Benzaldehyde

Toxicity to reproduction

Administrative data

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:

2022-MARCH-02 to 2022-NOV-XX

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information will be submitted later based on ECHA communication/decision number CCH-D-2114332899-33-01/F. According to ECHA communication CCH-D-2114332899-33-01/F (attached below and in Section 13 of the dossier) received on 13 June 2016, ECHA has requested an Extended One-Generation Reproductive Toxicity Study (Annex IX/X, Section 8.7.3; test method: OECD TG 443) in rats, oral route, with the registered substance. The outcome of a Board of Appeal (20 March 2020) states that the registrant is required to submit the requested information in an updated registration dossier by 1 October 2022.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals : 10 weeks

- Basis for dose level selection: Dose levels were selected after examining existing toxicity data on benzaldehyde and on the basis of results from a pilot OECD 443 study performed at Labcorp (Labcorp Study No. 8461365). The high dose level of 600 mg/kg/day in males and 450 mg/kg/day in females was selected as these dose levels were expected to produce some toxicity such as a reduction in body weight gain and/or food consumption but not excessive lethality that would prevent meaningful evaluation. 600 mg/kg/day was considered to be above the Maximum Tolerated Dose (MTD) in the pilot OECD 443 study for females due to issues with littering and pup survival. The mid-dose levels of 300 mg/kg/day in males and 225 mg/kg/day in females were the approximate geometric means between the high and low-doses which were expected to produce minimal to moderate toxicity. The low-dose level of 100 mg/kg/day was expected to produce no observable indications of toxicity.

Following commencement of direct dosing of the F1 generation on Day 21 of age, two males given 600 mg/kg/day were euthanized for welfare reasons following one or two doses and showed bodyweight losses and poor general clinical health. The remaining F1 males that received one or two consecutive dosages also generally showed bodyweight losses between Day 21-22 of age. In consultation with the Sponsor, the dose volume of Group 4 males was temporarily lowered to 7.5 mL/kg (equivalent to a dose level of 450 mg/kg/day) to ascertain the survival of the F1 males. The progression of these F1 animals was reassessed again prior to the formal start of the F1 generation (nominally Day 28 of age) where the animals were once again exposed to the test material at a dose volume of 10 mL/kg (equivalent to a dose level of 600 mg/kg/day).

- Inclusion/exclusion of extension of Cohort 1B : Cohort 1B included: Spare Cohort (treated from weaning to approximately 20 weeks of age; F1 animals mated to produce F2 generation).

- Termination time for F2 : Day 22 of age

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Not included

- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Not included

- Route of administration : Oral gavage

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]: The Han Wistar rat (sexually mature, virgin) was selected since it is widely accepted by regulatory agencies. Additionally, historical control data are available at the CRO. Acceptable models that do not use live animals currently do not exist and currently studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. The number of animals used will be the minimum that is consistent with scientific integrity and regulatory acceptability, consideration having been given to the welfare of individual animals in terms of the number and extent of procedures to be carried out on each animal. The oral route was selected since it is the most relevant to possible human exposure.

Test material

Test material information

Constituent 1

Reference substance name:

Benzaldehyde

EC Number:

202-860-4

EC Name:

Benzaldehyde

Cas Number:

100-52-7

Molecular formula:

C7H6O

IUPAC Name:

benzaldehyde

Test material form:

liquid

Details on test material:

Test material: Benzaldehyde
CAS No. 100-52-7
EC No. 202-860-4
Batch No. #2101-4
Expiration date: 04 January 2023
Purity: > 99.5% w/w
Storage: Stored in darkness at ambient temperature (approximately 21°C). All samples were stored under nitrogen.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Emerald Kalama Chemical, B.V.; Batch# #2101-4
- Purity, including information on contaminants, isomers, etc.: 99.5% w/w

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in darkness at ambient temperature (approximately 21°C). All samples were stored under nitrogen.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: formulations in the range 1 to 250 mg/mL were determined to be stable for 1 day at ambient temperature (15 to 25°C); 15 days when stored refrigerated (2 to 8°C)

FORM AS APPLIED IN THE TEST : Liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST.

Details on species / strain selection:

Han Wistar rat (sexually mature, virgin) is widely accepted by regulatory agencies. Additionally, historical control data are available at the CRO.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x 4-5 wks; (F1) x 18-20 days
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study : Not specified
- Housing:

F0 generation (from acclimatization) and selected F1 generation (from weaning): up to 4 rats/sex/cage in polycarbonate cages

Pairing: 1 male and 1 female rat per cage in in polycarbonate cages

Male rats to termination: up to 4 male rats/cage in polycarbonate cages

Female rats after mating (from Day 0 after mating): Individually in polycarbonate cages

Female rats during littering (from Day 20 after mating): Individually in polycarbonate cages along with the litter

Female rats to termination (after weaning): up to 4 female rats/cage in polycarbonate cages

- Use of restrainers for preventing ingestion (if dermal): No
- Diet (e.g. ad libitum): SDS VRF1 Certified, pelleted diet ad liibitum (except overnight for blood sampling for hematology, blood chemistry, urinalysis and thyroid hormone investigations for F0 and F1 Cohort 1A animals)
- Water (e.g. ad libitum): Potable water from the public supply ad libitum via polycarbonate bottles with sipper tubes (except overnight for blood sampling for hematology, blood, chemistry, urinalysis and thyroid hormone investigations for F0 and F1 Cohort 1A animals)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2022-MARCH-02 To: 2022-NOVEMBER-XX

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose with 0.5% Tween-80 in purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A series of formulations at the required concentration were prepared weekly and in advance of the first day of dosing under yellow light by dilution of individual weighings of the test material with vehicle (0.5% methylcellulose with 0.5% Tween-80 in purified water) to the final weight. Formulations were prepared in a container of suitable size to accommodate the whole formulation volume, thus avoiding multiple transfer steps. The formulation was mixed using a high shear homogenizer until fully homogeneous, and further mixed for a minimum of 20 minutes using magnetic stirring.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% methylcellulose with 0.5% Tween-80 in purified water (Justification not specified)
- Concentration in vehicle: 10, 30, or 60 mg/mL for the 100, 300, and 600 mg/kg bw/day dose groups, respectively (males) and 10, 22.5, or 45 mg/mL for the 100, 225, and 450 mg/kg bw/day dose groups, respectively (females)
- Amount of vehicle (if gavage): 10 mL/kg but temporarily reduced (2022-JULY-02 to 2022-JULY-06) for Group 4 (600 m/kg bw/day) males to 7.5 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 2 weeks, no mating change-overs
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- unsuccessful pairing replacement of first male by another male with proven fertility not undertaken.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability:
The homogeneity and stability of formulations during storage were determined as part of Labcorp Study No. 8462384. Formulations in the range 1 to 250 mg/mL were determined to be stable for 1 day at ambient temperature (15 to 25°C); 15 days when stored refrigerated (2 to 8°C).

Concentration analysis:
At specified intervals during treatment, the test formulations were analysed for achieved concentration of the test material using an analytical method that involved the dilution of the test formulations with a suitable solvent followed by a chromatographic assay. Acceptance criteria was = +10%/-15% of nominal concentration.

Duration of treatment / exposure:

F0 animals: For 10 weeks before pairing until termination after litters were weaned

F1 animals: Direct treatment of F1 offspring commenced at weaning (Day 21 of age)

Cohort 1A: Treated from weaning to approximately 13 weeks of age

Cohort 1B: Treated from weaning to approximately 20 weeks of age.

Cohort 1C: Treated from weaning to completion of sexual maturation (approximately 8 weeks of age; Groups 1, 2, and 3 only)

Unselected F1 offspring: No direct treatment

Frequency of treatment:

Once daily

Details on study schedule:

- F1 parental animals not mated until 14 weeks after selected from the F1 litters.

- Selection of parents from F1 generation when pups were 18-20 days of age.

- Age at mating of the mated animals in the study: (F0): 14-15 weeks; (F1): 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

F0 and F1 males and females: Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

F0 and F1 males and females: Group 2 (Low dose)

Doses / concentrations 3

Dose / conc.:

225 mg/kg bw/day (nominal)

Remarks:

F0 anf F1females: Group 3 (Intermediate dose)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

F0 and F1 males: Group 3 (Intermediate dose)

Doses / concentrations 5

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

F0 and F1 females: Group 4 (High dose)

Doses / concentrations 6

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

F1 males: Group 4 (High dose - Dose volume reduced from 10 mL/kg to 7.5 mL/kg (~450 mg/kg/day) from 2022-JULY-02 to 2022-JULY-06. Dose volume will be reduced from 10 mL/kg to 7.5 mL/kg (equal to 450 mg/kg/day). Dose volume returned to 10 mL/kg (~600 mg/kg/day) from 2022-JULY-07.

Doses / concentrations 7

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

F0 and F1 males: Group 4 (High dose)

No. of animals per sex per dose:

F0: 24/sex/dose
F1: 20/sex/dose

Specific details provided in Tables 1, 2, and 3 in 'Any other information on materials and methods incl. tables'.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected after examining existing toxicity data on benzaldehyde and on the basis of results from a pilot OECD 443 study performed at Labcorp (Labcorp Study No. 8461365). The high dose level of 600 mg/kg/day in males and 450 mg/kg/day in females was selected as these dose levels were expected to produce some toxicity such as a reduction in body weight gain and/or food consumption but not excessive lethality that would prevent meaningful evaluation. 600 mg/kg/day was considered to be above the Maximum Tolerated Dose (MTD) in the pilot OECD 443 study for females due to issues with littering and pup survival. The mid-dose levels of 300 mg/kg/day in males and 225 mg/kg/day in females were the approximate geometric means between the high and low-doses which were expected to produce minimal to moderate toxicity. The low-dose level of 100 mg/kg/day was expected to produce no observable indications of toxicity.

Following commencement of direct dosing of the F1 generation on Day 21 of age, two males given 600 mg/kg/day were euthanized for welfare reasons following one or two doses and showed bodyweight losses and poor general clinical health. The remaining F1 males that received one or two consecutive dosages also generally showed bodyweight losses between Day 21-22 of age. In consultation with the Sponsor, the dose volume of Group 4 males was temporarily lowered to 7.5 mL/kg (equivalent to a dose level of 450 mg/kg/day) to ascertain the survival of the F1 males. The progression of these F1 animals was reassessed again prior to the formal start of the F1 generation (nominally Day 28 of age) where the animals were once again exposed to the test material at a dose volume of 10 mL/kg (equivalent to a dose level of 600 mg/kg/day).

- Rationale for animal assignment (if not random): XXX

- Fasting period before blood sampling for clinical biochemistry: overnight food and water deprivation (access to water for a minimum period of one hour prior to blood sampling since blood sampling coincided with urine collection)

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals visually inspected at least twice daily for evidence of reaction to treatment or ill-health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0 and F1 (selected animals): once a week; F0 and F1 Cohort 1B females: on Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation

Animals were assessed for physical condition and behavior during handling outside the cages. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Additionally, detailed observations were performed on F0 and formal F1 generation animals to establish and confirm a pattern of signs associated with dosing according to the following minimum schedule: Week 1: Daily; Weeks 2 to 4: twice weekly (middle and end of the week); Week 5 onward: once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 and F1 Cohort 1B females).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Males:
- Day that treatment commenced followed by once a week; and on the day of necropsy

F0 Females:
- Day that treatment commenced followed by once a week until mating detected and then on Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 after mating; Days 1, 4, 7, 14, and 21 of lactation and on the day of necropsy.

F1 selected animals:
- on Days 21, 23, 25, 27, and 29 of age (as appropriate). From nominal Week 4 of age, twice during Week 1 of the formal F1 generation and weekly thereafter. For F1 Cohort 1B females on Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 after mating, and Days 1, 4, 7, 14 and 21 of lactation. Body weights were also measure on the day of attainment of sexual maturation and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
F0 Males:
- Once a week until termination (except when paired for mating).

F0 Females:
- Once a week until paired for mating and on Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating and on Days 1-4, 4-7, 7-14 and 14-21 of lactation.

F1 selected animals
- From nominal 4 weeks of age, twice during Week 1 of the formal F1 generation and weekly thereafter. For F1 Cohort 1B females on Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating, and on Days 1-4, 4-7, 7-14 and 14-21 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OTHER:

PARTURITION OBSERVATIONS
From Day 20 after mating, animals were checked three times daily for evidence of parturition.

HEMATOLOGY (F0):
At necropsy, following overnight deprivation of food, blood samples (0.5 mL) were collected from 10 male and 10 female F0 rats per dose group under light general anaesthesia (Isoflurane). Blood sampling coincided with urine collection and animals were therefore deprived of water overnight, but had access to water for a minimum period of one hour prior to blood sampling.

The hematology parameters listed in Table 4. were evaluated.

CLINICAL CHEMISTRY (F0):
At necropsy, following overnight deprivation of food, blood samples (0.7 mL) were collected from 10 male and 10 female F0 rats per dose group under light general anaesthesia (Isoflurane). Blood sampling coincided with urine collection and animals were therefore deprived of water overnight, but had access to water for a minimum period of one hour prior to blood sampling.

The clinical chemistry parameters listed in Table 5. were evaluated.

URINALYSIS (F0):
Urine samples were collected from 10 male and 10 female F0 rats per dose group overnight in individual metabolism cage with deprivation of food and water.
Routine urinalysis was conducted by qualitative, semi-quantitative and quantitative methods as presented in Table 6.

The deposit obtained from centrifugation of the urine samples was microscopically examined for epithelial cells, leucocytes, erythrocytes, crystals, casts, spermatozoa and precursors, and other abnormal components.

THYROID HORMONE ANALYSIS (F0):
At necropsy, following overnight deprivation of food, blood samples (1.0 mL) were collected from 10 male and 10 female F0 rats per dose group under light general anaesthesia (Isoflurane). Blood sampling coincided with urine collection and animals were therefore deprived of water overnight, but had access to water for a minimum period of one hour prior to blood sampling.

Samples were kept at ambient temperature (15 to 25⁰C) for a minimum of 30 minutes prior to centrifugation at 4ºC for 10 minutes at 2000g. 2 aliquots (tubes) were taken per animal (one for T3/T4 and one for TSH). Samples from F0 adults were assessed for levels of T3/T4 and TSH as detailed below:

T3/T4 analysis:
Serum samples were analyzed using LC-MS/MS (method number BM/2016/0632).

TSH analysis:
Analytical procedures followed were based upon the research paper by Lee et al (2006), ‘Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement’, Pharmaceutical Res., 23, 312-328. Serum samples were analyzed using Millipore Luminex multiplex kits for levels of thyroid stimulating hormone using validated method IAI/0017 (Validated in Covance Study Numbers SL13SG and HLS0980). The concentration of TSH was determined by immunoassay.

Oestrous cyclicity (parental animals):

Dry smears: For 15 days before pairing, using cotton swabs

Wet smears: Daily after pairing until evidence of mating confirmed, using pipette lavage; On the day of scheduled necropsy.

For females showing no evidence of mating, following completion of the pairing period, the females were separated from the male and vaginal smearing continued for up to five days or until the first estrus smear was seen.

• If a female showed an estrus smear during this period, the female was killed as soon as practically possible and subject to macroscopic examination. If necropsy was not possible on the day of the estrus smear, smears continued until the morning of necropsy.

• If a female did not show an estrus smear, a wet smear was taken on the morning of necropsy (Day 25 after removal from pairing where Day 0 = day of removal from pairing)

Sperm parameters (parental animals):

Parameters examined in all P and F1(Cohort 1A) male parental generations:
1) Vas deferens (from left side) – each animal in each group: For all males in all groups, sperm sample (200 spermatozoa where possible) assessed for motility using a computer assisted sperm analyzer (CASA).

2) Cauda epididymis (from left side): The cauda epididymis was weighed and homogenized and the number of sperm counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4.

3) Testis (from left side): The testis was homogenized and the number of homogenization-resistant spermatids counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

Groups 1 to 3: When possible, three male and three female offspring were selected from each selected litter, with one of each sex assigned to Cohorts 1A, 1B and Cohort 1C. If more were required, up to four males or four females were selected from each selected litter.

Group 4: When possible, up to two male and two female offspring were selected from each selected litter to be assigned to Cohort 1A. All remaining offspring from selected litters were assigned to Cohort 1B.

Selected animals were microchipped on Day 18-21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age.

Up to three male and three female F1 offspring per group (Groups 1-3 only) were retained as spares to provide potential replacement in the event of any mortalities. Body weights and clinical signs were monitored weekly and they were terminated after commencement of the F1 generation.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, eye opening, pupil reflex response

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

OTHER:

CAGE SIDE OBSERVATIONS (F1)
Animals visually inspected at least twice daily for evidence of reaction to treatment or ill-health.

CLINICAL OBSERVATIONS (F1)
Animals observed approximately 24 hours after birth and then daily for evidence of ill-health or reaction to treatment. On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity, and reaction to handling. Detailed clinical observations were made once each week for select F1 generation animals and on F1 Cohort 1B females on Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.
Additionally, detailed observations were performed on formal F1 generation animals to establish and confirm a pattern of signs associated with dosing according to the following minimum schedule: Week 1: Daily; Weeks 2 to 4: twice weekly (middle and end of the week); Week 5 onward: once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F1 Cohort 1B females).

BODY WEIGHTS:
F1 selected animals - on Days 21, 23, 25, 27, and 29 of age (as appropriate). From nominal Week 4 of age, twice during Week 1 of the formal F1 generation and weekly thereafter. For F1 Cohort 1B females on Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 after mating, and Days 1, 4, 7, 14 and 21 of lactation. Body weights were also measure on the day of attainment of sexual maturation and on the day of necropsy.

FOOD CONSUMPTION:
F1 selected animals - From nominal 4 weeks of age, twice during Week 1 of the formal F1 generation and weekly thereafter. For F1 Cohort 1B females on Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating, and on Days 1-4, 4-7, 7-14 and 14-21 of lactation.

PARTURITION OBSERVATIONS (F1)
From Day 20 after mating, animals were checked three times daily for evidence of parturition.

SEXUAL MATURATION (F1)
All males were examined daily from Day 38 of age for the completion of balano-preputial separation and body weight was recorded on day of completion of separation.

All females were examined daily from Day 25 of age until vaginal opening occurred and body weight was recorded on the day of vaginal opening.

For F1 Cohort 1A females only, a wet smear was taken daily from the day of vaginal opening until first estrus.

HEMATOLOGY (F1- COHORT 1A):
At necropsy, following overnight deprivation of food, blood samples (0.5 mL) were collected from 10 male and 10 female F1 rats per dose group under light general anaesthesia (Isoflurane). Blood sampling coincided with urine collection and animals were therefore deprived of water overnight, but had access to water for a minimum period of one hour prior to blood sampling.

The hematology parameters listed in Table 4. were evaluated.

CLINICAL CHEMISTRY (F1 – COHORT 1A):
At necropsy, following overnight deprivation of food, blood samples (0.7 mL) were collected from 10 male and 10 female F1 rats per dose group under light general anaesthesia (Isoflurane). Blood sampling coincided with urine collection and animals were therefore deprived of water overnight, but had access to water for a minimum period of one hour prior to blood sampling.

The clinical chemistry parameters listed in Table 5. were evaluated.

URINALYSIS (F1 – COHORT 1A):
Urine samples were collected from 10 male and 10 female F1 rats per dose group overnight in individual metabolism cage with deprivation of food and water.
Routine urinalysis was conducted by qualitative, semi-quantitative and quantitative methods as presented in Table 6.

The deposit obtained from centrifugation of the urine samples was microscopically examined for epithelial cells, leucocytes, erythrocytes, crystals, casts, spermatozoa and precursors, and other abnormal components.

THYROID HORMONE ANALYSIS (F1):

F1 Cohort 1A:
At necropsy, following overnight deprivation of food, blood samples (1.0 mL) were collected from 10 male and 10 female F1 Cohort 1A rats per dose group under light general anaesthesia (Isoflurane). Blood sampling coincided with urine collection and animals were therefore deprived of water overnight, but had access to water for a minimum period of one hour prior to blood sampling.

F1/F2 offspring:
At necropsy, samples (maximum volume possible) were collected from F1/F2 Offspring on Day 4 (10 litters per group – pooled litter sample of pups culled on Day 4 of age) and Day 22 (10 males and 10 females per group on Day 22 of age from as many litters as possible (1 male or 1 female per litter where possible)) of age following decapitation.

Samples were kept at ambient temperature (15 to 25⁰C) for a minimum of 30 minutes prior to centrifugation at 4ºC for 10 minutes at 2000g. 2 aliquots (tubes) were taken per offspring on Day 22 of age (one for T3/T4 and one for TSH). For offspring Day 4 of age, a single aliquot was taken for T3/T4 analysis (all available blood collected). Samples from unselected F1/F2 offspring on Day 22 of age and F1 animals (Cohort 1A) were assessed for levels of T3/T4 and TSH.

T3/T4 analysis:
Serum samples were analyzed using LC-MS/MS (method number BM/2016/0632).

TSH analysis:
Analytical procedures followed were based upon the research paper by Lee et al (2006), ‘Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement’, Pharmaceutical Res., 23, 312-328. Serum samples were analyzed using Millipore Luminex multiplex kits for levels of thyroid stimulating hormone using validated method IAI/0017 (Validated in Covance Study Numbers SL13SG and HLS0980). The concentration of TSH was determined by immunoassay.

COHORT SPECIFIC IN-LIFE OBSERVATIONS:

F1 Cohort 1A: Estrous Cycle Monitoring:
Wet smears: Following onset of vaginal patency until the first cornified (estrus) smear was recorded and on the day of scheduled termination.
Dry smears: For 14 days from nominal Day 75 of age

F1 Cohort 1B: Estrous Cycle Monitoring:
Wet smears: Daily after pairing until evidence of mating confirmed, using pipette lavage, and on the day of scheduled necropsy. For females showing no evidence of mating, following completion of the pairing period the females were separated from the male and vaginal smearing continued for up to five days or until the first estrus smear was seen. If a female showed an estrus smear during this period, the female was killed as soon as practically possible and subject to macroscopic examination. If necropsy was not possible on the day of the estrus smear, smears continued until the morning of necropsy. If a female did not show an estrus smear, a wet smear was taken on the morning of necropsy (Day 25 after removal from pairing where Day 0 = day of removal from pairing).

Postmortem examinations (parental animals):

SACRIFICE
Animals 14 days and older: carbon dioxide. Each animal was subsequently exsanguinated; Animals less than 14 days of age: intraperitoneal injection of sodium pentobarbitone.

- Male animals: All surviving animals - After at least 18 weeks of treatment and after weaning of the F1 animals, after confirmation that no further mating was required.
- Maternal animals:

- F0 females failing to mate: If an estrus smear was seen following completion of the pairing period, animals were terminated as soon as logistically possible. If no estrus smear was seen, animals were terminated on Day 25 after the last day of pairing where the day of separation was considered Day 0.

- F0 females failing to produce a viable litter: On or after Day 25 after positive indication of mating

- F0 females with total litter loss: On the day of total litter loss

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 7 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE: Animals 14 days and older: carbon dioxide. Each animal was subsequently exsanguinated; Animals less than 14 days of age: intraperitoneal injection of sodium pentobarbitone; Offspring culled on Day 4 of age requiring thyroid hormone sampling: decapitation.

- F1 Cohort 1A animals: At approximately 13 weeks of age

- F1 Cohort 1B animals:
Females – Day 22-25 postpartum

Males – After termination of the F2 litters, after confirmation that no further mating is required

F1 Cohort 1B females failing to mate:
If an estrus smear was seen following completion of the pairing period, animals were terminated as soon as logistically possible. If no estrus smear was seen, animals were terminated on Day 25 after the last day of pairing where the day of separation was considered Day 0.

F1 Cohort 1B females failing to produce a viable litter:
On or after Day 25 after positive indication of mating

F1 Cohort 1B females with total litter loss:
On the day of total litter loss

- F1 Cohort 1C animals:
After completion of sexual maturation (approximately 6-8 weeks of age)

- Unselected F1/F2 offspring:
On Day 4 and Day 22 of age

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 8 - 11 were prepared for microscopic examination and weighed, respectively.

Immunophenotyping of Spleen Leucocytes – F1 Cohort 1A Animals

Ten males and ten females per group from F1 Cohort 1A animals, from as many litters as possible, were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter. The spleen (whole) was weighed and then a portion placed in a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice. Cell suspensions from each tissue section were prepared by mechanical dissociation, following method IAI/0304, and the samples stained with a cocktail of directly conjugated monoclonal antibodies. Any contaminating red cells were removed using lysing buffer and the samples fixed by re-suspension in phosphate buffered saline containing 1% formaldehyde and analyzed immediately or stored at 2-8°C until analysis. Each sample was analyzed for the following cell markers using the combination of antibody markers and results reported in Table 12. Analysis was conducted using a Flow cytometer.

Statistics:

Data types
The following data types were analyzed at each timepoint separately, where required, in support of interpretation: Body weight; Food consumption; Estrous cycles; Vaginal opening to first estrus and pre-coital interval; Mating performance and fertility; Gestation length; Litter size and survival indices; Ano-genital distance (using cubed-root of body weight as covariate); Eye opening; Pupil reflex; Sexual maturation; Age and body weight at completion; Clinical pathology (hematology, blood chemistry, urinalysis); Thyroid hormone analysis; immunophenotyping; Organ weights (absolute and/or bodyweight relative); Sperm analysis (motility, morphology and counts); and Corpora lutea and ovarian primordial follicle counts.

Methods
For categorical data, the proportion of animals were analyzed for each treated group (as appropriate) versus the control group. For continuous data, Bartlett’s test was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary.

Reproductive indices:

Mating performance and fertility

Group values calculated for males and females separately:

1) Percentage mating = (Number of animals mating / Animals paired) x 100

2) Conception rate = (Number animals achieving pregnancy / Animals mated) x 100

3) Fertility index = (Number animals achieving pregnancy / Animals paired) x 100

Calculated for each group:

4) Gestation index = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

Survival indices (%)

Individual litter values calculated:

1) Post-implantation survival index = (Total number of offspring born / Total number uterine implantation sites) x 100

2) Live birth index = (Number live offspring on Day 1 after littering / Total number of offspring born) x 100

3) Viability index 1 = (Number live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100

4) Viability index 2 = (Number live offspring on Day 7 after littering / Number live offspring on Day 4 after culling) x 100

5) Viability index 3 = (Number live offspring on Day 14 after littering / Number live offspring on Day 7 after littering) x 100

6) Viability index 4 = (Number live offspring on Day 21 after littering / Number live offspring on Day 14 after littering) x 100

7) Lactation index = (Number live offspring on Day 21 after littering / Number live offspring on Day 4 (after culling)) x 100

8) Cumulative survival index = ((Number of pups Day 21 / No. pups Day 4 after culling) x (No. pups Day 4 before culling / No. of pups born)) x 100

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Remarks on result:

other: Study currently on going. Results will be presented subsequent to receipt of the final report from the CRO.

Remarks:

As per ECHA’s final decision (CCH-D-2114378524-42-01/F), an Extended One-Generation Reproductive Toxicity Study (OECD Guideline 443) study was commissioned and is currently ongoing. As per the letter from the CRO (Labcorp Early Development Laboratories Limited) dated August 19, 2022 (provided under ‘attached justification’ in this RSS) due to current laboratory capacity as well as challenging chemistry the contracted OECD 443 study commissioned will be completed by June 2023. This RSS will be updated upon receipt of the final report from the CRO and the endpoint updated accordingly followed by a spontaneous update by the lead registrant.

Results: F1 generation

Effect levels (F1)

Remarks on result:

other: Study currently on going. Results will be presented subsequent to receipt of the final report from the CRO.

Remarks:

As per ECHA’s final decision (CCH-D-2114378524-42-01/F), an Extended One-Generation Reproductive Toxicity Study (OECD Guideline 443) study was commissioned and is currently ongoing. As per the letter from the CRO (Labcorp Early Development Laboratories Limited) dated August 19, 2022 (provided under ‘attached justification’ in this RSS) due to current laboratory capacity as well as challenging chemistry the contracted OECD 443 study commissioned will be completed by June 2023. This RSS will be updated upon receipt of the final report from the CRO and the endpoint updated accordingly followed by a spontaneous update by the lead registrant.

Overall reproductive toxicity

Reproductive effects observed:

not specified