrophy and/or vacuolation. Hepatocellular degeneration/necrosis was recorded in males at 15 mg/kg/day and was considered to be adverse, although seen in only 1/20 males at minimal severity. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.

Cohort 1B: No test item-related mortality and no organ weight differences or gross findings were noted. Test item-related degenerative/inflammatory findings were noted in the kidneys of males treated at ≥ 1 mg/kg/day.

Cohort 1C: There was no test item-related mortality. There were no test item-related gross findings.

Non selected pups from PND22: There were no test item-related organ weight differences or gross findings.

Conclusion

The test item, AZDN, was administered daily by oral gavage, at dose level of 0, 1, 3 or 15 mg/kg/day, to sexually mature male and female rats [parental (P) generation] from 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity.

Systemic toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be:

• 1mg/kg/day in parental (P) generation (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at ≥ 3 mg/kg/day and on adverse degeneration/necrosis in liver of males and females at 15 mg/kg/day).


• Lower than 1 mg/kg/day in Cohort 1A (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at ≥ 1mg/kg/day and on adverse degeneration/necrosis in liver of males at 15 mg/kg/day).

Reproductive/developmental toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 15 mg/kg/day in the Sprague-Dawley rats, based on the absence of adverse effects at this dose level.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2021 - 14 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Study performed according to the specifications described in:
• ECHA decision No. CCH-D-2114516984-40-01/F (https://echa.europa.eu/documents/10162/e837b1d8-09e7-9aaa-6381-38cbe16a8c41, accessed on 09 Jun 2021).

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®)

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected as background data from previous studies are available at Charles River Laboratories Evreux.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: (P) males and females were 7 and 6 wks old respectively.
- Weight at study initiation: (P) Males: 153-261 g; Females: 128-163 g; (F1).
- Fasting period before study: No
- Housing: individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing appropriate bedding. Each cage contained two objects (rat hut and nylabone/cocoon) for environmental enrichment. Cohorts 1A, 1B and 1C animals were group housed. They were housed in polycarbonate cages with stainless steel lids (in 2000P cages: up to 4/sex per cage) containing autoclaved sawdust (Le Comptoir des Sciures, Meyzieu, France).
- Diet: SSNIFF rat/mouse pelleted maintenance diet ad libitum (SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water: tap water (filtered with a 0.22 um filter) ad libitum
- Acclimation period: 8 days
The batches of diet, sawdust and wood shavings were analyzed by the suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which may be expected to interfere with or prejudice the outcome of the study.

ENVIRONMENTAL CONDITIONS SET TO MANTAIN
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 8 to 15. Filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h
IN-LIFE DATES: From: 29 July 2021 To: 7 Feb 2022

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Done according to Study No. 48855 VAS (Robin, 2021) (stability testing) describing the preparation procedure for a range of concentrations [0.1 mg/mL - 10 mg/mL] covering the lowest and highest used in this study. The frequency of preparation dosing formulations was also based on test item dose formulation stability and vehicle expiry (i.e. for up to 10 days in corn oil after preparation of the formulations).
Dose formulations were stored in the refrigerator set at +5°C and protected from light.
Dose formulations (vehicle and test-item formulations) were stirred at least 30 minutes before administration. The formulations were maintained under continuous magnetic stirring throughout the dosing procedure

VEHICLE
- Justification for use and choice of vehicle: selected in agreement with the Sponsor, based on previous experimental work and/or preliminary study(ies)
- Concentration in vehicle: 0, 0.2, 0.6 and 3 mg/mL or the control low, mid and high dose group, respectively
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Lot/batch no.: MKCN9742 and MKCN3364

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days had elapsed.
- Proof of pregnancy: presence of a vaginal plug or sperm in a vaginal lavage. The day of confirmed mating was designated as post-coitum (p.c) day 0.
- When mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using High Performance Liquid Chromatography with UV detection (HPLC/UV) and a validated analytical procedure (Test Facility Study No. 48855 VAS) .
Eight dosages: once in each P-premating, P gestation, P lactation periods, and then five times spread over the F1 postweaning period. A sample was taken from control and test item dose formulations and analyzed using the validated method.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for solutions of target concentration.

Duration of treatment / exposure:

P: males and females 10 weeks before mating and during mating; males until the day before euthanasia (after weaning of the F1 pups); females during gestation and lactation (day 21 post-coitum); until euthanasia for females with no evidence of mating or no delivery (24-26 days after the last day of the mating period).
F1:
Cohort 1A: males and females from weaning day 22 post-natal until day 90-93
Cohort 1B: males and females from weaning day 22 post-natal until day 120-122
Cohort 1C: males and females from weaning day 22 post-natal until day 85-124 or until preputial separation was observed (males) and until day 126 or until vaginal opening was observed (females).

Frequency of treatment:

Once daily (pups during lactation were not dosed directly)

Dose / conc.:

1 mg/kg bw/day

Dose / conc.:

3 mg/kg bw/day

Dose / conc.:

15 mg/kg bw/day

No. of animals per sex per dose:

P: 24 males + 24 females per group
F1: 20 males + 20 females per group and per Cohort

Control animals:

yes, concurrent vehicle

Details on study design:

RATIONALE FOR DOSE LEVEL SELECTION:
The dose levels were selected on the basis of the results of the following previous studies:
-A 13-week toxicity study in rats (Covance reference No. 8266542, summary provided by the Sponsor): In this study, the test item was administered daily by gavage to males and females at dose levels of 0.5, 2 or 10 mg/kg bw/day for at least 90 days. The administration was well tolerated. There was no evidence of neurotoxicity. There were slight changes in some blood clinical chemistry and urine parameters at all dose levels. The liver and kidney were identified as target organs based on increased weights (at all dose levels in males) and microscopic findings of hepatocyte hypertrophy (at all dose levels in males and at 2 mg/kg bw/day in females) and focal nephropathy/tubular basophilia and hyaline droplets in males at all dose levels. The liver hypertrophy was considered as an adaptive change commonly associated with metabolic burden and the changes in the kidney were considered specific to the male rat as confirmed by immunohistochemical staining. The No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 10 mg/kg bw/day.
-An OECD 422 study in rats (Reference No. R-95-007, summary provided by the Sponsor): In this study, the test item was administered daily by gavage to males and females at dose levels of 2, 10 or 50 mg/kg bw/day until Day 43 for males and Day 4 post-partum for females. The test item had no effects on the copulation index or fertility index at 50 mg/kg bw/day or lower doses. The test item did not affect the length of the gestation period or delivery index in maternal animals, either. No abnormal parturition was observed. Abnormal nursing behavior was noted in 3 animals from the 50 mg/kg bw/day group. The findings in offspring showed no effects on the parturition index, live pup delivery index, overall delivery index, or the sex ratio and body weight on Day 0 at all dose levels. In the 50 mg/kg bw/day group, the offspring viability index and body weight on Day 4 of lactation showed a tendency to decrease. No offspring from the AZDN groups showed any morphological anomaly. Based on these results, the No-Observed-Effect-Level (NOEL) for reproductive and developmental toxicity was 50 mg/kg bw/day in males and 10 mg/kg bw/day in females and in pups.
-An OECD 414 study in rats (Covance reference No. 8266543, summary provided by the Sponsor): In this study, the test item was administered daily by gavage to pregnant females at dose levels of 1, 5 or 20 mg/kg bw/day from Day 6 p.c. to Day 19 p.c. Administration of the test item induced maternal toxicity from 5 mg/kg bw/day upwards based on transient effects of reduced body weight gain and reduced mean food intake while no effects on fetal development were observed. At 1 mg/kg bw/day, no adverse effects were observed in both parental and fetal development. There was no evidence of embryotoxicity at any dose level tested. The maternal NOAEL was 1 mg/kg bw/day based on a significant decrease in maternal body weight gain and food consumption. The embryo-fetal NOAEL was 20 mg/kg bw/day.
-AZDN - Reproduction/Developmental Toxicity Screening Test by Oral Route (Gavage) in Rats (Charles River Laboratories Evreux/Study No. 48747 RSR (Papineau)): In this OECD 421 study, the test item was administered daily by oral gavage to male and female Sprague-Dawley rats for 15 days before mating, during mating, throughout gestation and, for females, until Day 21 p.p., at dose levels of 0, 5, 20 or 50 mg/kg bw/day. No unscheduled deaths occurred in males. Two high dose females were prematurely euthanized or found dead before mating (on Study Day 8), due to poor clinical condition. Poor clinical conditions (emaciated appearance, piloerection, round back and/or hypoactivity) were observed in 1 out of 8 surviving females at 50 mg/kg bw/day during the pre-mating period. Ptyalism was also observed in test item treated males and females during the whole treatment period. At all dose levels and in both sexes, there were no adverse effects on body weight, body weight change or food consumption as changes observed in males and/or females were transient and/or of minimal to slight magnitude. There were no effects on estrous cycles, pairing, mating or fertility data. From 20 mg/kg bw/day and in male parents, liver centrilobular hepatocellular hypertrophy correlating with macroscopic enlargement and increased liver weight was observed in both sexes. At 50 mg/kg bw/day, there was also hepatocellular degeneration/necrosis and periportal vacuolation in males only. The hypertrophic changes were considered to be adverse at 50 mg/kg bw/day in view of their severity and association with degenerative changes. In the kidneys, in males only, hyaline droplet accumulation and adverse tubular degenerative changes consistent with a2µ globulin nephropathy were observed from 5 mg/kg bw/day. These findings are considered to be non-relevant for humans. At 50 mg/kg bw/day and in pups, there were adverse low mean body weights in males and females after culling (down to -16% vs. controls on Day 8 p.p., p<0.01). There were no effects on viability or lactation indexes and no effects on sex ratio. At pups' external examination, there were no areolae/nipples in male pups and no external abnormalities in any pups. There were no effects on mean plasma TSH or T4 concentration levels in male parents and Day 21 p.p. pups. Therefore, 15 mg/kg bw/day was selected as the high-dose level taking into account the increase of exposure duration in the OECD 443 and, the macroscopic and/or microscopic findings observed from 20 mg/kg bw/day. The low-dose and mid-dose were selected using ratios representing 3- to 5-fold intervals (i.e. 1 and 3 mg/kg bw/day).

Positive control:

N.A.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice a day (before and after treatment), at approximately the same time of day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week until the end of the study, including at least once before the first treatment. During the treatment period, detailed clinical examination was performed after dosing and replaced the cage side observation. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also evaluated.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly including mating period; Mated females on day 0, 4, 7, 10, 14, 17 and 20 p.c. and, on Days 1, 4, 7, 14 and 21 p.p. (post-partum).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly; Mated females for the intervals: Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the intervals: Days 1-4, 4-7, 7-14, and 14-21 p.p. Food consumption was not measured for males or females during the mating period.

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes at least 14 hours
- How many animals: 10 males + 10 females per group
- Parameters: Red blood cells (RBC), Mean cell volume (MCV), Haematocrit (PCV), Haemoglobin (HB), Mean cell haemoglobin concentration (MCHC), Mean cell haemoglobin (MCH), Thrombocyte (platelet) count (PLT), White blood cells (WBC), Differential white cell count with cell morphology [Neutrophils (N), Lymphocytes (L), Monocytes (M), Eosinophils (E), Basophils (B), Reticulocytes (RTC), large unstained cells (LUC)], Coagulation parameters [Prothrombin time (blood clotting time) (PT), Fibrinogen (FIB), Activated partial thromboplastin time (APTT)]

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: At least 14 hours
- How many animals: 10 males + 10 females per group
- Parameters checked : Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus (PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Bile acids (BIL.AC).

THYROID HORMONES: Yes
- Time schedule for collection of blood: at termination (between 07:30 and 10:00 a.m.)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: serum of 10 males + 10 females per group
- Parameters checked : TSH, T4

URINALYSIS: Yes
- Time schedule for collection of urine: on day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: At least 14 hours
- How many animals: 10 males + 10 females per group
- Parameters: Volume, pH, Specific gravity, Bilirubin, Protein, Ketones, Glucose, Blood, Appearance and Color.

Oestrous cyclicity (parental animals):

Daily vaginal lavage from 14 days prior to mating period and during mating until evidence of copulation was observed. On day of termination vaginal lavage of all surviving females to allow correlation with reproductive organs histopathology.

Sperm parameters (parental animals):

Sperm count in epididymides, sperm (progressive) motility, sperm morphology were measured on all P males from the control and high-dose groups (groups 1 and 4) from left epidydimis sampled after anesthesia and before euthanasia.
Sperm morphology was determined from a smear, after eosin staining, counting 200 spermatozoa per slide, including •Total number of sperm cells evaluated (n)
• Number of normal sperm cells (n)
• Number of normal sperm cells / Total number of sperm cells evaluated (%)
• Total number of abnormal sperm cells (n)
• Number of sperm cells with head abnormalities (n)
• Number of sperm cells with detached head (n)
• Number of sperm cells with abnormal midpiece (n).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- The size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible five males and five females per litter. Whenever necessary, partial adjustment (for example six males and four females) was permitted.

PARAMETERS EXAMINED IN F1 OFFSPRING:
- On day 1 p.p.: External visible abnormalities, cleft palate, subcutaneous hemorrhages, abnormal skin color or texture, presence of umbilical cord, lack of milk in the stomach, presence of dried secretion, sex of each pup, qualitative assessment of body temperature, activity, reaction to handling and anogenital distance (AGD) (all pups before culling; normalized to the cube root of body weight recorded on Day 1 p.p.),
- Body weight on Days 1, 4, 7, 14 and 21 p.p.; on the first day of treatment (Day 1); then at least once a week until euthanasia.
- Food Consumption was measured once a week from the first day of treatment until euthanasia.
- Sexual development was assessed from Day 35 p.p. until preputial separation or euthanasia in males, and from Day 25 p.p. until vaginal opening or euthanasia in females.
- Number of nipples and of areolae in male pups on Day 12 p.p.
- Animals were observed for clinical signs daily during lactation. After weaning, each animal was observed for clinical signs, at least twice a day.
- After weaning, detailed clinical examinations were performed on all animals at least once a week until the end of the study.
- Number and sex of pups, stillbirths, live births, postnatal mortality (at least twice a day)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes at least 14 hours
- How many animals: 10 males + 10 females of Cohort 1A
- Parameters: Red blood cells (RBC), Mean cell volume (MCV), Haematocrit (PCV), Haemoglobin (HB), Mean cell haemoglobin concentration (MCHC), Mean cell haemoglobin (MCH), Thrombocyte (platelet) count (PLT), White blood cells (WBC), Differential white cell count with cell morphology [Neutrophils (N), Lymphocytes (L), Monocytes (M), Eosinophils (E), Basophils (B), Reticulocytes (RTC), large unstained cells (LUC)], Coagulation parameters [Prothrombin time (blood clotting time) (PT), Fibrinogen (FIB), Activated partial thromboplastin time (APTT)]

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: At least 14 hours
- How many animals: 10 males + 10 females of Cohort 1A
- Parameters checked : Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus (PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Bile acids (BIL.AC).

THYROID HORMONES: Yes
- Time schedule for collection of blood: at termination (between 07:30 and 10:00 a.m.)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: serum of 10 males + 10 females of Cohort 1A
- Parameters checked : TSH, T4

URINALYSIS: Yes
- Time schedule for collection of urine: on day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: At least 14 hours
- How many animals: 10 males + 10 females of Cohort 1A
- Parameters: Volume, pH, Specific gravity, Bilirubin, Protein, Ketones, Glucose, Blood, Appearance and Color.

SPERM PARAMETERS: Yes
- Sperm count in epididymides, sperm (progressive) motility, sperm morphology were measured on all P and Cohort 1A males from the control and high-dose groups (groups 1 and 4) from left epidydimis sampled after anesthesia and before euthanasia.
Sperm morphology was determined from a smear, after eosin staining, counting 200 spermatozoa per slide, including •Total number of sperm cells evaluated (n)
• Number of normal sperm cells (n)
• Number of normal sperm cells / Total number of sperm cells evaluated (%)
• Total number of abnormal sperm cells (n)
• Number of sperm cells with head abnormalities (n)
• Number of sperm cells with detached head (n)
• Number of sperm cells with abnormal midpiece (n).

LYMPHOCYTE SUBTYPING: Yes
- For the investigation of pre- and post-natal induced immunotoxic effects, 10 males and 10 females from each group in Cohort 1A animals were subjected to a splenic lymphocyte subpopulation analysis [T lymphocytes, CD4+ and CD8+ T lymphocytes, B lymphocytes, Natural Killer (NK) cells and NKT cells] using one half of the spleen (weight determined at necropsy), the other half of the spleen was preserved for histopathological evaluation. The lymphocyte subtyping by flow cytometry followed the method validated in Study No. 44564 RDR (Hamel, 2020) and the internal procedures.

Postmortem examinations (parental animals):

SACRIFICE:
On completion of the treatment period, all surviving P animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- Male animals: after weaning of the F1 progeny.
- Maternal animals: on Day 23 p.p. (Days 24-26 p.c. if they did not deliver [euthanasia by inhalation of carbon dioxide gas followed by cervical dislocation], 25 days after the end of the mating period for a female with no evidence of mating [euthanasia by inhalation of carbon dioxide gas followed by cervical dislocation])

GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all P animals. Gross necropsy consisted of examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were recorded for females euthanized on Day 23 p.p. The numbers of corpora lutea and implantation sites were recorded when possible for females euthanized on Days 24 26 p.c. due to no delivery.

HISTOPATHOLOGY / ORGAN WEIGTHS
- The organs specified in Table 2 (under ''Any other information on materials and methods incl. tables'') were weighed wet as soon as possible after dissection.
-A microscopic examination was performed on:
• all tissues listed in Table 2 (under ''Any other information on materials and methods incl. tables'') from animals of the control and high-dose groups (groups 1 and 4) from P generation.
• reproductive organs from animals that did not mate or conceive, from pregnant females that did not deliver, from females with abnormal estrous cycles and from males with abnormalities at sperm analysis, to investigate possible causes.
• all macroscopic lesions of all groups, including those from prematurely euthanized animals.

Postmortem examinations (offspring):

SACRIFICE:
Animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- Pups whose mother died were ethanised as soon as possible,
- Pups not selected on Day 4 p.p.
- Pups not selected at weaning: on Day 22 p.p.
- On completion of the treatment period for all surviving F1 adults
• F1 males (Cohort 1A): on Days 90-93 p.p.
• F1 females (Cohort 1A): on Days 90-94 p.p.,
• F1 males and females (Cohort 1B): after euthanasia of Cohort 1A animals
• F1 males and females (Cohort 1C): after cleavage of the balanopreputial groove or vaginal opening. After euthanasia of Cohort 1A animals for animals with negative sexual development landmarks.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Moribund and prematurely euthanized pups were subjected to macroscopic post-mortem examination of the principal thoracic and abdominal organs.
- A complete macroscopic post-mortem examination was performed on all adult F1 animals. Gross necropsy included the same examinations performed for the P generation.

HISTOPATHOLOGY / ORGAN WEIGTHS
- The organs specified in Table 2 (under ''Any other information on materials and methods incl. tables'') were weighed wet as soon as possible after dissection.
-A microscopic examination was performed on:
• all tissues listed in Table 2 (under ''Any other information on materials and methods incl. tables'') from animals of the control and high-dose groups (groups 1 and 4) from cohort 1A. Microscopic examination in Cohort 1B was conducted if results from Cohort 1A are equivocal or in cases of suspected reproductive or endocrine toxicants.
• reproductive organs from from cohort 1A males with abnormalities at sperm analysis, to investigate possible causes.
• all macroscopic lesions of all F1 cohorts.

Statistics:

-Statistical analyses of body weight, food consumption and reproductive data was performed using Provantis or Reprotox softwares.
-PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01) according to the sequence in section 5.12.2 of the attached study report.
- Statistical analysis of Splenic Lymphocyte Immunophenotyping was performed using the SAS Enterprise Guide software according to the sequence in section 5.12.3 of the attached study report.
- Provantis software was used to perform the statistical analyses (according to the sequence in section 5.12.4 of the attached study report) of: Number of Primary Follicles/Corpora Lutea, Ano-genital Distance, Number of Nipples and Areolaes, Time of Preputial Separation/Vaginal Opening, Time to First Estrous After Vaginal Opening/Patency, Seminology, Hematology, Coagulation, Blood Biochemistry, Urinalysis, Thyroid Hormones, Post-implantation Loss, Sex Ratio, Live Birth, Viability and Lactation Indexes.

Reproductive indices:

• Post-implantation loss = [(Number of implantation sites - Number of live pups)/ Number of implantation sites] x 100,
• Mating index = (Number of mated animals / Number of paired animals) x 100,
• Fertility index = (Number of pregnant female partners / Number of mated pairs) x 100,
• Gestation index = (Number of females with live born pups / Number of pregnant females) x 100,

Offspring viability indices:

• Live birth index = (Number of live pups on Day 1 p.p. / Number of delivered pups) x 100,
• Viability index on Day 4 p.p. = (Number of surviving pups on Day 4 p.p. (before culling) / Number of delivered pups) x 100,
• Lactation index = [(Number of surviving pups on Day 21 p.p. / Number of surviving pups on Day 4 p.p. (after culling)] x 100,
• Normalized Ano-Genital Distance (Norm. AGD) = AGD / ³vBody weight.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs recorded in terminated as scheduled P Generation males (i.e. unscheduled deaths
excluded) are presented in table 3 under ''Any other information on results incl. tables'':
In males:
-At 15, 3 and 1 mg/kg bw/day, there were no test-item related adverse clinical signs.
Clinical signs recorded in terminated as scheduled P Generation females (i.e. unscheduled deaths excluded) are presented in table 4 under ''Any other information on results incl. tables''
In females:
-At 15 mg/kg bw/day, loud breathing and/or erected fur were recorded in one animal for 1 or 2 days. While a test-item relationship cannot be excluded, these findings were considered to be non-averse based on their incidence and duration.
-At 3 and 1 mg/kg bw/day, there were no test-item related adverse clinical signs.
Hypersalivation was observed in all treated groups. This finding is commonly observed after a gavage procedure and was considered to be test item treatment-related but not adverse.
Other findings observed with a low and/or similar incidence across groups (including controls) were those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

-One mid-dose male was prematurely euthanized on Study Day 99. Lateral recumbency, chromodacryorrhea, chromorhynorrhea, erected fur, cold to the touch and abdominal breathing were observed before euthanasia. At necropsy, no observations were noted. This death was not considered to be test item related.
-One control female was prematurely euthanized on Day 24 p.c. (Study Day 97) for delivery difficulties. Thin appearance, pallor (extremities and eyes), hunched posture, erected fur and abdominal breathing were recorded before death. At necropsy and microscopic examination, there was an imperforate vagina. In addition, 4 dead fetuses and 8 scars were recorded in uterus.
-One mid-dose female was prematurely euthanized on Day 23 p.c. (Study Day 96) due to delivery difficulties. No clinical signs were observed before death. At necropsy, 8 scars were present in the right horn, and 2 dead fetuses, 3 placentae and 4 scars were present in the left horn. No macroscopic findings were noted. These isolated reproduction troubles (difficulties to deliver) were considered to be unrelated to the test item administration.
-One high-dose female was prematurely euthanized on Study Day 70 on humane grounds. Soiled coat, pallor (extremities and eyes), hunched posture, erected fur and swelling (shoulder and forelimb) were observed before euthanasia. At necropsy, induration, red discoloration and gelatinous left forelimb muscle was noted. This was considered to be of accidental origin (trauma).
-A second high-dose female was prematurely euthanized on Day 14 p.c. (Study Day 89). Pallor (eyes and extremities), erected fur, half-closed eyes, decreased activity, thin fur and vaginal discharge were observed before death. At necropsy, this female had 10 alive concepti and 9 early resorptions. There was red content in vagina that correlated with hemorrhage in lumen. These isolated reproduction troubles (difficulties to deliver) were considered to be unrelated to the test item administration.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to controls and despite a few statistically significant differences, there were no dose-related effects on mean body weight or mean body weight change in P generation males or females during the premating period.
Despite a statistically significant difference (+5 vs. +9 g at 15 mg/kg/day on Days 4 to 7 p.c. p<0.05), there were no adverse effects on mean body weight or mean body weight change in P generation females during the pregnancy period.
There were no adverse effects on mean body weight or mean body weight change in P generation females during the lactation period either. At 15 mg/kg/day, the overall net body weight gain was higher than controls (+43 vs.+23 g, p<0.001). This difference was considered to be test-item treatment-related but non-adverse (mean body weight returning to control values during the post-partum period).
Therefore, any relationship to a test item treatment was considered to be unlikely. See tables 18 to 20 in the attached study report.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

When compared to controls and despite a statistically significant difference (+13% at 15 mg/kg bw/day in males on Days 113 to 120, p<0.05), there were no dose-related effects on mean food consumption in P generation males or females during the premating period.
There were no effects on females mean food consumption during the pregnancy period and lactation periods either.
Therefore, any relationship to a test item treatment was considered to be unlikely. See tables 21 to 23 in the attached study report.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on hematology or coagulation.
The few statistically significant differences (increased number of monocytes in high dose females and decreased APTT in high dose males) were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of Historical Control Data. See table 3 under ''Any other information on results incl. tables''

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on blood biochemistry in P generation animals.
The statistically significant differences in male potassium and cholesterol levels and female urea levels were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of Historical Control Data. See table 3 under ''Any other information on results incl. tables''

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no effects on TSH or T4 plasma levels

Urinalysis 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):

Test item-related changes were noted in the kidneys, liver and thyroid glands (See also table 3 under ''Any other information on results incl. tables'')
- Kidneys
Minimal to marked, dose-related changes were noted in the kidneys of males treated at
= 1 mg/kg bw/day, including tubular degeneration/regeneration, increased severity/incidence of accumulation of hyaline droplets, tubular casts, tubular basophilia and infiltrate of mononuclear inflammatory cells. The increased severity/incidence of tubular dilatation was seen only at
15 mg/kg bw/day.
The tubular degeneration/regeneration was characterized by the presence of exfoliated tubular cells with hypereosinophilic cytoplasm and/or shrunken boundaries (degeneration/necrosis) and enlarged tubules with clear cytoplasm and thickened basal membrane. The basophilic tubules were characterized by tubular cells with basophilic cytoplasm.
In females, a few occasional findings were noted but were seen with similar incidence and severity in control and treated females.
- Liver
Minimal to moderate hepatocellular hypertrophy in centrilobular zone was noted at
= 1 mg/kg bw/day, together with minimal degeneration/necrosis at 15 mg/kg bw/day in 2/24 males and 1/24 females and minimal to slight microvesicular vacuolation (mainly periportal) at = 1 mg/kg bw/day.
- Thyroid glands
Minimal follicular hypertrophy was noted in 3/24 males treated at 15 mg/kg bw/day. Although seen with low incidence and severity, this change was considered to be test item-related in view of the increased thyroid gland weight and biological evidence (see discussion under ''details on results(P0)'').
There were no test item-related findings in the reproductive tract of males and females.


The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
This included the minimal fibrosis in the parathyroid from 1/22 males treated at 15 mg/kg bw/day, minimal infiltrate of mononuclear inflammatory cells in the heart from 3/24 males and
1/22 females treated at 15 mg/kg bw/day, minimal vacuolation of cortical adrenal glands in
1/24 males and 1/22 females treated at 15 mg/kg bw/day and the hemorrhage/necrosis in the uterus of 1/22 females treated at 15 mg/kg bw/day.
These observations were seen with minimally increased incidence in some test item treated animals when compared to controls. In view of their low incidence and of their common occurrence in untreated rats, these changes were considered to be part of the spontaneous background.

ENUMERATION OF CORPORA LUTEA IN HE-STAINED SLIDES:
At enumeration of corpora lutea in the ovary of high-dose group and control group, there were no statistical differences with a mean number of corpora lutea of 7.10 (± 3.13) and 7.77 (± 4.08) per animal, respectively.

ENUMERATION OF THE NUMBER OF PRIMORDIAL FOLLICLES ON PCNA-STAINED SLIDES:
There were no statistical differences between the high-dose and control groups, with a mean number of 8.28 (± 4.68a) and 7.77 (± 5.95) primordial follicles per animal on PCNA-stained slides, respectively.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

See table 3 under ''Any other information on results incl. tables''

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects on motility, morphology and sperm cells count.

Reproductive performance:

no effects observed

Description (incidence and severity):

The following parameter were investigated: enumeration of corpora lutea in HE-stained slides , enumeration of the number of primordial follicles on PCNA-stained slides, pre-coital interval, mating index, pregnancies, fertility index, gestation length, gestation index, live litters born, live birth index, number of implantations, mean percent of post-implantation loss, number of pups delivered, litter size, mean number of live pups at birth, offspring bw and pup sex ratio.
See table 3 under ''Any other information on results incl. tables''

PATHOLOGY DISCUSSION:

There was no test item-related mortality in this study.
In the P generation, adverse degeneration/regeneration was noted in the renal tubules of males treated at 3 or 15 mg/kg bw/day together with hyaline droplets accumulation (that correlated with increased kidney weights in males treated at = 1 mg/kg bw/day), tubular basophilia, casts, dilatation and inflammatory cells infiltrate. The inflammatory/degenerative lesions correlated with macroscopic kidney enlargement, granular aspect and tan discoloration at necropsy. At 1 mg/kg bw/day, the renal findings were considered to be non-adverse in view of their morphology, their limited distribution and their low magnitude and incidence.
The hyaline droplet accumulation was probably consistent with Alpha-2-u-globulin although no immunohistochemical staining was performed and was highly suggestive of hyaline droplet nephropathy, a male rat specific change of no relevance to human.
In the liver, hepatocellular hypertrophy and periportal vacuolation were seen at = 1 mg/kg/day in males and females, that correlated with increased weight and accentuated lobular pattern at necropsy in males treated at 15 mg/kg/day. Isolated minimal hepatocellular degeneration/necrosis was also noted at 15 mg/kg/day (2/24 males and 1/24 females). Although there were no noteworthy clinical pathology correlates and in spite of the low magnitude of these changes (minimal), the hepatocellular degeneration/necrosis was considered to be adverse.
In the thyroid glands, minimal follicular hypertrophy correlated with increased weights in males treated at 15 mg/kg/day. In view of the low grade of this change and the association with liver hypertrophy, this was considered to be non-adverse.

Under the conditions of this study, the liver hypertrophy was considered rather an adaptive change (Greaves, 2012). This was consistent with microsomal enzyme induction that resulted in increased turnover of T4 (thyroxine) and secondary thyroid gland hypertrophy due to stimulation of the pituitary-thyroid-endocrine axis (Hall et al, 2012). This was partially confirmed by the TSH increase trend noted in P generation males and in spite no decrease in T4 was observed.
At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and control groups. There were no test item-related macroscopic or microscopic lesions in male or female reproductive organs. This was consistent with the estrous cycle, mating, fertility and delivery results.

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: based on adverse degeneration/necrosis in liver of males and females at 15 mg/kg/day

Key result

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

other: kidneys

Remarks on result:

other: based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 3 mg/kg/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

-Lactating pups (between Days 1 to 21 p.p., before weaning): At 15 mg/kg bw/day and when compared to controls, 1 to 2 litters had pups with dehydration, emaciated appearance, cold to the touch, generalized pallor and/or absence of milk in the stomach (up to 14 pups). A test-item relationship cannot be excluded but these findings were considered to be non-adverse based on absence of any effect on pup body weight and survival during the lactation period). At 3 and 1 mg/kg bw/day, there were no adverse test item-related findings. The remaining findings were recorded in controls, observed with low incidences and no dose relationship and/or are common findings in this species and strain maintained in the experimental conditions of this study. See also table 4 under ''Any other information on results incl. tables''

-Cohort 1A: There were episodes of hypersalivation in both sexes (from 1 mg/kg bw/day in males and from 3 mg/kg bw/day in females). This finding, commonly observed after a gavage procedure, was considered to be test-item related but non-adverse (See also table 5 under under ''Any other information on results incl. tables''). Other findings observed with a low and/or similar incidence across groups (including controls) were those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related. See also table 5 under under ''Any other information on results incl. tables''.

-Cohort 1B: There were episodes of hypersalivation in both sexes (from 15 mg/kg bw/day in males and from 3 mg/kg bw/day in females). This finding, commonly observed after a gavage procedure, was considered to be test-item related but non-adverse. Additionally, in females, instances of reflux at dosing, decreased activity and fur erected starting at 3 mg/kg bw/day were observed. Other findings observed with a low and/or similar incidence across the groups (including controls) are those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related. See also table 6 under under ''Any other information on results incl. tables''.

-Cohort 1C: There were instances of hypersalivation (after treatment), alopecia and/or thin fur, chromodacryorrhea and/or chromorhynorrhea, scabs (head, dorsal region and/or nose) and tail bent. Other findings observed with a low and/or similar incidence across groups (including controls) are those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related. See also table 7 under under ''Any other information on results incl. tables''.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

-Lactating pups (between Days 1 to 21 p.p., before weaning): There were no effects on the pre-culling Day 4 p.p. survival index, lactation index or sex-ratio at weaning. At 15 mg/kg bw/day, sex-ratio was lower than controls (41% vs. 50%, not statistically significant). However, a test item relationship was considered to be unlikely as the percentage was close to the lower values of the Historical Control Data. See also table 4 under ''Any other information on results incl. tables''.

-Cohort 1A: At 1 mg/kg bw/day group, one female was prematurely euthanized on Day 8 on humane grounds. Thin appearance, hunched posture, erected fur and swelling (abdomen) were observed before euthanasia. At necropsy, the ileum, cecum and colon were distended with gas. This death was unrelated to the test item administration since it was an isolated death seen in low-dose females at the beginning of the treatment period with undetermined cause of death. See also table 5 under under ''Any other information on results incl. tables''.

-Cohort 1B: In the control group one male was prematurely euthanized on Day 94 (Week 14). Chromorhynorrhea was observed on Days 48 to 50. Alopecia (forelimbs) was noted from Day 86, scabs (right forelimb) were noted from Day 92 and swelling (right forelimb) was noted on the day of euthanasia. At necropsy, a fracture of right forelimb was noted. This death was not considered to be test item related. A control females was found dead on Day 23 (Week 4). No clinical signs were noted. Perforation of the esophagus was noted at necropsy. A brown thick content was present in the lungs and thoracic cavity. An issue during the gavage procedure was considered to be the cause of death. In the low dose group one male was found dead on Day 40 (Week 6). No clinical signs were noted. No findings were noted at necropsy. The cause of death was not evident. Another low dose male was found dead on Day 52 (Week 8). Reflux at dosing was observed on Day 8. No findings were noted at necropsy. Finally, one high dose female was prematurely euthanized on Day 83 (Week 12). No clinical signs were noted. A fracture of left hindlimb was observed at necropsy. This death was not considered to be test item related. See also table 6 under under ''Any other information on results incl. tables''

-Cohort 1C: One high-dose male was prematurely euthanized after falling from the cage on Day 23 (Week 4). Before euthanasia, loud / abdominal breathing, ventral recumbency and decreased activity were observed. A series of cranial and brain traumas were observed at necropsy. This death was accidental and therefore not test-item related. There were no unscheduled deaths in Cohort 1C females. See also table 7 under under ''Any other information on results incl. tables''.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

-Lactating pups (between Days 1 to 21 p.p., before weaning): No effects (between Days 1 to 21 p.p., before weaning). See table 32 in the attached study report.

-Cohort 1A: There were no adverse effects on mean body weight or mean body weight changes.
When compared to controls, the few statistically significant differences were recorded without any dose relationship and with minimal amplitudes. Therefore, any test item relationship was considered to be unlikely. See table 37 in the attached study report.

-Cohort 1B: There were no effects on mean body weight or mean body weight change. The few statistically significant differences (recorded without a dose relationship and with minimal amplitude when compared to controls) were considered to be fortuitous. See table 45 in the attached study report.

-Cohort 1C: There were no effects on mean body weight or mean body weight changes. When compared to controls, the statistically significant difference was recorded without any dose relationship and with only minimal amplitudes. Therefore, any test item relationship was considered to be unlikely. See table 49 in the attached study report.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

-Cohort 1A: There were no adverse effects on mean food consumption. When compared to controls, the few statistically significant differences were recorded without any dose relationship and/or with minimal amplitudes. Therefore, any test item relationship was considered to be unlikely. See table 38 in the attached study report.

-Cohort 1B: In males, there was no evidence of an effect on mean food consumption. The increased observed at 15 mg/kg bw/day in week 6 (+9.3% vs. controls, p<0.01) was of mild amplitude and isolated; therefore a test-item relationship was considered to be unlikely. In females, from 3 mg/kg bw/day and mainly in weeks 12 to 14, there were dose-related increases in mean food consumption (up to +18.3% vs. controls in Week 14, p<0.001). A test item relationship cannot be excluded, but taking into account the amplitude of the difference, this finding was considered to be non-adverse (less than 20%). See table 46 in the attached study report.

-Cohort 1C: There were no effects on mean food consumption in Cohort 1C animals. See table 50 in the attached study report.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

-Cohort 1A: There were no test item-related effects. A statistically significant difference was recorded in low-dose females (RTC: 0.20 vs. 0.17 T/L in controls, p<0.05) without a similar finding in males or a dose relationship. See table 5 under ''Any other information on results incl. tables''.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

-Cohort 1A: There were no adverse effects on blood biochemistry in Cohort 1A animals.
The statistically significant differences in chloride, calcium, urea, cholesterol, triglycerides, alkaline phosphatase 2C, aspartate aminotransferase, total proteins and albumin were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of Historical Control Data. See table 5 under ''Any other information on results incl. tables''.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

-In Cohort 1A: There were no adverse effects on urinalysis parameters. The statistically significant differences in pH were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of Historical Control Data (see table 5 under ''Any other information on results incl. tables'')

Sexual maturation:

no effects observed

Description (incidence and severity):

In males (all cohorts combined), the mean age at which balanopreputial separation occurred and the mean body weight on the day of occurrence are detailed in table 8 under ''Any other information on results incl. tables''.
There were no effects on mean age at balanopreputial separation or on mean body weight on the day of occurrence.
In females (all cohorts combined), the mean age at which vaginal opening occurred and the mean body weight on the day of occurrence are detailed in table 8 under ''Any other information on results incl. tables''.
There were no effects on mean age at vaginal opening or on mean body weight on the day of occurrence.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

On Day 1 p.p and when compared to controls, there were no effects on mean anogenital distance (AGD) or normalized AGD. See also table 4 under under ''Any other information on results incl. tables''.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no nipples or areolae in male pups examined on Day 12 p.p.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

-Cohort 1A: Increased absolute and/or relative-to-body kidney weights were noted in males at = 1 mg/kg bw/day (up to +43% in relative-to-body weights for males treated at 15 mg/kg bw/day; p<0.01). This correlated with enlargement, tan discoloration and irregular color at gross observation and microscopic degenerative/inflammatory lesions in males. The differences were considered to be related to the test item administration in males. In females, a relationship was considered to be unlikely as the differences were seen only in relative-to-body weights and had no microscopic correlates. Increased absolute and/or relative-to-body liver weights were noted in males and females treated at = 3 mg/kg bw/day (up to +45% in relative-to-body weights for females treated at 15 mg/kg bw/day; p<0.01). This correlated mainly with microscopic hepatocellular hypertrophy in males and females. Increased absolute and/or relative-to-body pituitary gland weights were noted in females treated at = 1 mg/kg bw/day. In view of the poor dose-relationship and absence of microscopic correlates, a relationship with the test item administration was considered to be equivocal. Increased absolute and relative-to-body adrenal gland weights were noted in males treated at = 3 mg/kg bw/day (up to +28% in relative-to-body weights for males treated at 15 mg/kg bw/day; p<0.01). In the absence of microscopic correlates, a relationship with the test item administration was considered to be equivocal. The few other organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings. This included the increased relative-to-body spleen and testes weights in males treated at 15 mg/kg bw/day (respectively +13%; p<0.05 and +10%; p < 0.01). The differences were considered to be unrelated to the test item administration as they had no microscopic correlates and were of low magnitude. See also table 5 under ''Any other information on results incl. tables''.

-Cohort 1B: There were no test item-related organ weight differences. The few organ weight changes were not considered to be test item-related because they were of insufficient magnitude and/or were not dose-related.

-No organ weights were measured in cohort 1C.

-In pups non selected to conform cohorts 1A, 1B or 1C, there were no test item-related organ weight differences. The few organ weight changes were not considered to be test item-related because they were of insufficient magnitude and/or were not dose-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

-There were no test-item related findings at necropsy of pups found dead during weaning.
At 15 mg/kg bw/day, one litter had 4 found dead pups with no milk in the stomach. An absence of maternal care may have led to these deaths. However, finding is common in rats of this strain and was limited to a single litter. Therefore a test item relationship was considered to be unlikely. See also table 4 under under ''Any other information on results incl. tables''

-No test item related macroscopic post-mortem observations in pups euthanized after weaning.
The few findings were observed with low incidences, no dose level relationship and/or are common findings in this species and strain maintained in the experimental conditions of this study.

- Cohort 1A:There were test item-related findings in kidneys of males. Tan discoloration, enlargement and/or irregular color were noted in the kidneys from some males and females treated at = 1 mg/kg bw/day. These changes were considered to be test item-related in males and correlated with increased weights and microscopic degenerative/inflammatory lesions in this sex. In females, the tan discoloration in one female at 1 mg/kg bw/day and in one other female treated at 15 mg/kg bw/day had no microscopic correlates and thus was unrelated to the test item administration. The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age. This included the dilated pelvis in kidneys from 4/20 males and 1/20 females treated at 15 mg/kg bw/day, and the tan or white discoloration in the adrenal glands from 2/20 females treated at 15 mg/kg bw/day considered to be incidental. See also table 5 under ''Any other information on results incl. tables''.

-Cohort 1B: There were no test item-related findings. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age. This included the abnormal teeth growth in 1/18 males treated at 1 mg/kg bw/day, the dilated renal pelvis or ovarian cyst in 2/20 females treated at 3 mg/kg bw/day and in 2/19 females treated at 15 mg/kg bw/day.

-Cohort 1C: There were no test item-related findings. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age. This included the sternum deformation in 1/20 females treated at 15 mg/kg bw/day.

-Non Selected Pups: There were no test item-related gross changes. The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

-COHORT 1A: Test item-related changes were noted in the kidneys (males only treated at = 1 mg/kg bw/day) and in liver (males and females treated at = 1 mg/kg bw/day).
. Kidneys
Minimal to marked, dose-related changes were noted in the kidneys of males treated at
= 1 mg/kg bw/day, including tubular degeneration/regeneration, accumulation of hyaline droplets, increased severity/incidence of basophilic tubules, hyaline casts, infiltrate of mononuclear inflammatory cells and tubular/pelvic dilatation. No test item-related changes were noted in females.
. Liver
Minimal to slight hepatocellular hypertrophy in centrilobular zones was noted at = 1 mg/kg bw/day, together with minimal degeneration/necrosis in 1/20 males treated at 15 mg/kg bw/day and microvesicular periportal vacuolation in occasional females treated at = 1 mg/kg bw/day.
See also table 5 under ''Any other information on results incl. tables''
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
This included the minimal mononuclear cell infiltrate in the skeletal muscle from 1/20 males treated at 15 mg/kg bw/day, the vacuolation in the cortex of adrenal glands of 1/20 males treated at 15 mg/kg bw/day, the crust and ulcer in the skin of 1/20 males treated at 3 mg/kg bw/day and in 1/20 males treated at 15 mg/kg bw/day, the ventricular dilatation in the brain of 2/20 females treated at 15 mg/kg bw/day, a malformation (retinal disorganization) in the eyes from 1/20 females treated at 15 mg/kg bw/day, the retrobulbar hemorrhage in one male and one female treated at 1 mg/kg bw/day, one male and one female treated at 3 mg/kg bw/day and in one female treated at 15 mg/kg bw/day, the minimal tingible macrophages in the thymus of 1/20 females treated at 15 mg/kg bw/day, the minimal intrasinusoidal erythrocytes in the mandibular or mesenteric lymph node from 2/20 females treated at 15 mg/kg bw/day, minimal alveolar macrophages in the lungs of 7/20 females treated at 15 mg/kg bw/day, the minimal infiltrate of mononuclear cell infiltrate in the heart of 2/20 females treated at 15 mg/kg bw/day, the minimal tubular dilatation in the kidneys from 1/20 females treated at 15 mg/kg bw/day, the minimal to slight pelvic dilatation in the kidneys from 2/20 females treated at 15 mg/kg bw/day, the slight forestomach hyperplasia from 1/20 females treated at 15 mg/kg bw/day and the slight fracture in the sternum of 1/20 females treated at
3 mg/kg bw/day, that can be seen in untreated rats.
There were no test item-related changes in the male and female reproductive organs.
ENUMERATION OF CORPORA LUTEA IN HE-STAINED SLIDES
At enumeration of corpora lutea in the ovary of high-dose and control groups, there were no statistically significant differences, with a mean number of corpora lutea of 14.35(± 5.97) and 14.75 (± 6.95) per animal, respectively.
ENUMERATION OF THE NUMBER OF PRIMORDIAL FOLLICLES ON PCNA-STAINED SLIDES
At enumeration of primordial follicles in the ovary of high-dose and control groups, there were no statistically significant differences, with a mean number of primordial follicles of 17.77
(± 5.91) and 13.47 (± 6.57) per animal, respectively.

-COHORT 1B, Microscopic examination of macroscopic lesions: There were test item-related findings in kidneys of males treated at = 1 mg/kg bw/day, including accumulation of hyaline droplets, basophilic tubules, hyaline casts, infiltrate of mononuclear inflammatory cells and tubular dilatation. The few other microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.

-COHORT 1C, There were no test item-related findings.: The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls. This included the fracture of sternum seen in one female treated at 15 mg/kg bw/day.

Other effects:

no effects observed

Description (incidence and severity):

-There were no effects on the mean time (number of days) to first estrous after vaginal opening in Cohort 1A females. There were no effects on mean estrous cycle parameters in Cohort 1A females. When compared to controls, a statistically significant difference (see table 5 under ''Any other information on results incl. tables'') was recorded without any dose relationship and with no impact on associated estrous stages. Therefore, any test item relationship was considered to be unlikely. There were no effects on mean estrous cycle parameters in Cohort 1A females. When compared to controls, a statistically significant difference (2.0 vs. 2.4 mean number of days of cycles at 3 mg/kg bw/day, p<0.05) was recorded without any dose relationship and with no impact on associated estrous stages. Therefore, any test item relationship was considered to be unlikely.
- In Cohort 1A animals, when compared to controls, there were no effects on mean plasma TSH or T4 levels.
-No effects on sperm parameters (motility, morphology and sperm cells count) of Cohort 1A animals.

Developmental immunotoxicity:

effects observed, treatment-related

Description (incidence and severity):

LYMPHOCYTE SUBTYPINGIN COHORT 1A:
-Relative counts: A statistically significant difference was observed in terms of NK cell relative counts between the control and high dose group in females. A difference was also observed for groups 3 and 4 versus the male control group. However, while an increase was observed in treated females (+71%) a decrease was observed in males (-35% and -31% in group 3 and 4, respectively) when compared to their respective control groups. In males only, a statistically significant difference was observed in terms of B cell relative counts between group 4 and the control group (+14%).
Absolute counts: In terms of absolute counts, a statistically significant difference was observed in NK cells in female and male animals, but in opposite directions, as for NK cell relative counts.
A dose-related higher mean value for NK cells was observed in females (+40%, +49% and +74% in groups 2, 3 and 4, respectively, compared to control females), while a dose-related lower mean value was observed in males (-21%, -34% and -39% in group 2, 3 and 4, respectively, compared to control males). A test item-related effect on NK cells was found, however, as both relative and absolute counts were found to be increased in females, while they were both decreased in males.
See also table 5 under ''Any other information on results incl. tables''

PATHOLOGY DISCUSSION:
In cohort 1A, similar lesions were noted: No test item-related mortality was observed. Increased kidney weights were noted in males treated at = 1 mg/kg/day. This correlated with enlargement, irregular color and tan discoloration at gross observation and microscopic degenerative/inflammatory lesions in these males (considered as adverse at = 1 mg/kg/day). Increased liver weights were noted in males and females treated at = 3 mg/kg/day. This correlated with microscopic non-adverse hypertrophy and/or vacuolation. Hepatocellular degeneration/necrosis was recorded in males at 15 mg/kg/day and was considered to be adverse, although seen in only 1/20 males at minimal severity. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.
No test item-related changes were noted in the thyroid glands of cohort 1A animals.
At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and control groups. This was consistent with the time to first estrous and estrous cycle results (see main report).
In cohort 1B, no test item-related mortality, no organ weight differences and no gross findings were noted. In the selected organs submitted for microscopic examination, there were test item-related findings in kidneys of males treated at = 1 mg/kg/day, including accumulation of hyaline droplets, basophilic tubules, hyaline casts, infiltrate of mononuclear inflammatory cells and tubular dilatation, similar to P generation and Cohort 1A.
In cohort 1C, no test item-related mortality and no gross findings were noted.
There were no test item-related organ weight differences or gross findings in non-selected pups.

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: on adverse degeneration/necrosis in liver of males

Key result

Dose descriptor:

LOAEL

Generation:

F1 (cohort 1A)

Effect level:

< 1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 1 mg/kg/day

Remarks on result:

other: based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 1 mg/kg/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 mg/kg bw/day (nominal)

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Key result

Reproductive effects observed:

no

Chemical Analysis of the Dose Formulations

The dose formulations prepared once in the P-premating, P-gestation and P-lactation periods and five times over the F1-postweaning period were all found to be within the acceptance criteria (± 15% of the nominal concentrations). No test item was found in control dose formulations. Dose formulation homogeneity was also demonstrated, as the Relative Standard Deviation (RSD) was found to be ≤ 10% for the group 2 formulation tested in the P-premating period.

The results were as follows:

Group	Theoretical Concentration (mg/mL)	Sampling Location	Measured Concentration (mg/mL)	Bias (%)	RSD (%)
1	0	Middles	Nd	-	-
2	0.2	Top	0.190	-4.9	2.4
			0.200	0.1
		Middle	0.195	-2.6
			0.204	1.9
		Bottom	0.200	-0.2
			0.199	-0.3
		Mean	0.198	-1.0	-
3	0.6	Middle	0.610	1.7	-
4	3	Middle	2.96	-1.3	-

 

 

Table 3: Results P (males until termination after mating, females until weaning F1)

Dose (mg/kg bw/day)		0		1		3		15
Endpoint		M	F	M	F	M	F	M	F
Mortality		0/24	1/24	0/24	0/25	1/24	1/24*	0/24	2/24**
Relevant clinical signs		NTRE
·       Hypersalivation (before treatment)					2/24	1/23		4/24	2/22
·       Hypersalivation (after treatment				3/24	5/24	5/23		8/24	7/22
·       Loud breathing									1/22
·       Fur erected									1/22
Body weight		NTRE
Body weight gain		NTRE
Food consumption		NTRE
Haematology
·       M (G/L)									↑29%  (Mean values within HCR
·       APTT (sec)								↓11% (mean value within HCR)
Clinical chemistry
·       K+ (mmol/L)				↑6%		↑7%		↑4%
·       UREA (mmol/L)									↑18% (mean value within HCR)
·       CHOL (mmol/L)								↑28% (mean value within HCR)
Urinalysis		NTRE
Thyroid hormones -TSH		NTRE
-T4		NTRE
Organ weights	Kidneys			↑9% abs. / ↑10% rel.	↑4% abs. (NTR)	↑16% abs. / ↑16% rel.	↑5% abs. (NTR)	↑25% abs. / ↑29% rel.	↑6% abs. / ↑11% rel. (NTR).
	Liver					↑10% rel.		↑26% abs. / ↑31% rel.	↑14% abs. / ↑20% rel.
	Thyroid glands							↑11% asb.
	Adrenal glands								↑15% abs. / ↑22% rel. (NTR).
Macroscopy	-Liver: accentuated lobular pattern							14/24
	-Kidney: Enlarged, granular and/or discolorated			1/24		1/23		4/24
Microscopy	Kidneys; degeneration/regeneration; tubule			4/24	NA	14/23	NA	24/24
	Kidneys; accumulation; hyaline droplets	1/24		24/24	NA	23/23	NA	24/24
	Kidneys; basophilic tubules	5/24	1/23	20/24	NA	20/23	NA	23/24	2/22
	Kidneys; tubular casts	4/24	1/23	8/24	NA	14/23	NA	21/24	2/22
	Kidneys; infiltrate of mononuclear cells	8/24	5/23	20/24	NA	13/23	NA	18/24	4/22
	Kidneys; dilatation; tubule	1/24		1/24	NA	2/23	NA	6/24	1/22
	Liver; degeneration/necrosis							2/24	1/22
	Liver, hepatocellular hypertrophy			2/24	3/24	16/23	6/23	24/24	22/22
	Liver; vacuolation			5/24		8/23		10/24	1/22
	Thyroid gland; follicular hypertrophy				NA		NA	3/24
Reproductive data
Sperm parameters		NTRE
Estrous circle		NTRE
Enumeration of corpora lutea in HE-stained slides		NTRE
Enumeration of the number of primordial follicles on PCNA-stained slides		NTRE
Pre-coital interval		NTRE
Mating index (% mated)		95.8		100.0		100.0		95.8
Pregnancies		20		22		22		22
Fertility index		87.0		91.7		91.7		95.7
Gestation length		NTRE
Gestation index (%)		95.0		100		95.5		90.9
Live litters born		19		22		21		20
Live birth index (%)		99.0		100.0		99.7		100.0
Implantations		14.9		15.1		14.7		14.4
Mean percent of post-implantation loss (%)		7.8		8.7		10.0		9.7
Number of pups delivered (total)		262		304		276		262
Litter size (before cull)		13.8		13.8		13.1		13.1
Mean number of live pups at birth (before cull)		13.6		13.8		13.1		13.1
Offspring bw (g, on days 1 and 4 p.p before culling)		NTRE
Sex ratio (% M before cull)		49		52		49		43

NTRE= no treatment related effects

M = monocytes

APTT = Activated Partial Thromboplastin Time.

↑/↓= significantly increased/decreased

% compared to controls

* due to delivery difficulties on day 23

** On day 14 p.c. At necropsy, this female had 10 alive concepti and 9 early resorptions. There was red content in vagina that correlated with hemorrhage in lumen.

 

Table 4. Results F1 (between Days 1 to 21 p.p., before weaning)

Dose (mg/kg bw/day)		0		1		3		15
Endpoint		M	F	M	F	M	F	M	F
Mortality
·       Number of litters with found dead pups necropsied, Nb. (%)		4 (21)		2 (9)		2 (10)		1 (5)
·       Number of found dead pups necropsied (%)		4 (1.5)		2 (0.7)		4 (1.4)		6       (2.3)
-        pups with absence of milk in stomach, P/L		0/0		0/0		0/0		4/1
-        pups with autolysis, P/L		4/4		1/1		2/2		2/1
Viability index (%) (day 1-4 , before cull)		99.0		98.6		100.0		97.9
Sex ratio (day 21 % males)		50		50		47		41
Lactation index (%)(Days 4-21 p.p, before cull)		100.0		99.5		98.6		97.4
Body weight day 21		NTRE
Body weight gain		NTRE
Relevant clinical signs
·       - dehydratation, P/L								10/2
·       - cold to the touch, P/L		14/2						14/1
·       - emaciated appearance, P/L								9/2
·       - pallor (generalized), P/L								8/1
·       - absence of milk in stomach, P/L								1/1
AGD (corrected for cube root of BW, mm)		1.82	0.93	1.90	0.99	1.83	0.91	1.80	0.91
Nipple number on day 12	NTRE (0 nipples in males)
Macroscopy	NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

AGD: Anogenital Distance.

Nb.: Number;

P/L: Nb. of pups / Nb. of litters.

HCR: Historical control range

 

 

 

Table 5: Results F1 until termination COHORT 1A

Dose (mg/kg bw/day)		0		1		3		15
Endpoint		M	F	M	F	M	F	M	F
Mortality					1/20*
Relevant clinical signs: Hypersalivation (after treatment)				1/20		1/20	1/20	3/20	3/20
Body weight day 21		NTRE
Body weight gain		NTRE
Food consumption		NTRE
Time to first estrous		NTRE
Mean Estrous Cycle Parameters
·       Mean number of days of diestrus (± SD)		NTRE
·       Mean number of days of proestrus (± SD)		NTRE
·       Mean number of days of estrus (± SD)		NTRE
·       Mean number of days of metestrus (± SD)		NTRE
·       Mean number of cycles (± SD)			2.5		2.4		↓2.0		2.5
·       Mean duration of cycles (days) (± SD)		NTRE
·       Mean percent of females cycling normally (%)		NTRE
Haematology and coagulation:
·       RTC					↓17%
Clinical Biochemistry
·       Cl-						↑2%	↑2%	↑2%	↑2%
·       Ca++									↑4%
·       UREA (mmol/L)								↑26% (mean value within HCR)
·       CHOL (mmol/L)									↑56% (mean value within HCR)
·       TRIG (mmol/L)									↑500% (mean value within HCR)
·       ALP2c (U/L)					↓21%		↓23%		↓28% (mean value within HCR)
·       ASAT (U/L)									↓23% (mean value within HCR)
·       PROT (g/L)							↑7%	↑6%	↑15% (mean value within HCR)
·       ALB (g/L)							↑5%	↑6%	↑14% (mean value within HCR)
Urinalysis
·       pH						↓6%		↓8%
Splenic lymphocytes						NK cells↓ (34% abs./35% rel.)	NK cells ↑(49% abs.)	NK cells↓ (39% abs./ 31% rel.) B cells ↑(14% rel.)	NK cells ↑(74% abs / 71% rel.)
Thyroid hormones -TSH -T4		NTRE
Organ weights
·       Kidneys				↑16% abs. / ↑16% rel.		↑23% abs. / ↑24% rel.	↑7% rel.	↑39% abs. / ↑43% rel.	↑16% rel.
·       Liver						↑7% rel.	↑11% rel.	↑18% abs. / ↑23% rel.	↑36% abs. / ↑45% rel.
·       Pituitary gland					↑26% abs. / ↑23% rel.		↑24% abs. / ↑24% rel.		↑19% rel.
·       Adrenal glands						↑15% abs. / ↑15% rel.		↑23% abs. / ↑28% rel.
Sperm analysis		NTRE
Macroscopy	-Kidney: Enlarged, granular and/or discolorated			2/20	1/20	2/20		3/20	1/20
Microscopy	Kidneys; degeneration/regeneration; tubule			4/20	NA	16/20	NA	12/20
	Kidneys; accumulation; hyaline droplets			20/20	NA	20/20	NA	20/20
	Kidneys; basophilic tubules	7/20	2/20	15/20	NA	20/20	NA	20/20	3/20
	Kidneys; tubular casts	2/20		4/20	NA	7/20	NA	12/20
	Kidneys; infiltrate of mononuclear cells	4/20	2/20	5/20	NA	6/20	NA	4/20	4/20
	Kidneys; dilatation; tubule	1/20		1/20	NA	2/23	NA	14/20	1/20
	Kidneys; dilatation; pelvis	4/20		5/20	NA	3/20	NA	14/20	2/20
	Liver; degeneration/necrosis							1/20
	Liver, hepatocellular hypertrophy			7/20	1/19	13/20	10/20	20/20	20/20
	Liver; vacuolation				1/19		2/20		1/20
Enumeration of corpora lutea in HE-stained slides		NTRE
Enumeration of the number of primordial follicles on PCNA-stained slides		NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

* prematurely euthanized on Day 8 on humane grounds

RTC: Reticulocyte Count.

Cl-: chloride; Ca⁺⁺: calcium; UREA: urea; CHOL: cholesterol; TRIG: triglycerides;  ALP2c: alkaline phosphatase 2C; ASAT: aspartate aminotransferase; PROT: total proteins; ALB: albumin.

HCR: Historical control reange

 

 

Table 6: Results F1 until termination COHORT 1B

Dose (mg/kg bw/day)	0		1		3		15
Endpoint	M	F	M	F	M	F	M	F
Mortality	1/20^(a)	1/20^(b)	2/20^(c)					1/20 ^(d)
Relevant clinical signs
-   Hypersalivation (after treatment)						2/20	1/20
-   Reflux at dosing						2/20		3/19
-   Decreased activity						1/20
-   Fur erected						1/20
-   Hunched posture						1/20
Body weight	NTRE
Food consumption	NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

^(a) Prematurely euthanized on Day 94. Chromorhynorrhea was observed on Days 48 to 50. Alopecia (forelimbs) was noted from Day 86, scabs (right forelimb) were noted from Day 92 and swelling (right forelimb) was noted on the day of euthanasia. At necropsy, a fracture of right forelimb was noted.

^(b) Found dead on day 23 . No clinical signs were noted. No findings were noted at necropsy. Perforation of the esophagus was noted at necropsy. A brown thick content was present in the lungs and thoracic cavity. An issue during the gavage procedure was considered to be the cause of death

^(c) Found dead on Days 40 and 52. In one male, no clinical signs were noted and no findings were noted at necropsy, hence the cause of death was not evident. The other male was found dead on Day 52, reflux at dosing was observed on Day 8 and no findings were noted at necropsy.

^(d) Prematurely euthanized on Day 83. No clinical signs were noted. A fracture of left hindlimb was observed at necropsy. This death was not considered to be test item related

 

 

Table 7: Results F1 until termination COHORT 1C

Dose (mg/kg bw/day)	0		1		3		15
Endpoint	M	F	M	F	M	F	M	F
Mortality							1/20*
Relevant clinical signs: hypersalivation (after treatment)					2/20	1/20	3/19	1/20
Body weight	NTRE
Food consumption	NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

* euthanized after falling from the cage on Day 23.

 

Table 8: Mean Age of Balanopreputial Separation and mean age at Vaginal Opening with Mean Body Weight on the Day of Occurrence in Males and females from all cohorts (1A, 1B and 1C)

Dose level (mg/kg bw/day)	0	1	3	15
Number of males	60	60	60	59
Number of positive responses	59	59	59	57
Number of negative responses	1	1	1	2
Mean age (days ± SD)	48.7 ± 2.67	49.2 ± 5.47	48.5 ± 3.22	48.7 ± 3.19
Mean body weight (g ± SD)	292.1 ± 32.74	296.4 ± 33.27	289.9 ± 34.53	294.5 ± 29.66
Number of females	60	59	60	60
Number of positive responses	60	59	58	59
Number of negative responses	0	0	2	1
Mean age (days ± SD)	33.3 ± 1.89	33.5 ± 2.40	34.9 ± 11.89	32.7 ± 1.66
Mean body weight (g ± SD)	125.0 ± 14.74	129.9 ± 17.70	131.2 ± 26.02	121.3 ± 13.20

No statistically significant differences vs. controls.

 

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be:
• 1 mg/kg/day in parental (P) generation (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 3 mg/kg/day and on adverse degeneration/necrosis in liver of males and females at 15 mg/kg/day).
• Lower than 1 mg/kg/day in Cohort 1A (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 1 mg/kg/day and on adverse degeneration/necrosis in liver of males at 15 mg/kg/day).

Executive summary:

The objective of this study was to provide general information concerning reproductive and developmental effects of the test item, AZDN, that may occur as a result of pre- and post-natal exposure. Systemic toxicity in pregnant and lactating females and young and adult offspring were also evaluated.

In the assay, sexually mature male and female rats [parental (P) generation] were exposed to the test item from 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were selected and assigned to cohorts of animals for reproductive/developmental toxicity testing (cohorts 1A, 1B and 1C, without extension to mate the Cohort 1B animals to produce an F2 generation).

The test item was also administered to the F1 offspring from weaning to adulthood or euthanasia. Clinical observations and pathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development of the offspring.

Methods

Treatment

P generation and F1 lactating offspring:

Three groups of 24 male and 24 female Sprague-Dawley rats (P generation) received the test item, AZDN (batch No. B3J1117), at 1, 3 or 15 mg/kg/day, daily for 10 weeks prior to pairing, during pairing and gestation, and through lactation until weaning of the F1 pups [Day 22
post-partum (p.p.)]. The test item was administered orally (gavage, 5 mL/kg). A control group of 24 males and 24 females received the vehicle alone (corn oil), under the same experimental conditions.

F1 generations:

The test item was administered orally, by gavage (5 mL/kg) to groups of 20 rats/sex (Cohorts 1A, 1B and 1C) receiving 0 (vehicle alone: corn oil), 1, 3 or 15 mg/kg/day. The treatment schedules were the following:

• Cohort 1A: daily from weaning (Day 22 p.) until euthanasia (on Days 90 to 94 p.p.),


• Cohort 1B: daily from weaning (Day 22p.) for at least 10 weeks before euthanasia (after necropsy of cohort 1A, pending no alteration of estrous cycle or sperm parameters, and no macroscopic findings after examination of the reproductive organs ),


• Cohort 1C: daily from weaning (Day 22 p.) until euthanasia (after completion of sexual development testing).

Examination of Parental and F1 generations

Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals.

P generation males and females were paired until mated or until 14 days had elapsed.

P generation females were allowed to deliver normally and rear their progeny. Pregnancy and litter parameters were recorded.

During lactation, the F1 pups were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. In F1, the size of each litter was adjusted on Day 4 p.p. to obtain ten pups per litter. Pup physical development was assessed at designated time-points.

Examination of Cohorts

Cohort 1A: animals were selected on Day 22 p.p. for assessment of general toxicity and effects on their reproductive system. Estrous cycle stages were determined daily for all females after the onset of vaginal patency, until the first cornified smear was recorded (estrus), and for 14 days before the end of the treatment period.

Cohort 1B: animals were selected on Day 22 p.p. to potentially obtain additional histopathology data.

Cohort 1C: additional animals were selected on Day 22 p.p. for assessment of sexual maturity in F1 animals.

Terminal examinations

A macroscopic post-mortem examination was performed on all P and F1 animals (including F1 pups culled on Day 4 p.p. and F1 pups not selected on Day 22 p.p.). This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues, and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated and organs were weighed wet as soon as possible after dissection. A microscopic examination was performed on all macroscopic lesions and tissues (complete list) from animals in all groups or in the control and high-dose groups.

P generation and Cohort 1A: the first 10 surviving animals/sex/group were fasted (food only) for an overnight period of at least 14 hours prior to blood sampling (for hematology, coagulation and blood biochemistry) and urine collection (for urinalysis) at termination. A series of sperm cell evaluations was performed on surviving males from all groups: motility, morphology, sperm cell head count in testicular/epididymal tissues.

Cohort 1A: splenic lymphocyte subpopulation analysis was performed on 10 surviving animals/sex/group.

Thyroid hormone levels (P generation, F1 culled pups and Cohort 1A animals): prior to blood sampling the animals were not fasted (except for animals from which samples were collected for hematology, blood biochemistry and urinalysis). Blood samples were taken on Day 4 p.p. (F1 pups) for measurement of thyroid hormone (T4) levels. Blood samples were also taken on Day 22 p.p. (F1 pups not selected for Cohorts) and at termination [the first ten surviving males/group and the first ten lactating females/group (P generation animals)] for measurement of thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) levels.

Results

The concentrations in the dose formulations prepared for treatment of the P Generation and Cohorts were all found to be within an acceptable range of variations (measured concentrations at ± 15% of the theoretical concentrations). No test item was found in the control dose formulation.

P generation

Mortality: there were no test-item related deaths.

Clinical signs: there were no test-item related adverse clinical signs

Mean body weight, mean body weight change and mean food consumption: there were no test item-related adverse effects.

Estrous cycle, mating, fertility and delivery: there were no effects

Pre-weaning F1 pups:

Mortality: there were no effects on the repartition of deaths in F1 lactating pups and no test item-related findings at macroscopic post-mortem examination of found dead pups.

F1 pup survival during lactation: there were no effects on the F1 pup viability and lactation indexes.

Clinical observations during lactation: there were no adverse findings.

Body weight and body weight change: there were no effects.

Pup development during lactation: there were no effects on the Day 1 p.p. mean anogenital distance (AGD) or normalized AGD in males or females. There were no nipples or areolae in male pups examined on Day 12 p.p.

Cohorts 1A and 1B

Mortality: there were no test item-related deaths.

Clinical signs: there were no adverse clinical signs.

Body weight, body weight change and food consumption: there were no adverse effects.

Time to first estrous and estrous cycles: there were no effects in Cohort 1A females.

Sexual development:

Males: there were no effects on the mean age at balanopreputial separation.

Females: there were no effects on the mean age at vaginal opening,

Laboratory investigations:

Hematology, coagulation, blood chemistry and urinalysis in P-generation and/or Cohort 1A animals: there were no adverse findings.

Thyroid hormones: there were no effects on TSH or T4 plasma levels.

Sperm analysis: there were no effects on sperm parameters (motility, morphology, sperm/testicular numerations and daily sperm production rate) in P-generation or Cohort 1A males.

Lymphocyte subtyping: there were no adverse findings in Cohort 1A animals.

 

 

Pathology:

P generation: No test item-related mortality was observed. Test item-related increased kidney weights were noted in males treated at ≥ 1 mg/kg/day. This correlated with enlargement, granular aspect and tan discoloration at gross observation and microscopic degenerative/inflammatory lesions in males (considered as adverse at ≥ 3 mg/kg/day).  Increased liver weights were noted in males treated at ≥ 3 mg/kg/day and in females treated at 15 mg/kg/day. This correlated with increased incidence of accentuated lobular pattern in males at ≥ 3 mg/kg/day and microscopic non-adverse hypertrophy and vacuolation. Hepatocellular degeneration/necrosis was recorded at 15 mg/kg/day and was considered to be adverse, although seen in only 2/20 males and 1/24 females at minimal severity. In addition, non-adverse minimal follicular hypertrophy was seen in the thyroid glands from males treated at 15 mg/kg/day and correlated with increased weights. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.

Cohort 1A: No test item-related mortality was observed. Increased kidney weights were noted in males treated at ≥ 1 mg/kg/day. This correlated with enlargement, irregular color and tan discoloration at gross observation and microscopic degenerative/inflammatory lesions in these males (considered as adverse at ≥ 1 mg/kg/day). Increased liver weights were noted in males and females treated at ≥ 3 mg/kg/day. This correlated with microscopic non-adverse hypertrophy and/or vacuolation. Hepatocellular degeneration/necrosis was recorded in males at 15 mg/kg/day and was considered to be adverse, although seen in only 1/20 males at minimal severity. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.

Cohort 1B: No test item-related mortality and no organ weight differences or gross findings were noted. Test item-related degenerative/inflammatory findings were noted in the kidneys of males treated at ≥ 1 mg/kg/day.

Cohort 1C: There was no test item-related mortality. There were no test item-related gross findings.

Non selected pups from PND22: There were no test item-related organ weight differences or gross findings.

Conclusion

The test item, AZDN, was administered daily by oral gavage, at dose level of 0, 1, 3 or 15 mg/kg/day, to sexually mature male and female rats [parental (P) generation] from 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity.

Systemic toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be:

• 1mg/kg/day in parental (P) generation (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at ≥ 3 mg/kg/day and on adverse degeneration/necrosis in liver of males and females at 15 mg/kg/day).


• Lower than 1 mg/kg/day in Cohort 1A (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at ≥ 1mg/kg/day and on adverse degeneration/necrosis in liver of males at 15 mg/kg/day).

Reproductive/developmental toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 15 mg/kg/day in the Sprague-Dawley rats, based on the absence of adverse effects at this dose level.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 February 2021 to 04 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD®(SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (F0) approximately 10 wks (males) and 11 wks (females)
- Weight at study initiation: (F0) Males: 341-436 g (mean 379 g); Females: 231-307 g (mean 265 g)
- Housing: F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust. Toward the end of gestation and during lactation, autoclaved wood shavings were provided to females and their litter as nesting material. Each cage contained rat hut (until delivery, then wooden log) and nylabone for environmental enrichment.
- Diet: SSNIFF rat/mouse pelleted maintenance diet ad libitum, batch No. 81971534 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water: ad libitum from bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: males were acclimated to the study conditions for 7 days before treatment, females were acclimated to the study conditions for 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.

CONTAMINANT ANALYSIS
No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS (TARGET)
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: about 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod: 12 h light/12 h dark

IN-LIFE DATES: From: 02 March 2021 To: 04 May 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Test item suspensions were prepared dissolving the substance in the vehicle according to Study No. 48855 VAS (Final report for stability testing) describing the preparation procedure for a range of concentrations [0.1 mg/mL - 10 mg/mL] covering the lowest and highest used in this study. The frequency of preparation of dosing solutions was based on the results of the Study No. 48855 VAS and the vehicle expiry.

ADMINISTRATION OF DOSING SOLUTIONS
The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight. The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the dosing procedure. The control dose formulation was stirred just before administration and the test item dose formulations for at least 30 minutes before administration. The formulations were maintained under continuous magnetic stirring throughout the dosing procedure.

VEHICLE
- Justification for use and choice of vehicle: the test item demonstrated to be soluble and stable in the vehicle which was also well-tolerated by animals.
- Concentration in vehicle: 1, 4 and mg/mL for the low-, mid- and high-dose groups, respectively.
- Amount of vehicle: 5 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurred
- Proof of pregnancy: vaginal plug or sperm in vaginal lavage referred to as Day 0 p.c.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of test item concentrations in dose formulations was performed by High Performance Liquid Chromatography with UV detection (HPLC/UV) according to a validated analytical method (Study No. 48855 VAS (Final report) prior to dose formulation analysis. Analyses were performed on three occasions, during weeks 1, 3 and 7. A sample was taken from control and test item dose formulations and analyzed using the validated method. The acceptance criterion was a measured concentration within a range of ± 15% of the nominal concentration.

Duration of treatment / exposure:

The dose formulations were administered daily according to the following schedule:

F0 males:
- for 2 weeks before mating,
- during the mating period (until evidence of mating),
- until euthanasia (i.e. after an overall 8-week treatment period),

F0 females:
- for 2 weeks before mating,
- during the mating period (until evidence of mating),
- during gestation,
- during lactation until Day 21 p.p. inclusive,
- until euthanasia for females with no delivery.

Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

daily

Dose / conc.:

5 mg/kg bw/day

Remarks:

low-dose group

Dose / conc.:

20 mg/kg bw/day

Remarks:

mid-dose group

Dose / conc.:

50 mg/kg bw/day

Remarks:

high-dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE
The dose levels were selected based on the results of previous studies:
- In a 13-week toxicity study (Covance reference No. 8266542) performed in the same species, the test item was administered daily by gavage to males and females at dose levels of 0.5, 2 or 10 mg/kg bw/day for at least 90 days. The administration was well tolerated. There was no evidence of neurotoxicity. There were slight changes in some blood clinical chemistry and urine parameters at all dose levels. The liver and kidney were identified as target organs based on increased weights (at all dose levels in males) and microscopic findings of hepatocyte hypertrophy (at all dose levels in males and at 2 mg/kg bw/day in females) and focal nephropathy/tubular basophilia and hyaline droplets in males at all dose levels. The liver hypertrophy was considered as an adaptive change commonly associated with metabolic burden and the changes in the kidney were considered specific to the male rat as confirmed by immuno-histochemical staining. Therefore, the nature of these histopathological changes was considered to be of no toxicological relevance and the No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg bw/day,
- In an OECD 422 study (Reference No. R-95-007) performed in the same species, the test item was administered daily by gavage to males and females at dose levels of 2, 10 or 50 mg/kg bw/day until Day 43 for males and Day 4 post-partum for females. The test item had no effects on the copulation index or fertility index at 50 mg/kg bw/day or lower doses. The test item did not affect the length of the gestation period or delivery index in maternal animals, either. No abnormal parturition was observed. Abnormal nursing behaviour was noted in 3 animals from the 50 mg/kg bw/day group. The findings in offspring showed no effects on the parturition index, live pup delivery index, overall delivery index, or the sex ratio and body weight on Day 0 at all dose levels. In the 50 mg/kg bw/day group, the offspring viability index and body weight on Day 4 of lactation showed a tendency to decrease. No offspring from the test item groups showed any morphological anomaly.
Based on these results, the NOEL for reproductive and developmental toxicity was 50 mg/kg bw/day in males and 10 mg/kg bw/day in females and in pups.
- In an OECD 414 study (Covance reference No. 8266543) performed in the same species, the test item was administered daily by gavage to pregnant females at dose levels of 1, 5 or 20 mg/kg bw/day from Day 6 p.c. to Day 19 p.c. Administration of the test item induced maternal toxicity from 5 mg/kg bw/day upwards based on transient effects of reduced body weight gain and reduced mean food intake while no effects on foetal development were observed. At 1 mg/kg bw/day, no adverse effects were observed in both parental and foetal development. There was no evidence of embryotoxicity at any dose level tested. The maternal no observed adverse-effect level (NOAEL) was 1 mg/kg bw/day based on a significant decrease in maternal body weight gain and food consumption. The embryo-foetal NOAEL was 20 mg/kg bw/day.
Therefore, 50 mg/kg bw/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing approximately a 2.5- to 4-fold interval (i.e. 5 and 20 mg/kg bw/day).

ANIMAL ASSIGNMENT / FASTING
- Rationale for animal assignment: selected according to body weight and clinical condition and allocated to groups (by sex) according to stratified procedure based on body weight, so that the average body weight of each group was similar. In addition, only females with regular estrous were allocated to the groups (regularity of estrous cycles was confirmed 1 day before the beginning of the treatment period).
- Fasting period before blood sampling for clinical biochemistry: no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality and morbidity were checked once a day before the treatment period and at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day from the start of the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c.(post-coitum) (and on the day of euthanasia for females which did not deliver) and on Days 1, 4, 8, 13, 17 and 21 p.p. Animals euthanized prematurely were weighed before euthanasia.

FOOD CONSUMPTION: Yes
- Measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8, 8-13, 13-17 and 17-21 p.p.
During the mating period, food consumption was not measured for males or females.

OTHER:
- Parturition: females were allowed to litter normally and rear their progeny until Day 21 p.p. Any sign of a difficult or prolonged parturition was recorded. The morning when parturition was completed was designated Day 1 p.p. The length of gestation was calculated.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
• during the 2 weeks of the pre-treatment period (including for the 2 supplementary females per group, whose data are not presented in the study report),
• from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
• on the day of final sacrifice before euthanasia.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); whenever necessary, partial adjustment (for example five males and three females) was permitted. No cross-fostering was performed. Pups not selected were discarded without further examination.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring during the lactation period starting at Day 1 p.p.: total litter size and sex of each pup were recorded on Day 1 p.p. Any gross external malformations in pups were noted. The litters were observed daily in order to note the number of live, dead and cannibalized pups.
Clinical signs and abnormal behaviour were recorded on a daily basis. The body weight was recorded on Days 1, 4, 8, 13, 17 and 21 p.p. The body weight of one selected pup/sex/litter euthanized on Day 21 p.p. was recorded before euthanasia. The anogenital distance (AGD) normalized to the cube root of body weight was recorded on Day 1 p.p. The AGD was determined as follows: AGD / body weight^(1/3). On Day 12 p.p. the number of nipples and of areolae in male pups was determined.

GROSS EXAMINATION OF DEAD PUPS:
yes, found dead pups were submitted to a detailed external examination (including orifices and buccal cavity), after euthanasia when applicable. Particular attention was paid to the external genital organs and to whether the pup had been fed (e.g. presence of milk in the stomach) when possible. Then, they were discarded without any further examination.

Postmortem examinations (parental animals):

SACRIFICE
Prematurely euthanized or found dead animals: a prematurely euthanized female of the high-dose group was euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination. A found dead female and the prematurely euthanized female both of the high-dose group were submitted for a complete macroscopic post mortem examination.
On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- males: after the end of the gestation period (at least 7 weeks of treatment in total),
- females: on Day 22 p.p.
The following F0 females were euthanized by the same way without overnight fasting:
- females which did not deliver: on Days 25-26 p.c. (after a body weight recording to check for a possible un-noticed delivery).

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all F0 animals including females that died during the study and euthanized prematurely. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 22 p.p. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY / ORGAN WEIGHTS
The body weight of each F0 animal euthanized as scheduled (after the end of the gestation period for males or on Day 22 p.p. for females) was recorded before euthanasia. For these animals, the organs specified in the Table 1 (refer to "Any other information on materials and methods incl. tables") were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids (when applicable). The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.
The tissues of F0 animals specified in Table 1 were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in Modified Davidson's fixative). A microscopic examination was performed on:
- all tissues listed in Table 1 from all animals of the control and high dose groups (groups 1 and 4) euthanized as scheduled (at the end of the mating period for males or on Day 22 p.p. for females),
- all macroscopic lesions of all groups.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Based on findings observed at microscopic examination, liver from all parent animals (males and females) were examined.

Postmortem examinations (offspring):

SACRIFICE
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on Day 4 p.p. when blood sampled), followed by exsanguination when the thyroids were sampled:
- pups not selected on Day 4 p.p.: on Day 4 p.p.
- surviving pups: on Day 21 p.p.

GROSS NECROPSY
Pups euthanized on Day 21 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination, or after sampling of thyroids with parathyroids for the selected pups.

HISTOPATHOLOGY
Thyroids with parathyroids of the selected pup/sex/litter euthanized on Day 21 p.p. were preserved in 10% buffered formalin.

Statistics:

HORMONES, ANOGENITAL DISTANCE, NIPPLES/AREOLAE, LIVE BIRTH INDEX(ES), SEX-RATIO AND POST-IMPLANTATION LOSS
Data were compared by analysis of variance in case of a negative result a one-way ANOVA was performed and tests for normality (Shapiro-Wilk test) and for homoscedasticity (Levene test) were performed. If data were not normally or homogenously distributed, a logarithmic or a rank transformation was performed. The transformed data were analyzed again by one-way ANOVA, tests for normality (Shapiro-Wilk test) and for homoscedasticity (Levene test) were performed. If data were normally distributed and homogeneous (p > 0.05) an F-test from previous ANOVA is run. In case a significant difference (p = 0.05) was observed, pairwise group comparisons were performed (Dunnett test). If dat a were not normally distributed or homogeneous, the overall group differences were analyzed using ranked values (Kruskal-Wallis test). In case a significant difference (p = 0.05) was observed, pairwise group comparisons using ranked values were performed (Dunn test).

ORGAN WEIGHT (LEVEL OF SIGNIFICANCE OF 0.05 OR 0.01)
Data were analyzed for normality (Kolmogorov test). If all groups showed a normal distribution, the homogeneity of variance was tested (Bartlett test / F-test). If variance was homogenous, group
differences were analyzed by one-way analysis of variance (if > 2 groups) and in case of a significant result (p < 0.05 or 0.01) a Dunnett test (if > 2 groups) or t-test (if 2 groups) were performed. In case of non-normal distribution in at least one group or a non-homogeneous variance, group differences were analyzed by Kruskal-Wallis test (if > 2 groups). If a significant result (p < 0.05 or 0.01) was achieved, a Dunn test (if > 2 groups) or a Wilcoxon test (if 2 groups) were performed.

Reproductive indices:

pre-implantation loss (%): (number of corpora lutea - number of implantation sites) / (number of corpora lutea) x 100
post-implantation loss (%): (number of implantation sites - number of live pups) / (number of implantation sites) x 100
Mating index (%) = (numbers of mated animals / number of paired animals) x 100
Fertility index (%) = (number of pregnant female partners / number of mated pairs) x 100
Gestation index (%) = (number of females with live born pups / number of pregnant females) x 100

Offspring viability indices:

Live birth index (%) = (number of live offspring on Day 1 p.p. / number of delivered pups) x 100
Viability index on Day 4 p.p. (%) = (number of surviving pups on Day 4 p.p. before culling / number of delivered pups) x 100
Lactation index on Day 13 p.p. (%) = (number of surviving pups on Day 13 / number of surviving pups on Day 4 p.p. after culling) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 50 mg/kg bw/day, 1/8 surviving females showed signs of poor clinical condition (emaciated appearance, piloerection, round back, and/or hypoactivity) between Days 5 and 16 of the premating period. These clinical signs were considered to be test item-related and adverse.
Ptyalism was noted in 1/10, 8/10 and 10/10 males at 5, 20 and 50 mg/kg bw/day, respectively. This finding was also observed in surviving females during the premating (i.e. 5/8 females
at 50 mg/kg bw/day), gestation (i.e. 1/9 females and 5/8 females at 20 and 50 mg/kg bw/day, respectively) and lactation (i.e. 1/9 females and 8/8 females at 20 and 50 mg/kg bw/day, respectively) periods.
Ptyalism is a finding commonly observed after gavage administration with oily dose formulations.
All other clinical signs recorded during the study (cutaneous observations (areas of hair loss, cutaneous lesions, scabs, desquamation), chromodacryorrhea and reflux at dosing) were considered to be unrelated to the test item as they were present both in control and test item-related animals, and/or were reported sporadically in only a few animals. In addition, these findings are common observations under these experimental conditions for this strain of animal.

Mortality:

mortality observed, treatment-related

Description (incidence):

Refer also to Table 2 under "Any other information on results incl. tables".

There were no unscheduled deaths in males.

At 50 mg/kg/day
- A female was prematurely euthanized on Day 8. Signs of poor clinical condition (emaciated appearance, piloerection, round back, hypoactivity and/or half-closed eyes from Day 5) were observed prior to death.
- Another female was found dead on Day 8. Signs of poor clinical condition (emaciated appearance, piloerection, round back, cold to the touch, hypoactivity, ptyalism and/or half-closed eyes from Day 5) were observed prior to death.
At microscopic examination of the liver, slight centrilobular hepatocellular hypertrophy was observed in both females. This finding was also observed at the end of the treatment period and was related to test item administration.
The cause of premature euthanasia or death of the two females on Day 8 was not evident at macroscopic or microscopic examination. At necropsy, the spleen and thymus were reduced in size, which correlated with decreased lymphoid cellularity and was suggestive of stress (Everds et al., 2013). Nevertheless, a relationship with the test item administration could not be excluded for the death/poor clinical status of these two females.

At 20 mg/kg bw/day
One female was euthanized on Day 25 p.c. due to absence of delivery. No clinical signs were observed during the gestation period and no necropsy findings were observed at macroscopic post-mortem examination. This female was not pregnant. As this pregnancy status was not dose related, it was not considered to be test item-related.
At microscopic examination of the liver, slight centrilobular hepatocellular hypertrophy was observed. This finding was also observed at the end of the treatment period and was related to test item administration.

At 5 mg/kg bw/day
Two females were euthanized on Days 26 and 25 p.c., respectively, due to absence of delivery. No clinical signs were observed during the gestation period. No necropsy findings were observed at the macroscopic post-mortem examination of one female, whereas the uterine horns of the other female were dilated with a translucent content. These females were not pregnant. As this pregnancy status was not dose related, it was not considered to be test item-related.

At 0 mg/kg bw/day
One female was euthanized on Day 25 p.c. due to absence of delivery. No clinical signs were observed during the gestation period and no necropsy findings were observed at macroscopic post-mortem examination. This female was not pregnant. This scheduled death was unrelated to the treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 3, Table 4 and Table 5 under "Any other information on results incl. tables".

PRE-MATING (MALES AND FEMALES) AND POST-MATING (MALES) PERIODS
In males at 50 mg/kg bw/day and when compared with controls, there was a low mean body weight gain over the first week of the treatment period (not statistically significant) that correlated with low mean food consumption. This effect was considered to be test item-related but not adverse as it was of low magnitude, transient and did not impact the mean body weight or the overall mean body weight change.
In females, from 5 mg/kg bw/day and when compared with controls, a dose-related mean body weight loss was recorded over the first week of the treatment period, reaching statistical significance at 50 mg/kg bw/day. Mean body weight gain returned towards control values in the second week of the premating period. This finding correlated with a low dose-related mean food consumption and resulted in an overall dose-related, statistically significant, low mean body weight gain at all dose levels throughout the premating period. This effect was therefore considered to be test item-related and non-adverse as it was limited to the first week of the premating period.

GESTATION PERIOD
From 5 mg/kg bw/day and when compared with controls, there were low mean body weight changes on Days 0 to 7 p.c. (+29 g with p<0.05, +29 g with p<0.05 and +19 g with p<0.001 vs. +40 g in controls at 5, 20 and 50 mg/kg bw/day, respectively). This trend was also noted on Days 14
to 20 p.c. at 50 mg/kg bw/day (+73 g vs. +93 g in controls; p<0.05). These effects contributed in an overall low mean body weight gain over the gestation period and a low mean body weight on Day 20 p.c. at all dose levels (-8% with p<0.05, -7% and -10% with p<0.01 vs. controls at 5, 20 and 50 mg/kg bw/day, respectively). These changes were attributed to the test item but not considered to be adverse in the absence of any test item-related effects on mean food consumption and as mean body weights were only slightly to minimally affected.

LACTATION PERIOD
From 5 mg/kg bw/day and when compared with controls, a low mean boy weight was observed from delivery until Day 17 p.p., whereas a high mean body weight gain was noted in females
at 50 mg/kg bw/day for Days 1-4 p.p. (+30 g vs. +9 g in controls; p<0.01). Although these differences were poorly dose-related, they were attributed to the test item but were not considered to be adverse due to their direction, and as they were transient and/or of minimal magnitude.
Other statistically significant differences from controls were recorded in the mean body weight gain (i.e. for Days 1-21 p.p. at 20 and 50 mg/kg bw/day). They were not considered to be of toxicological importance due to their direction and as they were mainly due to the mean body weight loss recorded in controls at the end of the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 6, Table 7 and Table 8 under "Any other information on results incl. tables".

PRE-MATING PERIOD
In males, at 50 mg/kg bw/day and when compared with controls, low mean food consumption
(-10%; p<0.05) was noted on Study Days 1-8. This correlated with a low mean body weight gain. This effect was therefore considered to be test item-related but not adverse as it was of low magnitude, transient and did not impact the mean body weight or overall mean body weight change.
In females, from 5 mg/kg bw/day and when compared with controls, a dose-related lower mean food consumption was recorded on Study Days 1-8 (down to 33% at 50 mg/kg bw/day; p<0.001). This correlated with a dose-related body weight loss. This dose-related effect was therefore considered to be test item-related but non adverse as mean values returned towards control values thereafter.

GESTATION PERIOD
There were no effects on mean food consumption during the gestation period.

LACTATION PERIOD
There were no test item-related effects on mean food consumption during the lactation period. At 50 and 20 mg/kg bw/day, when compared with controls, there was a low mean food consumption on Study Days 4-8 p.p. and/or Days 8-13 p.p. As these differences were sporadic and/or did not resulted in low mean body weight gains, they were considered to be incidental.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer also to Table 9 under "Any other information on results incl. tables".

There were no test item-related effects on mean T4 or TSH levels in F0 males. In F0 males and when compared with controls or HCD, variations in T4 and TSH concentrations were not dose-related, were not consistent with each other, were of small magnitude and were within the range of the HCD. They were therefore considered to be fortuitous.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 12 and Table 13 under "Any other information on results incl. tables".

Substance-related microscopic observations were observed in the kidneys and liver. No test item related microscopic findings were observed in the male and female reproductive organs.

In the kidneys examined because of macroscopic findings, test item related accumulation of hyaline droplets was observed in males from 5 mg/kg bw/day. This finding was characterized by the accumulation of dense eosinophilic droplets in proximal tubular epithelium. It was observed with higher severity (moderate or marked) than that usually observed spontaneously, and was associated with tubular degeneration/necrosis, dilatation, casts, and interstitial inflammation. Unilateral or bilateral pelvic dilatation was also noted in many of the kidneys examined, and although this is a common spontaneous finding in control rats, a relationship with the test item could not be excluded. The hyaline droplets, occasionally seen in untreated male rats, are consistent with a2u globulin and are known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2012). The prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover. The renal changes were considered to be adverse in the current study in view of the tubular degenerative findings. Although humans excrete proteins of a similar nature, they are found in only trace amounts and therefore this finding is considered to be non-relevant for humans.

In the liver, substance-related minimal to marked centrilobular hypertrophy of hepatocytes was observed in males from 5 mg/kg bw/day and in females from 20 mg/kg bw/day. In addition, at 50 mg/kg bw/day, minimal or slight periportal hepatocellular vacuolation was noted with increased incidence in males compared to controls, and hepatocellular degeneration/necrosis characterized by coagulative necrosis and/or hydropic degeneration was present in a few males and one female. The hypertrophic changes were considered to be adverse in both sexes at 50 mg/kg bw/day in view of their severity and association with degenerative changes.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Refer also to Table 14 under "Any other information on results incl. tables".

There were no test item-related effects on estrous cycle during the pre-mating period.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 15 and Table 16 under "Any other information on results incl. tables".

There were no effects on mating or fertility indices and all groups had similar pre-coital time intervals (number of days taken to mate).
In all test item-treated groups and when compared with controls, there were no effects on the mean duration of gestation.
At 50 mg/kg bw/day and when compared with controls, a low mean number of pups was delivered (13.5 vs. 15.8) as a consequence of low mean numbers of corpora lutea (15.6 vs. 17.6) and implantation sites (15.3 vs. 17.2) and a high mean post-implantation loss (11.1% vs. 7.5%). Therefore a test item relationship could not be excluded. These findings were not considered to be adverse based on the low magnitude and as they were within or close to the upper limit of the HCD. At 20 and 5 mg/kg bw/day, there were no effects on reproductive or litter data.

Key result

Dose descriptor:

LOAEL

Remarks:

parental systemic toxicity

Effect level:

5 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

maternal systemic toxicity

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 17 under "Any other information on results incl. tables".

At 50 mg/kg bw/day and when compared with controls, one litter of the high-dose group had eight pups with thinning of hair over Days 13-21 p.p., this clinical sign was considered to be non-adverse as it was not associated with any other findings.
At 20 and 5 mg/kg bw/day, there were no test item treatment-related findings.

Other findings (thin appearance, generalized pallor, dehydration, hematoma, increased size of neck, wound, scab and/or short tail) recorded in pups during the lactation period were not
dose-related, were of isolated occurrence and/or were common observations in this species and strain of rats maintained under the experimental condition of this study.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Refer also to Table 18 and Table 19 under "Any other information on results incl. tables".

The high number of cannibalized pups at 20 mg/kg bw/day was mainly due to one litter (eight cannibalized pups on Day 2 p.p. without clinical signs prior to death). As this was isolated and not dose-related, the incidence was considered to be fortuitous.

There were no effects on pup viability. The low viability index at 20 mg/kg bw/day was mainly due to the litter of the high-dose group where 8 pups were cannibalized on Day 2 p.p. As this was isolated and not dose-related, this was considered to be incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 20 and Table 21 under "Any other information on results incl. tables".

At 50 mg/kg bw/day, in both sexes and when compared with controls, there were lower mean pup body weights after culling (down to -16% on Day 8 p.p., p<0.01) along with lower mean body weight gain (statistically significant up to p<0.001 for Days 4-8 p.p.). Taking into account the magnitude of the effects, they were considered to be adverse.
At 20 and 5 mg/kg bw/day, there were no effects on mean pup body weight or body weight change.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Refer also to Table 22 under "Any other information on results incl. tables".

There were no test item-related effects on mean T4 or TSH levels in Day 21 p.p. pups.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Refer also to Table 23 and Table 24 under "Any other information on results incl. tables".

At 50 mg/kg bw/day in males and when compared with controls, the lower mean anogenital distance was considered to be related to the lower mean body weight, as confirmed by the AGD/BW cube root ratio. As values remained within the range of the HCD, they were not considered to be test item-related.
At 50 mg/kg bw/day in females and at 20 and 5 mg/kg bw/day in both sexes, there were no effects on mean anogenital distance (or normalized AGD).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Refer also to Table 25 under "Any other information on results incl. tables".

There were no test item-related effects on areolae or nipples in male pups on Day 12 p.p.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No relevant external abnormalities were observed at any dose level.

Other effects:

no effects observed

Description (incidence and severity):

Refer also to Table 26 under "Any other information on results incl. tables".

There were no effects on sex ratio (mean percentage of males) at any dose level.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Chemical Analysis of the Dosage Forms: Concentration

The test item concentrations in the administered dose formulations analyzed in Weeks 1, 3 and 7 remained within an acceptable range of variations (-10.8% to +14.3%) when compared to the nominal values (± 15% of the nominal concentrations).

No test item was observed in the control dose formulations.

 

 

Table 2 - Mortality in Females

Dose level (mg/kg bw/day)	0	5	20	50
Humane reasons	0	0	0	1
No delivery	1(npg)	2 (npg)	1 (npg)	0
Found dead	0	0	0	1
Dead litter	0	0	0	0
Total surviving animals	9/10	8/10	9/10	8/10

npg: not pregnant.

 

 

Table 3 - Mean Body Weight and Mean Body Weight Change (g) (pre-mating and post-mating periods)

Sex	Male				Female
Dose level (mg/kg bw/day)	0	5	20	50	0	5	20	50
Mean body weight (g)
. Day 1	377	383	380	374	264	260	264	269
. Day 8	417	422	424	404	272	253	257	254
. Day 15	445	451	452	431	280	260 (-7)	265 (-5)	270 (-4)
. Day 22	464	472	477	455	/	/	/	/
. Day 29	485	495	500	475	/	/	/	/
. Day 36	514	515	521	495	/	/	/	/
. Day 43	526	527	534	507	/	/	/	/
. Day 50	547	547 (0)	554 (+1)	530 (-3)	/	/	/	/
Mean body weight change (g)
. Days 1 to 8	+41	+38	+44	+29	+8	-6	-7	-14**
. Days 8 to 15	+27	+29	+28	+27	+8	+7	+7	+7
. Days 15 to 22	+19	+21	+25	+24	/	/	/	/
. Days 22 to 29	+22	+23	+23	+20	/	/	/	/
. Days 29 to 36	+28	+20	+21	+20	/	/	/	/
. Days 36 to 43	+13	+12	+13	+12	/	/	/	/
. Days 43 to 50	+20	+20	+20	+23
. Days 1 to 15	+68	+68	+72	+57	+15	0*	0*	+1*
. Days 1 to 50	+170	+164	+174	+156	/	/	/	/

(): in brackets, percentage (%) difference vs. controls.

/: not applicable.

Statistical significance: *: p<0.05, **: p<0.01.

 

 

Table 4 - Mean Body Weight and Mean Body Weight Change (g) (gestation period)

Female
Dose level (mg/kg bw/day)	0	5	20	50
Mean body weight (g)
. Day 0 p.c.	276	266 (-4)	270 (-2)	277 (0)
. Day 7 p.c.	316	295 (-7)	299 (-5)	296 (-6)
. Day 14 p.c.	349	325 (-7)	324 (-7)	324 (-7)
. Day 20 p.c.	442	407* (-8)	412 (-7)	397** (-10)
Mean body weight change (g)
. Days 0 to 7 p.c.	+40	+29*	+29*	+19^#
. Days 7 to 14 p.c.	+33	+30	+25	+28
. Days 14 to 20 p.c.	+93	+81	+88	+73*
. Days 0 to 20 p.c.	+166	+141*	+142*	+121^#

(): in brackets, percentage (%) difference vs. controls.

Statistical significance: *: p<0.05, **: p<0.01, ^#: p<0.001.

 

 

Table 5 - Mean Body Weight and Mean Body Weight Change (g) (lactation period)

Female
Dose level (mg/kg bw/day)	0	5	20	50
Mean body weight (g)
. Day 1 p.p.	336	309 (-8)	309* (-8)	291** (-13)
. Day 4 p.p.	345	319 (-8)	317 (-8)	321 (-7)
. Day 8 p.p.	358	331 (-8)	333 (-7)	325* (-9)
. Day 13 p.p.	374	349 (-7)	351 (-6)	346 (-7)
. Day 17 p.p.	369	358 (-3)	362 (-2)	359 (-3)
. Day 21 p.p.	362	349 (-4)	354 (-2)	359 (-1)
Mean body weight change(g)
. Days 1 to 4 p.p.	+9	+10	+8	+30**
. Days 4 to 8 p.p.	+13	+12	+15	+4
. Days 8 to 13 p.p.	+16	+18	+19	+20
. Days 13 to 17 p.p.	-6	+9	+11	+13
. Days 17 to 21 p.p.	-6	-9	-7	0
. Days 1 to 21 p.p.	+26	+39	+45*	+68^#

(): in brackets, percentage (%) difference vs. controls.

Statistical significance: *: p<0.05, **: p<0.01, ^#: p<0.001.

 

 

Table 6 - Mean Food Consumption (g/animal/day) (pre-mating period)

Sex	Male				Female
Dose level (mg/kg bw/day)	0	5	20	50	0	5	20	50
. Days 1 to 8	30	30 (0)	30 (0)	27* (-10)	18	15* (-17)	14** (-22)	12^# (-33)
. Days 8 to 15	27	32 (+19)	28 (+4)	29 (+7)	17	17 (0)	16 (-6)	18 (+6)

(): in brackets, percentage (%) difference vs. controls.

Statistical significance: *: p<0.05; **: p<0.01 and ^#: p<0.001.

 

 

Table 7 - Mean Food Consumption (g/animal/day) (gestation period)

Female
Dose level (mg/kg bw/day)	0	5	20	50
. Days 0 to 7 p.c.	21	20	21	22
. Days 7 to 14 p.c.	21	19	20	21
. Days 14 to 20 p.c.	26	25	26	25

 

 

Table 8 - Mean Food Consumption (g/animal/day) (lactation period)

Female
Dose level (mg/kg bw/day)	0	5	20	50
. Days 1 to 4 p.p.	36	34 (-6)	32 (-11)	36 (0)
. Days 4 to 8 p.p.	50	50 (0)	47 (-6)	43 (-14)
. Days 8 to 13 p.p.	66	64 (-3)	60* (-9)	59* (-11)
. Days 13 to 17 p.p.	71	78 (+10)	71 (0)	72 (+1)
. Days 17 to 21 p.p.	83	86 (+4)	80 (-4)	77 (-7)

(): in brackets, percentage (%) difference vs. controls.

Statistical significance: *: p<0.05.

 

 

Table 9 - Mean T4 (ng/mL) and TSH Concentrations (pg/mL) (F0 animals)

Dose level (mg/kg bw/day)	0	5	20	50	HCD [min.; max.]
T4
. F0 males at termination	39.94±5.51	38.86±5.30 (-2.7)	41.90±3.05 (+4.9)	35.73±4.16 (-10.6)	[25.8; 54.2]
TSH
. F0 males at termination	2046±1006	1213±615 (-40.7)	1427±737 (-30.3)	1589±738 (-22.3)	[302; 4687]

HCD: Historical Control Data [n = 30 males and n = 85 to 102 litters on Day 13 and/or 21 p.p. (referring to ‘female’ litter data in HCD)].

(): in brackets, percentage (%) difference vs. controls.

No statistical significance vs. controls.

 

 

Table 10 - Mean Organ Weight Changes (% Changes from Controls) at Main Necropsy

	Male Dosage (mg/kg bw/day)			Female Dosage (mg/kg bw/day)
	5	20	50	5	20	50
Liver
Absolute weight	+14	+40**	+83**	+2	+13**	+39**
Ratio relative to body weight	+14	+38**	+90**	+13	+26**	+53**
Kidneys
Absolute weight	+20	+34**	+35**	0	-1	+2
Ratio relative to body weight	+19	+33**	+40**	+11	+10	+13

Statistically significant from controls: **: p<0.01

The significance concerned the organ weights values and not the percentages.

 

 

Table 11 - Group Incidences of Macroscopic Observations at Main Necropsy

Finding	Male Dosage (mg/kg bw/day)				Female Dosage (mg/kg bw/day)
	0	5	20	50	0	5	20	50
Kidneys
Enlarged	-	2	4	6	-	-	-	-
Dilated pelvis	-	4	3	3	1	-	2	-
Tan discoloration	-	-	1	1	-	-	-	-
Liver
Enlarged	-	-	2	9	-	-	-	-
Accentuated lobular pattern	-	-	-	1	-	-	-	-

-: finding not recorded.

 

 

Table 12 - Group Incidences (with Severities) of Microscopic Observations in the Kidneys in Males at Main Necropsy

Finding	Male Dosage (mg/kg bw/day)
	0	5	20	50
Kidneys (No. Examined)	1	4	5	6
Accumulation; hyaline droplets
Minimal (grade 1)	1	-	-	-
Moderate (grade 3)	-	2	3	1
Marked (grade 4)	-	2	2	5
Degeneration/necrosis; tubule
Minimal (grade 1)	-	2	2	2
Slight (grade 2)	-	-	1	2
Moderate (grade 3)	-	2	2	2
Cast; tubular
Minimal (grade 1)	-	1	2	5
Slight (grade 2)	-	1	1	1
Dilatation; tubule
Minimal (grade 1)	-	1	2	1
Slight (grade 2)	-	1	1	1
Moderate (grade 3)	-	-	-	2
Inflammation
Minimal (grade 1)	-	1	3	4
Slight (grade 2)	-	1	1	1
Moderate (grade 3)	-	-	-	1
Dilatation; pelvis
Minimal (grade 1)	-	-	1	1
Slight (grade 2)	-	3	1	1
Moderate (grade 3)	-	-	1	1

-: finding not recorded.

 

 

Table 13 - Group Incidences (with Severities) of Microscopic Observations in the Liver at Main Necropsy

Finding	Male Dosage (mg/kg bw/day)				Female Dosage (mg/kg bw/day)
	0	5	20	50	0	5	20	50
Liver (No. Examined)	10	10	10	10	10	10	10	8
Hypertrophy; hepatocyte
Minimal (grade 1)	-	9	-	-	-	-	2	-
Slight (grade 2)	-	1	3	-	-	-	7	1
Moderate (grade 3)	-	-	7	5	-	-	1	6
Marked (grade 4)	-	-	-	5	-	-	-	1
Degeneration/necrosis; hepatocyte
Minimal (grade 1)	-	-	-	2	-	-	-	1
Slight (grade 2)	-	-	-	2	-	-	-	-
Vacuolation; periportal
Minimal (grade 1)	1	1	3	3	-	-	-	-
Slight (grade 2)	-	1	-	4	-	-	-	-

-: finding not recorded. NA: not applicable.

 

 

Table 14 - Mean Estrous Cycles

Female
Dose level (mg/kg bw/day)	0	5	20	50
. Number of cycles	2.9	2.7	2.9	3.0
. Cycle length (days)	4.0	4.2	4.0	4.0
. Rats cycling normally.	10/10 (100%)	9/10 (90%)	8/10 (80%)	7/8 (88%)
. Number of females with all stages	9 (90%)	9 (90%)	10 (100%)	7 (88%)

 

 

Table 15 - Summary of Mating and Fertility Data

Dose level (mg/kg bw/day)	0	5	20	50
Number of animals paired (M+F)	10+10	10+10	10+10	8+8 ^(a)
Number of males mated	10	10	10	8
Number of females mated	10	10	10	8
Mean number of days taken to mate ^(b)	2.5	2.4	2.1	2.4
Mating index (%)	100	100	100	100
Number of pregnant females	9	8	9	8
Fertility index (%)	90.0	80.0	90.0	100.0

M: male; F: female.

^(a): two females were prematurely euthanized or found dead before mating (two males from this group were therefore not paired).

^(b): no statistically significant difference vs. controls.

 

 

Table 16 - Summary of Reproductive and Litter Data

Dose level (mg/kg bw/day)	0	5	20	50	HCD [min.; max.]
Number of pregnant females	9	8	9	8	30
Number of females which delivered	9	8	9	8	30
Mean duration of gestation (days ± SD)	21.4±0.5	21.5±0.5	21.6±0.5	21.4±0.5	[22.0; 22.2]
Mean number of corpora lutea ± SD	17.6±2.7	16.6±1.8	16.8±1.9	15.6±1.6	[14.0; 16.6]
Mean number of implantation sites ± SD	17.2±2.8	15.9±1.1	16.8±1.9	15.3±1.7	[13.4; 14.4]
Mean pre-implantation loss (% ± SD)	1.9±4.3	3.9±7.8	0.0±0.0	2.4±4.8	[4.0; 14.7]
Mean number of pups delivered ± SD	15.8±2.3	14.4±1.5	15.2±2.4	13.5±2.0	[10.7; 12.3]
Mean post-implantation loss (% ± SD)	7.5±10.33	9.1±10.95	9.4±8.71	11.1±11.71	[16.7; 22.5]

HCD: Historical Control Data (OECD 421/422, Jan-2016 to Dec-2017, n = 3 studies).

No statistically significant differences vs. controls

 

 

Table 17 - Clinical Signs in Pups During Lactation (Number of Pups Affected per Group)

Dose level (mg/kg bw/day)	0	5	20	50
Thin appearance			1(1): Days 1-4 p.p.
Generalized pallor			1(1): Days 1-4 p.p.
Dehydration	1(1): Days 9-17 p.p.		1(1): Days 3-4 p.p
Hematoma (head, neck, limb, and/or back)		3(3): Days 1-3 p.p.	5(4): Days 1-5 p.p.	3(2): Days 1-4 p.p.
Increased in size (neck)		1(1): Days 2-3 p.p.
Wound (head)			1(1): Days 4-5 p.p.
Scab (limb, back, head and/or tail)	1(1): Days 9-18 p.p.	2(2): Days 12-17 p.p.	4(2): Day 1-19 p.p.	1(1): Days 13-16 p.p.
Short tail			1(1): Days 20-21 p.p.
Thinning of hair				8(1): Days 13-21 p.p.
Number of litters	9	8	9	8
Number of affected litters [%]	2 [22]	4 [50]	5 [56]	3 [38]

(): in brackets, number of litters.

Duration: first day of apparition until last day of presence.

No statistical analysis performed.

 

 

Table 18 - Incidence of Mortality

Dose level (mg/kg bw/day)	0	5	20	50
Number of litters	9	8	9	8
Total number of pups on Day 1 p.p.	142	115	137	108
Number of pups found dead	3	0	2	2
Number of pups cannibalized	0	2	10	1

 

 

Table 19 - Viability Indexes

Dose level (mg/kg bw/day)	0	5	20	50
Live birth index (%)	99.3	100.0	100.0	98.3
Day 4 p.p. viability index (%)	97.8	98.4	91.5	97.5
Day 21 p.p. lactation index (%)	100.0	100.0	100.0	100.0

 

 

Table 20 - Mean Body Weight (g)

Dose level (mg/kg bw/day)	0	5	20	50
Mean body weight (males and females)
. Day 1 p.p.	7.2	7.2 (0)	6.8 (-6)	6.7 (-7)
. Day 4 p.p. (preculling)	10.5	10.6 (+1)	9.9 (-6)	9.2* (-12)
. Day 8 p.p.	20.9	20.6 (-1)	19.4 (-7)	17.5** (-16)
. Day 13 p.p.	35.5	35.2 (-1)	33.0 (-7)	30.4^# (-14)
. Day 17 p.p.	45.5	46.7 (+3)	44.4 (-2)	40.7 (-11)
. Day 21 p.p.	61.8	63.3 (+2)	61.2 (-1)	55.0* (-11)

(): in brackets, percentage (%) difference vs. controls.

Statistical significance vs. controls: *: p<0.05; **: p<0.01; ^#: p<0.001.

 

 

Table 21 - Mean Body Weight Change (g)

Dose level (mg/kg bw/day)	0	5	20	50
Body weight changes (males and females)
. Days 1 - 4 p.p.	+3.3	+3.4	+3.1	+2.5*
. Days 4 - 8 p.p. (preculling)	+10.4	+10.1	+9.2	+8.3^#
. Days 8 - 13 p.p.	+14.6	+14.6	+13.6	+12.9*
. Days 13 - 17 p.p.	+10.0	+11.5	+11.3	+10.3
. Days 17 - 21 p.p.	+16.3	+16.6	+16.8	+14.2*

Statistical significance vs. controls: *: p<0.05; ^#: p<0.001.

 

 

Table 22 - Mean T4 (ng/mL) and TSH Concentrations (pg/mL) (F1 Day 21 p.p. pups)

Dose level (mg/kg bw/day)	0	5	20	50	HCD [min.; max.]
T4
. Day 21 p.p. pups	33.94±5.45	33.66±4.39 (-0.8)	36.48±5.94 (+7.5)	39.48±2.87 (+16.3)	[8.2; 49.9]
TSH
. Day 21 p.p. pups	823±301	764±190 (-7.2)	699±311 (-15.1)	522±203 (-36.5)	[421; 4038]

HCD: Historical Control Data [n = 30 males and n = 85 to 102 litters on Day 13 and/or 21 p.p. (referring to ‘female’ litter data in HCD)].

(): in brackets, percentage (%) difference vs. controls.

No statistical significance vs. controls.

 

 

Table 23 - AGD in Male Pups

Dose level (mg/kg bw/day)	0	5	20	50	HCD [min.; max.]
AGD (mm)	3.99	3.90 (-2.5)	3.90 (-1.9)	3.66 (-8.4)	[3.53; 5.95]
AGD / (BW)^1/3	2.05	2.00 (-2.8)	2.04 (0.0)	1.92 (-5.8)	[1.78; 3.00]

HCD: Historical Control Data (Anogenital Distance in F1 generation on Day 1 p.p., n = 65 male pups).

AGD: anogenital distance on Day 1 p.p.

(BW)^1/3: cube root of body weight recorded on Day 1 p.p.

(): in brackets, percentage (%) difference vs. controls.

 

Table 24 - AGD in Female Pups

Dose level (mg/kg bw/day)	0	5	20	50	HCD [min.; max.]
AGD	2.18	2.17 (-1.1)	2.05 (-6.6)	2.04 (-6.3)	[1.64; 3.90]
AGD / (BW)^1/3	1.14	1.13 (-0.6)	1.09 (-4.9)	1.10 (-2.8)	[0.82; 2.06]

HCD: Historical Control Data (Anogenital Distance in F1 generation on Day 1 p.p., n = 66 female pups).

AGD: anogenital distance on Day 1 p.p.

(BW)^1/3: cube root of body weight recorded on Day 1 p.p.

(): in brackets, percentage (%) difference vs. controls.

 

 

Table 25 - Mean Numbers of Areolae and Nipples in Male Pups on Day 12 p.p.

Dose level (mg/kg bw/day)	0	5	20	50
Mean number of areolae	0.0	0.0	0.0	0.0
Mean number of nipples	0.0	0.0	0.0	0.0

 

 

Table 26 - Sex Ratio (Mean Percentage of Male Pups)

Dose level (mg/kg bw/day)	0	5	20	50
Day 1 p.p. (%)	54	55	49	48
Day 21 p.p. (%)	53	49	52	50

Conclusions:

Under the experimental conditions of the study:
- The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg bw/day in female parents based on unscheduled mortality, signs of poor clinical condition and hypertrophic liver changes at 50 mg/kg bw/day and no NOAEL could be determined in male parents based on the tubular degenerative changes in the kidneys from 5 mg/kg bw/day,
- The No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 50 mg/kg bw/day, based on the absence of adverse findings at this dose level,
- The No Observed Adverse Effect Level (NOAEL) for F1 development was considered to be 20 mg/kg bw/day, based on the decreased pup body weight after culling at 50 mg/kg bw/day.

Executive summary:

The potential toxic effects of the test item was evaluated following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 21 post-partum (p.p.). The study was conducted in accordance with the OECD TG 421 and GLP principles.

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item daily by the oral route (gavage) at dose levels of 5, 20 or 50 mg/kg bw/day. Males were treated for 2 weeks before mating, throughout mating and then until the day before euthanasia (i.e. after an 8-week treatment period). Females were treated for an overall period of 8 to 10 weeks: 2 weeks before pairing, then throughout the mating and gestation periods until Day 21 p.p. inclusive.

Another group of 10 males and 10 females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg bw/day was used.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 3 and 7 were determined using a validated High-Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method.

Animals were checked daily for clinical signs and mortality. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from 2 weeks before the start of treatment until the females had mated, and then on the day before euthanasia.

The dams were allowed to deliver and rear their progeny until Day 21 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible four males and four females per litter. The pups were observed daily for clinical signs and abnormal behavior and were weighed at designated intervals. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p.

Thyroid hormone (TSH and T4) levels were determined in all F0 males at final euthanasia and in pups euthanized on Day 21 p.p.

F0 males were euthanized after completion of the gestation period (i.e. study Day 56). Dams were euthanized on Day 22 p.p.

A full macroscopic post-mortem examination was performed, and particular attention was accorded to the reproductive organs. Designated organs were weighed, and selected tissue specimens were preserved. A microscopic examination was performed on epididymides, oviducts, ovaries, testes, uterus and vagina from the control and high-dose groups on liver from all parent animals and on all macroscopic lesions.

A detailed external examination was performed after euthanasia of pups found dead or prematurely euthanized, or euthanized on Day 21 p.p., and particular attention was paid to the external genital organs.

The test item concentrations in the administered dose formulations analyzed in Weeks 1, 3 and 7 remained within an acceptable range of variations (-10.8% to +14.3%) when compared to the nominal values (± 15% of the nominal concentrations). No test item was observed in the control dose formulations.

There were no unscheduled deaths in males.

On Day 8, two high dose females with poor clinical condition were prematurely euthanized or found dead before mating. In both females, test item-related microscopic changes suggestive of stress were observed (decreased lymphoid cellularity in the thymus and/or spleen).

In surviving females at 50 mg/kg bw/day, adverse clinical signs (emaciated appearance, piloerection, round back and/or hypoactivity) were observed in 1 out of 8 animals during the pre-mating period.

At all dose levels and in both sexes, there were no adverse effects on body weight, body weight change or food consumption as changes observed in males and/or females were transient and/or of minimal to slight magnitude.

At all dose levels, there were no test item-related effects on the estrous cycle during the pre-mating period. There were no effects on mating or fertility index and all groups had similar pre-coital time intervals (number of days taken to mate).

In all test item-treated groups and when compared with controls, there were no effects on the mean duration of gestation

There were no adverse effects on the number of corpora lutea, implantation sites and delivered pups up to 50 mg/kg bw/day.

There were no effects on mean plasma TSH or T4 concentration levels in male parents.

In the liver, centrilobular hepatocellular hypertrophy was observed from 5 mg/kg bw/day in males and from 20 mg/kg bw/day in females. This correlated with increased liver weight in both sexes from 20 mg/kg bw/day and macroscopic enlargement in males from 20 mg/kg bw/day. At 50 mg/kg bw/day, there were also test item-related hepatocellular degeneration/necrosis in a few males and a single female and periportal vacuolation in males. The hypertrophic liver changes were considered to be adverse in both sexes at 50 mg/kg bw/day in view of the severity and their association with degenerative changes.

In the kidneys, in males only, hyaline droplet accumulation and adverse tubular degenerative changes consistent with α2u globulin nephropathy was observed from 5 mg/kg bw/day. This finding was considered to be non-relevant for human.

There were no effects on the incidence of pups found dead or cannibalized and no test item-related effects on pup viability, sex ratio, mean anogenital distance or areolae/nipples in male pups at any dose level.

There were no external abnormalities at any dose level and no effects on mean TSH or T4 plasma concentrations in Day 21 p.p. pups.

At 50 mg/kg bw/day, when compared with controls, there were lower mean body weights in male and female pups after culling (down to -16% on Day 8 p.p., p<0.01) as a consequence of lower mean body weight gain (statistically significant up to p<0.001 on Days 4-8 p.p). Taking into account the magnitude of the differences, these findings were considered to be adverse.

The test item was administered daily by the oral gavage to male and female Sprague-Dawley rats for 15 days before mating, during mating, and, for females, throughout gestation and until Day 21 p.p. at dose levels of 0, 5, 20 or 50 mg/kg bw/day.

Under the experimental conditions of the study:

- The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg bw/day in female parents based on unscheduled mortality, signs of poor clinical condition and hypertrophic liver changes at 50 mg/kg bw/day and no NOAEL could be determined in male parents based on the tubular degenerative changes in the kidneys from 5 mg/kg bw/day,

- The No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 50 mg/kg bw/day, based on the absence of adverse findings at this dose level,

- The No Observed Adverse Effect Level (NOAEL) for F1 development was considered to be 20 mg/kg bw/day, based on the decreased pup body weight after culling at 50 mg/kg bw/day.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not available in English language.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

No english data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

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

Frequency of treatment:

No English data

Details on study schedule:

No English data

Remarks:

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

Remarks:

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

Remarks:

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

No. of animals per sex per dose:

13

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

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

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

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

Sperm parameters (parental animals):

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

Litter observations:

STANDARDISATION OF LITTERS
no

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


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

Postmortem examinations (parental animals):

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


GROSS NECROPSY
no data.


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

Postmortem examinations (offspring):

SACRIFICE
- Supposed at day 4. Not accurately indicated.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
No

Statistics:

No data

Reproductive indices:

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

Offspring viability indices:

Viability index

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

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

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

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

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

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

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

Dose descriptor:

NOEL

Effect level:

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

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Effect level:

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

Sex:

female

Basis for effect level:

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

Dose descriptor:

NOEL

Effect level:

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

Sex:

male

Basis for effect level:

other: No effect on reproductive toxicity in male

Dose descriptor:

NOEL

Effect level:

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

Sex:

female

Basis for effect level:

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

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

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

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

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

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

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

Reproductive effects observed:

not specified

Summary of developement of pups

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

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

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

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

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

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

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

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

Conclusions:

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

Executive summary:

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

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

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

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

based on the OECD 443 Study (CRL, 2022), GLP and valid study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

1- There is a rat developmental study by oral route performed

according to regulatory guideline OECD 414 (J. Rhodes, 2014).

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

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

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

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

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

 

2-There is a second developmental toxicity study performed in a

second species according to OECD 414 in rabbits (Bentz, 2017)

Dose-levels of 3, 9 and 24 mg/kg/day were selected in the study.

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

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 OCTOBER 2012 to 24 JANUARY 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to current test guidelines and GLP compliant

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Details on mating procedure:

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

Duration of treatment / exposure:

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

Frequency of treatment:

Daily

Duration of test:

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

No. of animals per sex per dose:

22 females per dose concentration

Control animals:

yes, concurrent vehicle

Details on study design:

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

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

Maternal examinations:

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

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

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

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

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

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

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue. Dead foetuses were classified as those which appeared to have died shortly before necropsy.

Fetal examinations:

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

Statistics:

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

Indices:

Not applicable

Historical control data:

Not applicable

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

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

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

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

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

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

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

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

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

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

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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

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

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

Dose descriptor:

NOAEL

Effect level:

> 20 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no effect

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

not specified

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

* P<0.05

DR = significant dose response test

Conclusions:

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

Executive summary:

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

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

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

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

 

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

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

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

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

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

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

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

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

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

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

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

 

 

Justification for classification or non-classification

According to the criteria laid down in EU CLP 1272/2008 regulation, the test item,is not classified as toxic to reproduction or fetal development.