Benzaldehyde

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the overall read-across approach that benzaldehyde is rapidly metabolised to benzoic acid, and therefore the reproductive and developmental potential of benzaldehyde has been evaluated (and the data requirements fulfilled) as part of the "Benzoates" group, the following summary of the studies presented for IUCLID Section 7.8 for benzoic acid is presented for completeness.

The key study evaluating effects on fertility were by Kieckebusch and Lang (1960), which evaluated the effects of benzoic acid over 4 generations in rats via feeding. While this study does have some limitations when compared to the current OECD 443 EOGRTS, when supplemented by information on reproductive organs/tissues (sperm parameters, including epididymis/cauda epididimys/testis weights, sperm motility/density/abnormal sperm; Estrous cyclicity in females) from a 13 -week repeated dose study of benzyl acetate (a substances that is metabolized completely to benzoic acid), the apparent gaps in data from the current OECD 443 study design are filled.

A supporting dose range-finding toxicity (DRF) study (Labcorp Early Development Laboratories Ltd., 2022) was conducted to evaluate the general systemic toxic potential of the test material (Benzaldehyde; CAS# 100-52-7) in rats, including reproductive / developmental effects.

Based on results observed in this preliminary dose range-finding study, oral gavage administration of benzaldehyde at 600 mg/kg/day to F0 generation animals was concluded to exceed the maximum tolerated dose due to the death of a majority of offspring shortly after birth. This dose level was therefore considered unsuitable for further investigation and the recommended high dose levels for the main Extended One-Generation Study (OECD 443) were 600 mg/kg/day for male and 450 mg/kg/day for female rats.

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 the concerned RSS) due to current laboratory capacity as well as challenging chemistry the contracted OECD 443 study commissioned will be completed by June 2023. This summary 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. 

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:

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.

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.

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

225 mg/kg bw/day (nominal)

Remarks:

F0 anf F1females: Group 3 (Intermediate dose)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

F0 and F1 males: Group 3 (Intermediate dose)

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

F0 and F1 females: Group 4 (High dose)

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.

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)

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

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.

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.

Reproductive effects observed:

not specified

Reference 2

Endpoint:

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

Remarks:

Dose-Range Finding Study to set dose levels for an Extended One-Generation Reproductive Toxicity Study (OECD 443)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021-JUN-17 to 2021-SEP-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Dose-Range Finding Study conducted to set dose levels for an Extended One-Generation Reproductive Toxicity Study (OECD 443).

GLP compliance:

no

Remarks:

Dose Range-finding study

Limit test:

no

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 No. #2101-4
- Purity, including information on contaminants, isomers, etc.: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in darkness at ambient temperature (approximately 21°C) 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); and 15 days when stored refrigerated (2 to 8°C)

FORM AS APPLIED IN THE TEST: Liquid

Species:

rat

Strain:

Wistar

Remarks:

RccHan® WIST rat

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan®;WIST) strain was used because of the historical control data 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: Not specified
- Age at study initiation: (F0): Males: Males: 76 to 82 days; Females: 69 to 75 days; (F1) <21 days of age
- Weight at study initiation: (F0) Males: Males: 325 to 369 g; Females:181 to 208 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: Not specified
- Housing: Solid (polycarbonate) bottom cages and animals were housed as follows:

Acclimatization/Pre-pairing: up to four animals of one sex
Pairing: one male and one female
Males after mating: up to four animals
Gestation: Indivudually
Lactation: Individual dam with litter
F1 Generation: Siblings housed together

- Use of restrainers for preventing ingestion (if dermal): No
- Diet (e.g. ad libitum): SDS VRF1 Certified, pelleted diet ad liibitum
- Water (e.g. ad libitum): Potable water from the public supply ad libitum via polycarbonate bottles with sipper tubes
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2021-JUN-23 to 2021-SEP-11

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:
Formulations were stored refrigerated (2 to 8°C), prepared weekly under yellow light, and in advance of the first day of dosing.

Formulations administered in Weeks 1 and 2 (pre-pairing period):
The required amount of test material was weighed and starting with the lowest concentration, approximately 50% of the final volume of the vehicle was added to the test material. The formulation was mixed using a high shear homogeniser until fully homogenous and magnetically stirred for a minimum of 20 minutes before being transferred to the final container via syringe (while being magnetically stirred). A series of formulations at the required concentrations were prepared by dilution of individual weighing’s of the test material.

Formulations administered in Weeks 3 to 5 (from pairing to mid/late gestation):
The required amount of test material was weighed and the final volume of the vehicle added. The formulation was mixed using a high shear homogeniser until fully homogenous and magnetically stirred for a minimum of 20 minutes before being transferred to the final container via syringe (while being magnetically stirred). A series of formulations at the required concentrations were prepared by dilution of individual weighing’s of the test material.

Formulations administered in Week 6 onwards:
The required amount of test material was weighed into a suitable sized beaker to hold the whole formulation and vehicle was added to achieve the required weight. The formation was mixed using a high shear homogeniser until fully homogenous and magnetically stirred for a minimum of 20 minutes before being transferred to the final containers via syringe (while being magnetically stirred). A series of formulations at the required concentrations were prepared by dilution of individual weighing’s of the test material.

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: 0, 10, 30, or 60 mg/mL for the control, 100, 300, and 600 mg/kg bw/day dose groups, respectively
- Amount of vehicle (if gavage): 10 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
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the 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: Not specified
- 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:

Stability and homogeneity:
The homogeneity and stability of formulations during storage were confirmed as part of another study (Labcorp Study Number 8462384). Formulations in the range 1 to 250 mg/mL were determined to be stable for: One day at ambient temperature (15 to 25°C); 15 days when stored refrigerated (2 to 8°C).

Concentration analysis:
Samples of the first preparation formulations were analyzed for homogeneity of the test material. Samples of each of the first and last preparation formulations were analyzed for achieved concentration of the test item. Also, samples of each formulation prepared for administration in Weeks 3 and 5 of treatment and were analyzed for achieved concentration of the test material.

Duration of treatment / exposure:

F0 males; For two weeks before pairing until termination after litters were established (after minimum of eight weeks).

F0 females: For two weeks before pairing until Day 20 of lactation.

F1 animals: Day 21* to 34 of age.

* Although direct exposure started at weaning on Day 21 of age, all offspring had potential indirect exposure in utero and through the milk during lactation.

Frequency of treatment:

Once daily

Details on study schedule:

- F1 parental animals not mated

- Selection of parents from F1 generation when pups were less than 21 days of age

- Age at mating of the mated animals (F0) in the study: males: 13-14 weeks; females: 12-13 weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

F0 and F1: Group 1 (Control)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

F0 and F1: Group 2 (Low dose)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

F0 and F1: Group 3 (Intermediate dose)

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

F0: Group 4 (High dose)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels of 100, 300, and 600 mg/kg/day were selectd for the current study based on significant amount of toxicological information available on the benzaldehyde (NTP, 1990). The low dose level of 100 mg/kg/day was expected to produce no observable indications of toxicity. The mid dose level of 300 mg/kg/day (approximate geometric mean between the high and low dose), was expected to produce minimal to moderate toxicity. The high dose level of 600 mg/kg/day was selected as this dose level was expected to produce some toxicity, such as a reduction in body weight gain or food intake, but not excessive lethality that would prevent meaningful evaluation.

- Rationale for animal assignment (if not random): On arrival, with non-selective allocation to cages

- Fasting period before blood sampling for clinical biochemistry: Not specified

- Other: The oral route (via gavage) of administration was chosen as the route most relevant to possible human exposure and as the route specified in the Final Decision Letter from the European Chemicals Agency (ECHA).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health and during the acclimatization period, observations of the animals and their cages were recorded at least once per day. A viability check was performed near the start and end of each working day.

Signs associated with dosing:

Detailed observations were performed to establish and confirm a pattern of signs associated with dosing according to the following schedule:

- F0 animals: Week 1 – Daily; Weeks 2 to 4 - twice weekly; Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for F0 females).
- F1 animals: Daily from Days 21-34 of age.

Detailed observations were recorded in the treatment period one to two hours after completion of dosing and as late as possible in the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded in the treatment period one to two hours after completion of dosing and as late as possible in the working day.

A detailed physical examination was performed on each animal to monitor general health once each week for all F0 and selected F1 generation animals and on Days 0, 5, 12, 18, and 20 after mating and Days 1, 7, 14, and 21 of lactation for F0 females.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males: Before dosing on the day that treatment commenced (Day 1), weekly thereafter and on the day of necropsy.
F0 females: Before dosing on the day that treatment commenced (Day 1) and weekly thereafter until mating detected; Days 0, 7, 14, and 20 after mating; Days 1, 7, 14, and 21 of lactation; Before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
- F0 males and females: Weekly until paired for mating.
- For females after mating food consumption was performed on: Days 0-3, 3-7, 7-11, 11-14, 14-17 and 17-20 after mating; Days 1-4, 4-7, 7-11, 11-14, 14-18 and 18-21 of lactation.

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

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

OTHER: Parturition Observations and Gestation Length:

Duration of gestation: Time that elapsed between mating and commencement of parturition.

Parturition observations: From Day 20 after mating females were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded, and any difficulties observed were noted.

Oestrous cyclicity (parental animals):

Dry smears were taken for 15 days before pairing, using cotton swabs; Wet smears were taken after pairing until evidence of mating confirmed, using pipette lavage.

Sperm parameters (parental animals):

Not specified

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On Day 4 of age, litters containing more than eight offspring were reduced to eight by random culling, leaving, whenever possible, four male and four female offspring in each litter.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: F1 offspring were observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment; Sex ratios of each litter were recorded on Days 1, 4 (before and after culling) and on Day 21 of age; Individual offspring body weights were recorded on Days 1, 4 (before culling), 7, 11, 14, 17, and 21 of age.

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

Postmortem examinations (parental animals):

SACRIFICE
F0 males: After successful littering by females, after confirmation that no further mating was required.
F0 females:
- Scheduled kill: Day 21 of lactation.
- Failing to produce viable litter: Day 25 after mating.
- Litters dying before weaning: on day last offspring died.

All animals 14 days and older were sacrificed by Carbon dioxide asphyxiation. All animals less than 14 days of age were sacrificed via intraperitoneal injection of sodium pentobarbitone.

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

All adult F0 animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed and all external features and orifices examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

For F0 females, the following were recorded:
- Each uterine horn: Number of implantation sites. The number of uterine implantation sites were checked after staining technique, for females failing to produce a viable litter only.

- For female whose litter died before Day 21 of lactation: Mammary tissue appearance.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [2] were prepared for microscopic examination and weighed, respectively.

Organ weights:
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled termination, or (where possible) at premature termination of a group.

Histopathology:
Tissues listed in Table 2 were routinely preserved in 10% Neutral Buffered Formalin with the exception of testes which were preserved in modified Davidson’s fluid.

Postmortem examinations (offspring):

SACRIFICE
Unselected offspring:
- Culled: Day 4 of age.
- Scheduled kill: Day 21 of age.
- Selected spares after establishment of the F1 generation.

F1 selected offspring:
- Scheduled kill: Day 35 of age.

All animals 14 days and older were sacrificed by Carbon dioxide asphyxiation. All animals less than 14 days of age were sacrificed via intraperitoneal injection of sodium pentobarbitone.

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

Selected F1 animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed and all external features and orifices examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Premature deaths (excluding culled offspring):

Where possible, a fresh macroscopic examination with an assessment of stomach for milk content was performed. Abnormal tissues were retained in an appropriate fixative.

In the 600 mg/kg/day group, for litters which had been born, where possible all pups were retained in 10% Neutral Buffered Formalin for possible future examination. For those litters which had not been born, the fetuses were subjected to an external examination and were retained in Bouin’s solution for possible future examination.

Culled offspring on Day 4 of age:

Culled offspring with clinical signs on Day 4 of age were subject to complete macroscopic examination with assessment of stomach for presence of milk, where possible. Abnormal tissues retained in an appropriate fixative. Culled offspring with no clinical sign on Day 4 of age were killed and discarded without necropsy examination.

Unselected F1 offspring at scheduled kill (including selected spares):

Subject to complete macroscopic examination. Any abnormal tissues retained in appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not evaluated

Statistics:

For information on statistics please see 'Any other information on materials and methods'.

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

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

5) Sex Ratio: Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No signs of treatment-related clinical toxicity were observed among the F0 males or females at routine physical examination. Following dose administration, a low incidence of signs were observed in relation to dosing (not considered adverse treatment-related findings), primarily transient occasions of piloerection in males given 300 mg/kg/day, and during the post-mating and lactation periods in females given 100 or 300 mg/kg/day. There were no clinical signs observed at routine physical examination or post-dosing observations which were clearly attributable to treatment at 600 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One premature death occurred in the F0 generation in a male (no. 26) receiving 300 mg/kg/day. The animal was despatched to necropsy prior to dose administration on Day 20 of treatment for reasons of animal welfare due to signs of vocalization, piloerection, irregular breathing, sneezing, tremors (slight, whole body), and whole-body pallor. Subsequent macroscopic examination did not reveal any abnormalities and in the absence of similar signs in animals given 600 mg/kg/day, a relationship between this premature death and treatment with the test material was considered unlikely.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of F0 males given 100 mg/kg/day was slightly variable throughout the study, with overall mean weight gain being 12% lower than the corresponding control animals. Males given 300 mg/kg/day exhibited mean body weight stasis during Week 1 of treatment, compared to mean weight gain of 7 grams in the control animals. Thereafter, mean body weight gain was essentially similar to controls, such that overall mean weight gain during the 9-week treatment period (Day 1-64) was 13% lower than the control group. Group mean body weight gain for female rats given 100 or 300 mg/kg/day was unaffected throughout the 2-week pre-pairing period, and during gestation and lactation.

F0 males given 600 mg/kg/day displayed group mean weight loss of 5 grams during the first week of treatment compared with mean weight gain of 7 grams in the control animals, with the difference attaining statistical significance. Thereafter, mean body weight gain for males given 600 mg/kg/day was variable, but was generally slightly lower than control, such that overall mean body weight gain during Days 1-50 of treatment was 32% lower than the corresponding control group. The body weight performance of females given 600 mg/kg/day was not adversely affected during the 2-week pre-pairing treatment period or during Days 0-14 of gestation, with mean weight gain during these periods being similar to or slightly higher than controls. From Day 14 to Day 20 of gestation (the fetal growth period) however, mean body weight gain was statistically significantly lower than the control group (44 g versus 60 g in controls), such that overall mean weight gain during Days 0-20 of gestation was 16% lower than the corresponding control animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food consumption for males given 300 mg/kg/day was low when compared to the control group during Week 1 (10% lower than Control), correlating with the period of mean body weight stasis observed for these males. There was no effect of treatment on mean food intake in Week 2 for males given 300 mg/kg/day, or throughout the 2-week pre-pairing period for males given 100 mg/kg/day. Group mean food consumption for females given 100 or 300 mg/kg/day was unaffected throughout the 2-week pre-pairing period, and during gestation and lactation. Following the commencement of dose administration, F0 males given 600 mg/kg/day exhibited low mean food intake (14% lower than Control). Mean food consumption for females at 600 mg/kg/day was unaffected throughout the 2-week pre-pairing and gestation periods.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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, treatment-related

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment on estrous cycle regularity at 100 or 300 mg/kg/day with all females showing regular cycles during the 2-week pre-pairing treatment period. Pre-coital interval was similarly unaffected, with all mating pairs showing positive evidence of mating within four days of pairing.

During the 2-week pre-pairing treatment period, females given 600 mg/kg/day showed a clear disturbance to the regularity of estrous cycles, with one female showing irregular cycle lengths and three females being acyclic. Following pairing, however, 9/10 females given 600 mg/kg/day showed positive evidence of mating within 4 days (i.e. at the first estrous opportunity) with only one female (No. 87) having a pre-coital interval of 9 days.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating performance and fertility were considered unaffected by treatment. Three females in the 300 mg/kg/day group were not pregnant, however since all females in the 100 and 600 mg/kg/day groups were pregnant, the incidences of non-pregnancy in the 300 mg/kg/day group were considered incidental and unrelated to treatment. Gestation length and gestation index were unaffected by treatment at 100 or 300 mg/kg/day.

Conception rate and fertility index for the 600 mg/kg/day group were 100%. Of the first five females in the mating stagger to give birth, only one managed to maintain a live litter; the remaining four females experienced total litter loss prior to formal assignment to Day 1 of lactation. Consequently, for reasons of animal welfare, and to avoid any further pup deaths, all of the F0 females at 600 mg/kg/day were prematurely terminated. The five females which had not given birth were all pregnant with live fetuses. All ten females in the high dose group showed inactive mammary tissue at macroscopic examination. There was no clear effect on litter size (number of offspring born, or number of uterine implantation sites/fetuses).

Following the premature sacrifice of the F0 females, the F0 males at 600 mg/kg/day were also prematurely terminated, since it was clear that the dose level of 600 mg/kg/day exceeded the maximum tolerated dose for reproductive performance and was unsuitable for further investigation.

Key result

Remarks on result:

other: Dose range-finding study: Based on effects observed, the high dose level for the main OECD 443 study was recommended to be 600 mg/kg/day for males and 450 mg/kg/day for females.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs observed among the F1 offspring at 100 or 300 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Total offspring born, live offspring on Day 1 of age, and subsequent live litter size to Day 21 of age were unaffected by treatment at 100 or 300 mg/kg/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weights of male and female offspring in the 300 mg/kg/day group were marginally lower than Control on Day 1 of age (7-8% lower than Control), although differences did not attain statistical significance. Group mean body weight gain of the offspring between Day 1 and Day 21 of age was unaffected by parental exposure to the test material at 100 or 300 mg/kg/day.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic findings observed among the F1 offspring which died or were killed prematurely, were culled on Day 4 of age, or were killed at scheduled termination on Day 21 of age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

The mean number of uterine implantation sites, total offspring born, live offspring on Day 1 of age and subsequent live litter size to Day 21 of age were unaffected by benzaldehyde at 100 or 300 mg/kg/day.

Group mean post implantation survival in the 300 mg/kg/day group was slightly low when compared to Controls, although the difference did not attain statistical significance. There was no effect on live birth index, viability index to Day 4 of age or lactation index to Day 21 of age at 100 or 300 mg/kg/day.

Sex ratio, as assessed by the percentage of male offspring, was in line with expectation and considered unaffected by treatment.

Clinical signs:

no effects observed

Description (incidence and severity):

Direct administration of the test material at 100 or 300 mg/kg/day to selected F1 offspring for two weeks from Day 21 of age was well tolerated. No signs of clinical toxicity were observed in relation to dose administration and there were no treatment-related clinical signs observed at routine physical examination.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No premature deaths were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean absolute body weights for males and females on Day 1 of the formal F1 generation (Day 21 of age) were similar in all groups, and there was no adverse treatment-related effect on subsequent body weight gain during the 2-week dosing period.

Females administered 300 mg/kg/day showed slightly, but statistically significantly low mean body weight gain during Days 21-25 of age, such that overall mean body weight gain was 4% lower than the control females. Male rats administered 300 mg/kg/day showed marginally low mean body weight gain during Days 31-35 of age, such that overall mean body weight gain was 6% lower than the control animals. The extent of these differences was not considered to be adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Group mean food consumption for the selected F1 generation animals was unaffected by treatment.

Food efficiency:

not specified

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

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio, as assessed by the percentage of male offspring, was in line with expectation and considered unaffected by treatment.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy did not reveal any treatment-related macroscopic abnormalities at scheduled termination on Day 35 of age.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Remarks on result:

other: Dose range-finding study: Based on effects observed, the high dose level for the main OECD 443 study was recommended to be 600 mg/kg/day for males and 450 mg/kg/day for females.

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Table 3. Body weight and body weight change - group mean values (g) before pairing (F0 Males)
Dose Group (mg/kg/day)		Day										Change
		1	8	15	22	29	36	43	50	57	64	1-8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	1-50	1-64
Statistics test		Av	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Sh	Wi	Wi	Wi	Sh	Wi	Wi	Sh	Wi	Sh	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	349	356	370	382	398	410	420	430	442	453	7	13	12	16	13	9	10	11	11	81	104
	SD	11.0	9.6	13.4	13.3	17.2	20.4	23.8	27.0	31.3	33.7	17.3	7.2	7.0	6.2	4.5	9.3	5.3	6.8	4.8	33.3	39.0
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
Group 2 (100 mg/kg/day)	Mean	336**	345	361	370	382	393	398	409	419	428	9	16	8	12	11	5	11	10	9	73	92
	SD	9.0	7.5	8.7	12.6	13.7	18.5	16.6	15.8	17.7	20.3	2.3	2.8	7.4	4.7	7.2	8.4	4.9	2.6	5.8	12.3	17.0
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
Group 3 (300 mg/kg/day)	Mean	342	342*	355*	362*	380*	394	404	409	420	429	0	13	10	17	14	10	5	10	10	70	90
	SD	10.6	19.3	19.3	16.0	15.7	17.5	17.1	20.2	20.7	24.1	15.5	7.3	4.5	3.3	5.2	6.3	3.8	2.7	4.6	14.1	18.8
	N	10	10	10	9	9	9	9	9	9	9	10	10	9	9	9	9	9	9	9	9	9
Group 4 (600 mg/kg/day)	Mean	335**	330**	346**	356**	367**	368**	382**	390**			-5*	17	9	12	1*	13	8			55*
	SD	8.0	17.3	19.0	20.1	24.1	33.9	34.7	34.3			11.5	6.9	8.6	5.0	13.3	9.6	7.3			29.1
	N	10	10	10	10	10	10	10	10			10	10	10	10	10	10	10			10

* p<0.05

** p<0.01

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Sh: Treated groups compared with Control using Shirley’s test

Wi: Treated groups compared with Control using Williams’ test

Table 4. Body weight and body weight change - Group mean values (g) before Pairing (F0 Females)
Dose Group (mg/kg/day)		Day			Change
		1	8	15	1-8	8-15	1-15
Statistics Test		Av	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	196	199	204	3	5	8
	SD	9.2	9.2	9.1	3.9	3.7	4.9
	N	10	10	10	10	10	10
Group 2  (100 mg/kg/day)	Mean	191	196	203	4	8	12
	SD	6.1	4.4	6.4	4.1	3.7	5.6
	N	10	10	10	10	10	10
Group 3 (300 mg/kg/day)	Mean	193	195	202	1	8	9
	SD	6.4	7.3	8.9	4.8	4.1	5.5
	N	10	10	10	10	10	10
Group 4 (600 mg/kg/day)	Mean	190	197	207	8*	10*	17**
	SD	6.7	7.9	8.5	4.5	5.6	4.3
	N	10	10	10	10	10	10

* p<0.05

** p<0.01

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi: Treated groups compared with Control using Williams’ test

Table 5. Body weight and body weight change - group mean values (g) gestation (F0 Females)
Dose Group (mg/kg/day)		Day				Change
		0	7	14	20	0-7	7-14	14-20	0-20
Statistics Test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	208	229	252	312	21	23	60	105
	SD	10.8	11.0	12.2	17.8	5.1	3.4	7.6	12.7
	N	8	8	8	8	8	8	8	8
Group 2  (100 mg/kg/day)	Mean	204	226	250	309	22	24	58	104
	SD	7.4	6.7	9.5	15.0	3.5	6.1	6.9	11.6
	N	10	10	10	10	10	10	10	10
Group 3 (300 mg/kg/day)	Mean	206	225	251	308	19	25	57	102
	SD	6.1	5.9	8.9	21.5	5.3	4.4	13.7	18.7
	N	7	7	7	7	7	7	7	7
Group 4 (600 mg/kg/day)	Mean	207	229	250	294	22	21	44**	88*
	SD	9.6	8.8	11.8	17.1	7.7	8.1	11.7	16.3
	N	10	10	10	9	10	10	9	9

* p<0.05

** p<0.01

Wi: Treated groups compared with Control using Williams’ test

Table 6. Food consumption - group mean values (g/animal/week) before pairing (F0 Males)
Dose Group (mg/kg/day)		Week		Mean
		1	2	1-2
Statistics Test		Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	148	145	146
	SD	5.1	4.5	4.5
	N	3	3	3
Group 2  (100 mg/kg/day)	Mean	146	148	147
	SD	3.6	1.6	2.2
	N	3	3	3
Group 3 (300 mg/kg/day)	Mean	133	144	138
	SD	17.8	6.7	7.9
	N	3	3	3
Group 4 (600 mg/kg/day)	Mean	127*	152	139
	SD	8.6	7.7	7.3
	N	3	3	3

* p<0.05

Wi: Treated groups compared with Control using Williams’ test

Table 7. Food consumption - group mean values (g/animal/day) during Gestation (F0 Females)
Dose Group (mg/kg/day)		Day						Mean
		0-3	3-7	7-11	11-14	14-17	17-20	0-20
Statistics Test		Wi	Wi	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	17	19	19	21	21	22	20
	SD	1.3	2.0	1.6	2.0	0.9	1.7	1.2
	N	8	8	8	8	8	8	8
Group 2  (100 mg/kg/day)	Mean	16	18	19	20	21	20	19
	SD	1.7	1.3	2.1	1.3	2.4	2.0	1.3
	N	10	10	10	10	10	10	10
Group 3 (300 mg/kg/day)	Mean	16	18	19	21	21	21	19
	SD	2.3	1.8	1.8	1.5	1.3	2.8	1.3
	N	6	7	7	7	7	7	7
Group 4 (600 mg/kg/day)	Mean	18	19	19	20	23**	22	20
	SD	2.5	3.2	3.2	2.0	1.6	2.3	1.3
	N	10	10	10	10	9	9	10

** p<0.01

Wi: Treated groups compared with Control using Williams’ test

Table 8. Estrous cycles - group values (F0)
Dose Group (mg/kg/day)	Number of Animals		Regular 4 or 5 Day Cycles	Irregular Cycle l	Acyclic ᴪ
Statistics Test: Lt
Group 1 (Control – 0 mg/kg/day)	10	N	10	0	0
		(%)	(100)
Group 2  (100 mg/kg/day)	10	N	10	0	0
		(%)	(100)
Group 3 (300 mg/kg/day)	10	N	10	0	0
		(%)	(100)
Group 4** (600 mg/kg/day)	10	N	6	1	3
		(%)	(60)	(10)	(30)

lAt least one cycle of two, three or six to ten days

yAt least ten days without estrus

Table 9. Mating performance and fertility - Group values (F0)
Dose Group (mg/kg/day)	Number Paired	Number Mating	Number achieving pregnancy	Percentage Mating	Conception Rate (%)	Fertility Index (%)
Males
Group 1 (Control – 0 mg/kg/day)	10	10	9	100	90	90
Group 2  (100 mg/kg/day)	10	10	10	100	100	100
Group 3 (300 mg/kg/day)	10	10	7	100	70	70
Group 4 (600 mg/kg/day)	10	10	10	100	100	100
Females
Group 1 (Control – 0 mg/kg/day)	10	10	9	100	90	90
Group 2  (100 mg/kg/day)	10	10	10	100	100	100
Group 3 (300 mg/kg/day)	10	10	7	100	70	70
Group 4 (600 mg/kg/day)	10	10	10	100	100	100

Table 10. Gestation length and gestation index - Group values (F0 Females)
Dose Group (mg/kg/day)	Number of Pregnant Animals		Gestation length (days)				Number of complete live litters born	Gestation Index (%)
			22	22.5	23	23.5
Statistics Test			Lt					CA
Group 1 (Control – 0 mg/kg/day)	9A	N	2	4	2	0	8	89
		(%)	(25)	(50)	(25)
Group 2  (100 mg/kg/day)	10	N	2	6	2	0	10	100
		(%)	(20)	(60)	(20)
Group 3 (300 mg/kg/day)	7	N	0	6	1	0	7	100
		(%)		(86)	(14)
Group 4* (600 mg/kg/day)	10B	N	0	1	3	1	5	50*
		(%)		(20)	(60)	(20)

A Percentage distribution of gestation lengths calculated from eight animals - one pregnant female failed to litter

B Percentage distribution of gestation lengths calculated from five animals - five pregnant females euthanized for welfare reasons prior to parturition

* p<0.05

CA: Trend test using Cochran-Armitage test

Lt: Comparison of all groups using Linear by Linear tests

Table 11. Offspring survival indices - Group mean values (F1 Generation)
Dose Group (mg/kg/day)		Post Implantation Survival Index (%)	Live Birth Index (%)	Viability Index (%) Day 4	Lactation Index (%) Day 21
Statistics Test:		Wi	Ch	Ch
Group 1 (Control – 0 mg/kg/day)	Mean	96.5	100.0	99.0	100.0
	SD	7.26	0.00	2.72	0.00
	N	8	8	8	8
	N<100%	2	0	1	0
Group 2  (100 mg/kg/day)	Mean	93.5	100.0	99.2	100.0
	SD	7.11	0.00	2.43	0.00
	N	10	10	10	10
	N<100%	5	0	1	0
Group 3 (300 mg/kg/day)	Mean	90.6	99.0	100.0	100.0
	SD	11.76	2.70	0.00	0.00
	N	7	7	7	7
	N<100%	4	1	0	0

Table 12. Organ weights - group mean absolute values (g) for F0 Males
Dose Group (mg/kg/day)		Terminal
		Body Weight	Epididymides	Kidneys	Liver	Testes	Prostrate
Statistics Test		Wi	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	451.2	1.426	2.517	16.212	4.072	0.933
	SD	33.8	0.200	0.311	1.915	0.268	0.214
	N	10	10	10	10	10	10
Group 2  (100 mg/kg/day)	Mean	429.0	1.489	2.516	15.938	3.977	0.900
	SD	17.1	0.152	0.237	1.184	0.277	0.228
	N	10	10	10	10	10	10
Group 3 (300 mg/kg/day)	Mean	429.3	1.504	2.510	16.008	3.885	0.955
	SD	24.8	0.263	0.159	1.787	0.234	0.265
	N	9	9	9	9	9	9
Group 4 (600 mg/kg/day)	Mean	390.5**	1.311	2.564	17.636	3.581**	0.663*
	SD	34.3	0.144	0.238	1.656	0.288	0.214
	N	10	10	10	10	10	10

* p<0.05

** p<0.01

Wi: Treated groups compared with Control using Williams’ test

Table 13. Organ weights - group mean values relative (%) to body weight (g) for F0 Males
Dose Group (mg/kg/day)		Terminal
		Body Weight	Epididymides	Kidneys	Liver	Testes	Prostrate
Statistics Test		Wi	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	451.2	0.316	0.557	3.59	0.904	0.207
	SD	33.8	0.038	0.039	0.30	0.050	0.042
	N	10	10	10	10	10	10
Group 2  (100 mg/kg/day)	Mean	429.0	0.348	0.588	3.71	0.928	0.210
	SD	17.1	0.039	0.063	0.19	0.066	0.055
	N	10	10	10	10	10	10
Group 3 (300 mg/kg/day)	Mean	429.3	0.349	0.585	3.73	0.907	0.224
	SD	24.8	0.051	0.031	0.35	0.061	0.068
	N	9	9	9	9	9	9
Group 4 (600 mg/kg/day)	Mean	390.5**	0.339	0.658**	4.52**	0.921	0.170
	SD	34.3	0.053	0.051	0.31	0.079	0.055
	N	10	10	10	10	10	10

** p<0.01

Wi: Treated groups compared with Control using Williams’ test

Table 14. Organ weights - group mean absolute values (g) for Females on Day 21 of lactation (F0)
Dose Group (mg/kg/day)		Terminal
		Body Weight	Kidneys	Liver	Ovaries	Uterus
Statistics Test		Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	268.2	1.852	13.882	0.075	0.445
	SD	12.0	0.132	1.460	0.012	0.196
	N	8	8	8	8	8
Group 2  (100 mg/kg/day)	Mean	269.8	1.908	14.913	0.085	0.468
	SD	8.2	0.121	1.184	0.013	0.132
	N	10	10	10	10	10
Group 3 (300 mg/kg/day)	Mean	274.6	1.849	16.160*	0.082	0.505
	SD	10.1	0.206	2.175	0.013	0.179
	N	7	7	7	7	7

* p<0.05

Wi: Treated groups compared with Control using Williams’ test

Table 15. Organ weights - group mean values relative (%) to body weight (g) for Females on Day 21 of lactation (F0)
Dose Group (mg/kg/day)		Terminal
		Body Weight	Kidneys	Liver	Ovaries	Uterus
Statistics Test		Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	268.2	0.691	5.17	0.0281	0.168
	SD	12.0	0.041	0.41	0.0042	0.081
	N	8	8	8	8	8
Group 2  (100 mg/kg/day)	Mean	269.8	0.707	5.53	0.0316	0.173
	SD	8.2	0.036	0.40	0.0048	0.048
	N	10	10	10	10	10
Group 3 (300 mg/kg/day)	Mean	274.6	0.672	5.87*	0.0298	0.184
	SD	10.1	0.054	0.64	0.0053	0.066
	N	7	7	7	7	7

* p<0.05

Wi: Treated groups compared with Control using Williams’ test

Conclusions:

Based on results observed in this preliminary dose range-finding study, oral gavage administration of benzaldehyde at 600 mg/kg/day to F0 generation animals was concluded to exceed the maximum tolerated dose due to the death of a majority of offspring shortly after birth. This dose level was therefore considered unsuitable for further investigation and the recommended high dose levels for the main Extended One-Generation Study (OECD 443) were 600 mg/kg/day for male and 450 mg/kg/day for female rats.

Executive summary:

A supporting dose range-finding toxicity (DRF) study was conducted to evaluate the general systemic toxic potential of the test material (Benzaldehyde; CAS# 100-52-7) in rats, including reproductive / developmental effects. This DRF was commissioned as a preliminary study to assist with setting dose levels for an Extended One-Generation Reproductive Toxicity Study (EOGRTS, OECD 443).

 

The test material was administered to Han Wistar (RccHan®;WIST) rats (10/sex/dose) once daily via oral gavage in a 0.5% methylcellulose with 0.5% Tween-80 in purified water at doses of 0, 100, 300, or 600 mg/kg/day. Male rats were treated for two weeks before pairing, up to necropsy after litters were weaned and females were treated for two weeks before pairing, throughout pairing until Day 20 of lactation. Due to a very high incidence of total litter loss amongst the high dose F0 females shortly after birth, they were prematurely terminated on animal welfare grounds since this dose level clearly exceeded the maximum tolerated dose in female rats for reproductive/developmental effects and would be unsuitable for use in future studies. The F0 males in the high dose group were subsequently prematurely terminated since the primary purpose of these males (to assess fertility) had been achieved.

 

The F1 generation (10 male and 10 female progeny from each surviving group, including controls) animals were treated from weaning (Day 21 of age) at 0, 100 or 300 mg/kg/day until Day 34 of age, with terminal procedures conducted on Day 35 of age.

 

The parameters evaluated in the study for the F0 generation included: clinical condition; post dosing observations; body weight; food consumption; estrous cycles; pre coital interval; mating performance; fertility; gestation length; organ weight; and macroscopic pathology. For all F1 offspring, clinical condition; litter size and survival; sex ratio; body weight; and macropathology were evaluated. Additionally, after weaning, the F1 generation animals were assessed for clinical condition, post dosing observations, body weight, food consumption and macroscopic pathology investigations.

 

There were no adverse treatment-related effects observed at 100 or 300 mg/kg/day either in the F0 generation, the F1 litters from birth to weaning, or in the selected F1 generation. No treatment-related premature deaths or changes in general clinical condition in the F0 or F1 generations were observed through the study period. Post-dosing observations were limited to a low incidence of transient piloerection in the F0 generation animals. Non-adverse reductions in body weight gain were apparent in F0 males at 100 or 300 mg/kg/day, accompanied by low food intake in Week 1 of treatment for F0 males given 300 mg/kg/day. Estrous cycle regularity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment at 100 or 300 mg/kg/day. 

 

Administration of the test material at 600 mg/kg/day was shown to exceed the maximum tolerated dose in pregnant female rats under the conditions of this dose-range finding study. Effects in the parental animals were limited to mean body weight loss accompanied by low food intake in F0 males in Week 1; reduced mean body weight gain in F0 females during Days 14-20 of gestation (the fetal growth phase); disruption to estrous cycle regularity in the 2-week pre-pairing period (in the absence of any effect on pre-coital interval or mating performance and fertility), and increased kidney and liver weights in the F0 males. Following parturition, however, of the first five females to give birth, only one female maintained a live litter, with all pups in the remaining four litters either cannibalised, found dead or killed for reasons of animal welfare due to poor clinical condition or poor survival prognosis. Consequently, the remaining five females were killed for reasons of animal welfare to avoid any further pup deaths (all of these five females were pregnant with live fetuses). There was no clear effect on litter size (number of offspring born, or number of uterine implantation sites/fetuses). All ten parental females showed inactive mammary tissue at macroscopic examination (This finding is preliminary and it is unclear whether it is an incidental finding or a treatment-related effect. The main OECD 443 study, currently on going, will provide a conclusive assessment on the same and the dossier will be updated upon completion of the main study).

 

Potential treatment-related effects were observed on the F1 litters but limited to slightly low post-implantation survival and marginally low birth weights in the 300 mg/kg/day group. There were no treatment-related macroscopic abnormalities detected in the F0 generation animals, the F1 offspring or the selected F1 generation animals, and differences in organ weights for the F0 generation animals were limited to slightly, but dose dependently, high liver weights for females in the 100 or 300 mg/kg/day groups.

 

Based on results observed in this preliminary dose range-finding study, oral gavage administration of benzaldehyde at 600 mg/kg/day to F0 generation animals was concluded to exceed the maximum tolerated dose due to the death of a majority of offspring shortly after birth. This dose level was therefore considered unsuitable for further investigation and the recommended high dose levels for the main Extended One-Generation Study (OECD 443) were 600 mg/kg/day for male and 450 mg/kg/day for female rats.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

other: early study on fertility

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study pre-dates OECD guidelines, but full paper contains some details of study design.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Study Description from OECD SIDS document for Benzoates:

"A single study was conducted to examine the potential reproductive toxicity of benzaldehyde , and the report was available as a translation from Romanian. A group of 10 rats of breeding age were given 2 mg benzaldehyde in oil (type not specified) by gavage every other day for 32 weeks, equivalent to about 5 mg/kg bw per day. Ten controls were used. Two pregnancies in each rat, one at 75 days and one at 180 days, were studied. The end-points examined included the number of pregnant females, number of offspring born, pup body weight at days 7 and 21 post partum, and pup viability.

At the end of treatment, the body weights of control and treated rats were similar: 265 g and 260 g, respectively. It was reported that fewer females in the group given benzaldehyde than in the control group became pregnant; however, no data or statistical analyses were presented. The authors concluded that treatment did not significantly modify any of the parameters studied. No further details were available.

The NOAEL was about 5 mg/kg bw per day."

This description was confirmed against the original paper, now attached to this endpoint study record.

GLP compliance:

no

Limit test:

yes

Specific details on test material used for the study:

No details given on source or purity of test material.

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

No details given.

Route of administration:

oral: gavage

Vehicle:

other: oil, exact type not specified

Details on exposure:

The paper describes exposure as follows: "The animals received a farm ration and once every two days 0.1 ml of oil containing 2 mg of benzoic aldehyde." It is assumed that because the dosing it described as "per os", and the delivery of food is described as a separate event, that the dosing was by gavage, and not a feeding study.

Details on mating procedure:

There are no details in the paper. However, based on the description that each dam was followed through two pregnancy cycles, the animals were most likely co-habitated throughout the study in mating pairs.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The study describes parturition at 75 days and 180 days, and then describes animal weights at 233 days. This appears to have been the basis of the estimate in the OECD description of 32 weeks.

Frequency of treatment:

Once every two days.

Details on study schedule:

Exposure assumed to begin at cohabitation, and animals monitored throughout the course of the 233 days.

Dose / conc.:

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

Remarks:

2 mg substance every other day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Study Description from OECD SIDS document for Benzoates:

"A single study was conducted to examine the potential reproductive toxicity of benzaldehyde , and the report was available as a translation from Romanian. A group of 10 rats of breeding age were given 2 mg benzaldehyde in oil (type not specified) by gavage every other day for 32 weeks, equivalent to about 5 mg/kg bw per day. Ten controls were used. Two pregnancies in each rat, one at 75 days and one at 180 days, were studied. The end-points examined included the number of pregnant females, number of offspring born, pup body weight at days 7 and 21 post partum, and pup viability.

At the end of treatment, the body weights of control and treated rats were similar: 265 g and 260 g, respectively. It was reported that fewer females in the group given benzaldehyde than in the control group became pregnant; however, no data or statistical analyses were presented. The authors concluded that treatment did not significantly modify any of the parameters studied. No further details were available.

The NOAEL was about 5 mg/kg bw per day."

This description was confirmed against the original paper, now attached to this endpoint study record.

Positive control:

None stated

Parental animals: Observations and examinations:

Two pregnancies per rat were studied, one at 75 days and one at 180 days. The number of pregnant females was examined.

Oestrous cyclicity (parental animals):

Not described.

Sperm parameters (parental animals):

Not described.

Litter observations:

The parameters examined included the number of offspring born, pup body weights at days 7 and 21 postpartum, and pup vitality.

Postmortem examinations (parental animals):

Not described.

Postmortem examinations (offspring):

Not described.

Statistics:

Paper concludes "From the data on subacute and chronic toxicity it appears that in the doses used benzoic aldehyde did not produce significant changes in any of the indicators used," but statistical methods are not described.

Reproductive indices:

Number of pregnant females.

Offspring viability indices:

- Number of pups born
- Pup weight at birth, 7 and 21 days
- Pup viability throughout

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

Although not explicityl stated, assumption is that given the conclusion that benzaldehyde "did not produce significant changes in any of the indicators used", that no mortality was observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No difference was observed between the average weights of the control and treated groups, when evaluated at 223 days after initation of treatment.

Description (incidence and severity):

N/A, as this was not a feeding study.

Description (incidence and severity):

N/A, as this was not a feeding study.

Description (incidence and severity):

N/A, as this was not a drinking water study.

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Description (incidence and severity):

The paper describes minimal clinical biochemistry for the sub-acute studies described in the paper, but does not describe this for the chronic studies (which included reproduction).

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

From a gross evaluation, the lack of difference in the reproductive indices evaluated suggests that there was no impact on estrous cycle.

Reproductive function: sperm measures:

not specified

Description (incidence and severity):

From a gross evaluation, the lack of difference in the reproductive indices evaluated suggests that there was no impact on sperm measures.

Reproductive performance:

no effects observed

The number of pregnant females was lower in the group receiving benzoic aldehyde than in the control group, however no detail is given around this observation. The overall conclusion of the paper was that there was no significant changes between the control and treated groups, therefore the decrease in the number of pregnant females can be assumed to not be statistically significant.

Dose descriptor:

NOAEL

Effect level:

ca. 5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No indications for effects on fertility at limit dose; reference: OECD risk assessment, 2004

Remarks on result:

other: Generation: P and F (migrated information)

Clinical signs:

no effects observed

Description (incidence and severity):

Although there is no specific detail, "pup viability" can be assumed to represent clinical signs. There were no differences observed in viability between pups from the control and treated groups.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Although not explicityl stated, assumption is that given the conclusion that benzaldehyde "did not produce significant changes in any of the indicators used", that no mortality was observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference between pups from control and treated parental pairs at birth, 7 or 21 days post partum.

Food efficiency:

not specified

Description (incidence and severity):

N/A, as this was not a feeding study.

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

not specified

Description (incidence and severity):

N/A, as this was not a drinking water study.

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

No statistical difference in observations between F1 generations from control and treated F0 generation.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive Toxicity

Reproductive effects observed:

no

Conclusions:

In the one-generation reproduction study in rats, it was concluded that treatment did not affect reproduction (limited study design and reporting).

Executive summary:

Cited from OECD 2004: "A single study was conducted to examine the potential reproductive toxicity of benzaldehyde, and the report was available as a translation from Romanian. A group of 10 rats of breeding age were given 2 mg benzaldehyde in oil (type not specified) by gavage every other day for 32 weeks, equivalent to about 5 mg/kg bw per day. Ten controls were used. Two pregnancies in each rat, one at 75 days and one at 180 days, were studied. The end-points examined included the number of pregnant females, number of offspring born, pup body weight at days 7 and 21 post partum, and pup viability.

 

At the end of treatment, the body weights of control and treated rats were similar: 265 g and 260 g, respectively. It was reported that fewer females in the group given benzaldehyde than in the control group became pregnant; however, no data or statistical analyses were presented. The authors concluded that treatment did not significantly modify any of the parameters studied. No further details were available. The NOAEL was about 5 mg/kg bw per day."

 

(Sporn et al. 1967, OECD 2004)

Reference 4

Endpoint:

multi-generation reproductive toxicity

Remarks:

The effect of benzoic acid on reproduction was studied through four generations.

Type of information:

other: Read-across based on metabolic series

Remarks:

In a chronic feeding study, male and female rats were exposed to up to 1% benzoic acid in the diet through four generations.

Adequacy of study:

key study

Study period:

Benzoic acid was fed in the diet to male and female rats for 11-12 weeks before the first mating and then through four generations.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Please see remarks for details.

Remarks:

While the design of this four-generation study for reproductive toxicity does not exactly match the endpoints included within the EOGRTS, it does include almost all of the endpoints associated with the OECD Guideline 416 – two generation reproductive toxicity that was previously considered adequate to satisfy the reproductive toxicity endpoint under the REACh regulation. The only missing reproductive endpoints within the Kiekebusch and Lang publication are collection of sperm parameters or estrous cyclicity data. However, the endpoints that are missing from the four-generation study with benzoic acid are available for benzyl acetate, a chemical that is metabolized completely to benzoic acid. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain). Therefore, it appears that all of the reproductive toxicity endpoints have been collected, albeit in two separate studies on benzoic acid and benzyl acetate.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Study predates current published regulatory guidelines for GLP compliance and reproductive toxicity studies; study contains most of the same elements found in a present-day two generation reproductive toxicity test and continued exposure through four generations. The premating exposure intervals were at least 10 weeks and the dose levels used were comparable to the limit dose level of 1000 mg/kg/day required under the current regulatory guidelines. Endpoints examined included collection of body weight and feed consumption data, determination of effects on sexual maturity and fertility, collection of litter and pup data, necropsy data including organ weights and histopathology, and effect on onset of sexual senescence, and lifespan. The study did not collect sperm parameters and estrous cyclicity data but these endpoints are addressed in a separate 13-week NTP feeding study in rats with the analogue benzyl acetate.

Deviations:

yes

Remarks:

Deviations did not impact the scientific integrity and validity of the study

Principles of method if other than guideline:

A feeding study was performed, in which 4 generations of rats received the test substance (0.5% or 1%). The first generation was exposed for 8 weeks an then allowed to mate (1/1 for a period of 14 days). Mating was repeated in week 48 to raise a second litter. Survival of the first and second generation was measured. The third generation was terminated after 16 weeks and examined histopathologically. In this generation weights of brain, heart, liver, spleen, kidneys and testes were determined. The fourth generation was terminated after weaning of the pups. Body weights were determined in week 4, 8 and 12 weeks of each generation (week 12 males only). Feeding efficiency was measured in all generations after 2,4, 6 and 8 weeks. Some reproduction parameters were assessed: percentage of infertility, delayed sexual maturation, litter size, total pups, surviving pups. These parameters were assessed for all generations (summed) and for thefirst two generations separately.

GLP compliance:

no

Remarks:

Study pre-dated GLPs.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Not provided in publication.
- Expiration date of the lot/batch: Not provided in publication.
- Purity test date: Not provided in publicaton.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not provided in publication.
- Stability under test conditions: Not provided in publication.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not provided in publication.

Species:

rat

Strain:

not specified

Details on species / strain selection:

TEST ANIMALS
- Source: Farbwerken Bayer (Elherfeld)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Not provided in publication.
- Weight at study initiation: 40-50g
- Fasting period before study: Not provided in publication.
- Housing: Rats were kept in double cages in a simplified Columbus-type feed apparatus.
- Diet (e.g. ad libitum): For the first 8 weeks, they were fed according to the “paired feed” method, and then ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: Not provided in publication.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not provided in publication.
- Humidity (%): Not provided in publication.
- Air changes (per hr): Not provided in publication.
- Photoperiod (hrs dark / hrs light): Not provided in publication.

IN-LIFE DATES: From: To: Not provided in publication.

Sex:

male/female

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Not provided in publication.
- Mixing appropriate amounts with (Type of food): Feed mixtures were produced in a feed mixing machine made of stainless steel. Diet consisted of commercial standard rat feed made by Lutz (Euskirchen) with sufficient benzoic acid added to achieve feed concentrations of 0.5% and 1.0%
- Storage temperature of food: Not provided in publication.

Details on mating procedure:

- M/F ratio per cage: 1M/1F
- Length of cohabitation: 14 days
- Proof of pregnancy: Not provided in publication.
- After 14 days of unsuccessful pairing ,the pairing was repeated 8 weeks later to investigate delays in sexual maturity or full sterility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Not provided in publication.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Not provided in publication.

Duration of treatment / exposure:

11-12 weeks prior to mating and through 4 generations.

Frequency of treatment:

Feed containing test material provided ad libitum.

Details on study schedule:

Details on study schedule were not provided in the publication.

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Overall, the dose level from the 0.5% diet for the entire premating period was approximately 450 and 600 mg/kg/day for the male and female rats, respectively.

Dose / conc.:

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

Remarks:

Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

No. of animals per sex per dose:

20 males/group/generation
20 females/group/generation

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations were selected based upon a previous probe study where rats consuming 5% benzoic acid in the diet died within 3 weeks due to lack of palatability of the diet and corrosive effects of the free acid on the digestive tract.

Positive control:

None stated

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: During the first 8 weeks of the experiment, weight checks were done weekly; after that, weight was checked in 4-week intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined: Yes; feed consumption data were collected throughout the exposure period over the four generations; for reporting purposes in the publication, feed consumption data are presented as “Protein efficiency (weight increase per gram of dietary protein)”
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: calculated from body weight and feed consumption data include within the publication.

Oestrous cyclicity (parental animals):

Not examined in this study.

Sperm parameters (parental animals):

Testis weight was examined in the 3rd generation.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No; Litter size was recorded on the 1st and 2nd day as well as the number of surviving young on the 21st day.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Since there was no demonstratable effect of benzoic acid on reproduction and since the data from all four generations were similar, the data were combined. Parameters examined included total number of pups born, pup survival, and litter size.

GROSS EXAMINATION OF DEAD PUPS: No information provided.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The third generation of animals was necropsied after 16 weeks of exposure. Organ weights of the brain, heart, spleen, liver, kidneys and testis were collected. Organs were examined histopathologically.
- Maternal animals: The third generation of animals was necropsied after 16 weeks of exposure. Organ weights of the brain, heart, spleen, liver, and kidneys were collected. Organs were examined histopathologically.

HISTOPATHOLOGY / ORGAN WEIGHTS: The following organs from the third generation animals were prepared for microscopic examination and weighed, respectively.: brain, heart, spleen, liver, kidneys, testis.

Postmortem examinations (offspring):

None stated.

Statistics:

Not provided in publication.

Reproductive indices:

Not provided in publication.

Offspring viability indices:

No differences were observed between generations so all data were combined.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.

Food efficiency:

no effects observed

Description (incidence and severity):

Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathology was only reported for 3rd generation animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.

There were no differences in reproduction or development of the young in the 4 observed generations exposed to 1% benzoic acid in the diet. See Table 1 in "Any other information on results incl. tables".

Key result

Dose descriptor:

NOEL

Effect level:

> 1 other: %

Based on:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Generational data were not reported separately. At concentrations up to 1% benzoic acid in the diet, there were no adverse effects on parents or offspring through four generations of test substance administration. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

Key result

Critical effects observed:

no

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.

Food efficiency:

no effects observed

Description (incidence and severity):

Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.

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:

no effects observed

Description (incidence and severity):

Organ weights were only reported for the 3rd generation animals.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathology was only reported for 3rd generation animals.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.

There were no differences in reproduction or development of the young in the 4 observed generations exposed to 1% benzoic acid in the diet. See Table 1 in "Any other information on results incl. tables".

Key result

Dose descriptor:

NOEL

Effect level:

> 1 other: %

Based on:

other:

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Generational data were not reported separately. At concentrations up to 1% benzoic acid in the diet, there were no adverse effects on parents or offspring through four generations of test substance administration. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

Key result

Critical effects observed:

no

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.

Food efficiency:

no effects observed

Description (incidence and severity):

Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights were only reported for the 3rd generation animals.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Description (incidence and severity):

Histopathology was only reported for the 3rd generation animals.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

> 1 other: %

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

There were no adverse effects on viability, sexual maturation, mortality, body weight and weight gain, food consumption and efficiency, body weights, select organ weight and pathology for any of the four generations in this study receiving up to 1% benzoic acid in the diet. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

Key result

Critical effects observed:

no

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.

Food efficiency:

no effects observed

Description (incidence and severity):

Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights were only reported for the 3rd generation animals.

Gross pathological findings:

not specified

Histopathological findings:

no effects observed

Description (incidence and severity):

Histopathology was only reported for 3rd generation animals.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.

Key result

Dose descriptor:

NOEL

Generation:

F2

Effect level:

> 1 other: %

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

There were no adverse effects on viability, sexual maturation, mortality, body weight and weight gain, food consumption and efficiency, body weights, select organ weight and pathology for any of the four generations in this study receiving up to 1% benzoic acid in the diet. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Reproduction of rats given chronic feeding of benzoic acid over 4 generations

	% Benzoic Acid in Feed
	0	0.5	1.0
Total number of females used	80	78	80
Sterile females in %	14%	10%	4%
Females with delayed sexual maturity	7.5	17	9
Litter Size	9.0 + 0.33	9.5 + 0.26	9.6 + 0.29
Total number of pups born	625	688	741
Surviving young in % of number of pups born	74%	66%	65%
Age Pairing (2nd Generation)
Number of females used	37	38	38
Litter size	6.9	7.7	7.5
Total number of pups born	173	139	171
Surviving young in % of number of pups born	72%	61%	73%

 

Conclusions:

In a chronic feeding study, there were no adverse effects on reproductive parameters including fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size when male and female rats were fed up to 1% benzoic acid in the diet over four generations. Under conditions of this study, benzoic acid is not a reproductive toxicant.

Executive summary:

In a long-term feeding study, benzoic acid was added to standard feed to achieve concentration of 0, 0.5% or 1.0% in the diet. Diets were provided ad libitum to groups of 20 rats/sex through four generations. Body weights and feed consumption data were collected throughout the exposure period. Other endpoints examined included: onset of sexual maturity, evidence of permanent sterility, onset of menopause, litter sizes, number of pups born, surviving young, organ weights and histology, and effect on lifespan. Feed consumption, body weights, and weight gain were unaffected by exposure to benzoic acid at concentrations up to 1% in the diet. There was actually an unexplained statistically significant increase in the lifespan of rats in the 0.5% exposure group, i.e., a higher percentage of rats lived longer. There were no differences between the groups exposed to benzoic acid and the control group for fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size. In addition, organ weights and histopathologic findings were similar for all groups. Under conditions of this study, there were no dose-related adverse effects on either reproductive or developmental parameters in both sexes of rats fed 1% benzoic acid in the diet over four generations.

Reference 5

Endpoint:

reproductive toxicity, other

Remarks:

Evaluation of Rodent Sperm, Vaginal Cytology, and Reproductive Organ Weight Data from National Toxicology Program 13-Week Studies

Type of information:

other: read-across based on grouping of substances (category approach): Regarding the reproductive parameters missing from the Key Study (KS) for benzoic acid, data from a 13-week repeated dose study of benzyl acetate are presented.

Remarks:

In a chronic feeding study, male and female rats were exposed to up to 1% benzoic acid in the diet through four generations.

Adequacy of study:

key study

Study period:

Publication compiles reproductive parameters from 13-week repeated dose studies from 50 different substances.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data are derived from National Toxicology Program (NTP) testing program.

Qualifier:

no guideline followed

Version / remarks:

Data are derived from National Toxicology Program series of 13-week repeated dose studies. The publication states that "Sperm morphology and vaginal cytology examinations (SMVCEs), which include evaluations of motility, concentration and head morphology of sperm from the cauda epididymis, and male reproductive organ weight data, were developed by the National Toxicology Program as a screening system for reproductive toxicants.

Principles of method if other than guideline:

The studies were designed as standard repeated-dose (13-week) and carcinogenicity (2-year) assays, commensurate with accepted standards at the time.

GLP compliance:

yes

Remarks:

GLP Compliance confirmed in NTP TR 431

Limit test:

no

Justification for study design:

The studies were designed as standard repeated-dose (13-week) and carcinogenicity (2-year) assays, commensurate with accepted standards at the time.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Benzyl acetate was obtained in two lots (8743-84 and 845585) from GivaudanCorporation (Clifton, NJ). Identity and purity analyses were conducted by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, MO), and confirmed by the study laboratory. Purity was determined to be > 98% for both lots.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: Stability studics performed at the analytical chemistry laboratory indicated that benzyl acetate was stable as a bulk chemical for 2 weeks at temperatures as high as 60" C. The stability of the bulk chemical was monitorcd periodically by the study laboratory using infrarcd and spectroscopy gas ultraviolet and chromatography; no change in purity was observed.

Species:

other: both rat and mouse are reported by Morrissey et al.

Strain:

other: F344/N rats; B6C3F1 mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Facility (Frederick, MD)
- Age at study initiation: Average of 30 days old.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days prior to study initiation
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.6 +/- 3.0 degrees C
- Humidity (%): 46-65%
- Air changes (per hr): Minimum of 10 changes/hour.
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): diet with test material prepared weekly.
- Storage temperature of food: Dose formulations stored at -20 degrees C in sealed, double plastic bags.

Details on mating procedure:

Not Applicable: The study is a 13-week repeated dose study, and is included regarding the collection of data on effects on reproductive parameters, not including mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

"Periodic analyses of the dose formulations of benzyl acctate were conducted at the sludy laboratory and the analytical chemistry laboratory using gas chromatography. The stability of 330ppm dose formulations stored in the dark at -20 degrees C was established for at least 3 weeks. Dose formulations were analyzed four times for the 13-week studies and approximately every 6 to 8 weeks during the 2-year studies. All dose formulations for rats and mice were within 10% of the target concentrations throughout the studies." "The results of periodic referee analysis pcrformed by the analytical chemistry laboratory indicated agreement with the results obtained by the study laboratory."

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Feed containing test material provided ad libitum.

Details on study schedule:

The appropriate feed was supplied every 1 to 2 days. Feed and water were available ad libitum. Feed consumption was recorded daily by cage. Rats were housed five per cage and mice were housed individually. Clinical findings were recorded once weekly. The animals were weighed at the beginning of the studies, weekly, and at the end of the studies.

Dose / conc.:

0 ppm

Remarks:

Control for both rats and mice

Dose / conc.:

3 130 ppm

Remarks:

Resulted in 235 mg/kg bw/day in rats (average of Male and Female), and 537.5 mg/kg bw/day in mice (average of Male and Female).

Dose / conc.:

6 250 ppm

Remarks:

Resulted in 470 mg/kg bw/day in rats (average of Male and Female), and 1,140 mg/kg bw/day in mice (average of Male and Female).

Dose / conc.:

12 500 ppm

Remarks:

Resulted in 915 mg/kg bw/day in rats (average of Male and Female), and 2,490 mg/kg bw/day in mice (average of Male and Female).

Dose / conc.:

25 000 ppm

Remarks:

Resulted in 1,810 mg/kg bw/day in rats (average of Male and Female), and 4,000 mg/kg bw/day in mice (average of Male and Female).

Dose / conc.:

50 000 ppm

Remarks:

Resulted in 4,200 mg/kg bw/day in rats (average of Male and Female), and 8,650 mg/kg bw/day in mice (average of Male and Female).

No. of animals per sex per dose:

10 males/group
10 females/group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Not described in NTP report.

Positive control:

None stated

Parental animals: Observations and examinations:

N/A - This study endpoint record is focused on reproductive parameters as reported by Morrissey et al., 1988.

Oestrous cyclicity (parental animals):

Estrous cyclicity evaluated in mice and rats.

Sperm parameters (parental animals):

Sperm quality parameters were evaluated at the end of the 13-week in-life phase, including:
- Sperm motility
- Sperm count
- Sperm head staining for morphology

Litter observations:

Not examined in this study.

Postmortem examinations (parental animals):

Regarding male reproductive tissues, in the basic NTP study, the right testis and seminal vesicle was necropsied, and histopathology was examined on testis with epididymis, and seminal vesicle. For testis w/ epididymis, all doses were evaluated, not just the two high doses. Regarding female reproductive tissues, in the basic NTP study, the uterus was necropsied, and histopathology of the uterus was examined.

Postmortem examinations (offspring):

N/A - This study endpoint record is focused on reproductive parameters as reported by Morrissey et al., 1988.

Statistics:

For body, organ and relative organ weight, either William's or Dunnett's test was used. Because Dunnett's test makes no allowance for dose-response relationships, Jonckheere's test (a non-parametric test for dose-response relationship) was used to determine whether Dunnett's or Williams' would be used. Potential decreases in sperm motility and increases in percentage abnormal sperm were evaluated using Jonckheere's test. Sperm density was evaluated using the Kruskal-Wallis test.

Reproductive indices:

Not examined in this study.

Offspring viability indices:

Not examined in this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Overall toxicity findings were not discussed in Morrissey et al. (1988), but were discussed in the original NTP study. The discussion of clinical findings in rats was limited to the description of "tremors, ataxia and urine stains" in the high-dose animals. The discussion of clinical findings in mice was as follows:
"The significant clinical finding was tremors, which occurred only in females and was predominant in the 50,000 ppm group; however, one 12,500
ppm female and one 25,000 ppm female also exhibited tremors. The tremors occurred first on day 16 of the study in three females receiving 50,000
ppm, day 94 in one female receiving 25,000 ppm, and day 93 in one female receiving 12,500 ppm, and continued until the end of the study."

Mortality:

mortality observed, treatment-related

Description (incidence):

In rats, nine male and nine female animals receiving the highest dose died or were killed moribund between weeks 2 and 8 of the study. One 12,500 ppm female died under anesthesia during blood collection at the end of the study.

In mice, one 50,000 ppm male mouse died, and one 50,000 ppm female mouse was sacrificed as moribund before the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In rats, the mean body weight gain and the final mean body weight of 25,000 ppm males were significantly lower than control. The final mean body weight of 25,000 ppm males was 10% lower than that of controls, whereas the final body weights of the surviving 50,000 ppm male and female animals was less than 50% of controls. Final mean body weights of males and females of other exposed groups were similar to or slightly lower than those of controls.

In mice, statistically significant dose-related decreases in final mean body weights occurred in all groups of exposed male and female mice.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In rats, feed consumption was comparable to control in all but the 25,000 and 50,000 ppm groups, where reduced consumption was attributed to decreased palatability and/or toxicity.

In mice, the mean feed consumption was lower in all groups when compared to control.

Key result

Dose descriptor:

NOEL

Effect level:

> 50 000 ppm

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

> 50 000 ppm

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

> 50 000 ppm

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

> 50 000 ppm

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

50 000 ppm

Treatment related:

no

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

This citation is being presented to cover apparent gaps in the Kieckebusch and Lang (1960) study regarding effects on reproducitve tissues/organs. Table 1 of the Morrissey et al. paper provides the following information regarding benzyl acetate on male reproductive tissues, relative to control:

Species	Terminal Body Weight	Organ Weight						Sperm Parameters
		R. cauda		R. epididymis		R. Testis
		Absolute	Relative	Absolute	Relative	Absolute	Relative	Motility (%)	Density	Abnormal (%)
Mouse	Decrease	Decrease	Increase	Decrease	Increase	Decrease	Increase	No difference	No difference	No difference
Rat	Decrease	No difference	No difference	No difference	No difference	No difference	No difference	No difference	No difference	No difference

Regarding female reproductive tissue outcomes, benzyl acetate was noted to significantly increase the mean cycle length in the high-dose group of mice.

Conclusions:

These data supplement missing endpoints from the Kieckebusch and Lang (1960) Key Study, and fill the gap in that study design. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain).

Executive summary:

These data supplement missing endpoints from the Kieckebusch and Lang (1960) Key Study, and fill the gap in that study design. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Three key studies on benzoic acid along with one supporting DRF study on Benzaldehyde currently available for assessment. The Extended One-Generation Reproductive Toxicity Study (EOGRTS, OECD 443) requested by ECHA is currently ongoing and this summary and the dossier will be updated upon receipt of the final report from the CRO.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Benzoic Acid

In 1-generation study (Sporn 1967) with the test substance no effects reproduction were reported at a dose level of 5 mg/kg bw/day (limited report). From the studies that are available on repeated dose and carcinogenicity no report on effects on the reproductive organs/tissues in both male and female laboratory animals are reported.

Again, because this dossier relies on the same data presented for benzoic acid, the following information from the benzoic acid dossier is included for completeness.

From the attached "Review of Developmental Toxicology Data for Benzoic Acid":

Kiekebusch and Lang (1960) describes an experiment where 0, 0.5 and 1% benzoic acid was mixed into the feed and fed to rats (groups of 20 males and females/group) for a period of 11-12 weeks prior to breeding. The dietary concentrations were selected based upon a previous probe study where rats consuming 5% benzoic acid in the diet died within three weeks due to lack of palatability of the diet and corrosive effects of the free acid on the digestive tract. The weight range of the animals at the start of the four generation study was 40 -50 grams. Based on the mean value of the feed consumed, the authors reported that the rats in the 1% dietary group received “150 mg of benzoic acid/day”. Based upon the starting body weights and the mean body weight increases reported in Table 1 of the paper, the starting dose level in the males and female animals was in the range of 3750 mg/kg/day for the 1% dose level. After four weeks of feeding, the dose level decreased to 1304 and 1485 mg/kg/day in the male and female rats, respectively. At eight weeks, the dose levels decreased further to 802 and 867 mg/kg/day in the male and female rats, respectively (as the rats grew and consumed roughly the same amount of feed, the effective dose level decreased with the increase in body weight). At 12 weeks of age, the dose level in the male animals was 542 mg/kg/day. The dose level for the female animals was not calculated as they were pregnant by 12 weeks and not included in the body weight gain table. Overall, the dose level for the entire premating period was approximately 900 and 1176 mg/kg/day for male and female rats, respectively. This pattern of feed consumption, body weight gain and corresponding dose levels repeated itself over the four generations of rats consuming these diets within this study. These premating exposure intervals and dose levels are generally comparable to the ten week premating exposure period and limit dose level of 1000 mg/kg/day required under the current EOGRTS test guideline.

Body weights and feed consumption data were collected throughout the exposure periods over the four generations. The body weight data within this publication is presented as body weight “increase” (Table 1) and the feed consumption data is presented as “Protein efficiency (weight increase per gram of dietary protein)” in Table 2. There was no effect of feeding 0.5 or 1% benzoic acid in the diet on feed consumption (efficiency), body weight gain or body weights over the four generations of rats tested in this study.

Effects of benzoic acid on sexual maturity and fertility were determined by pairing male and female animals for 14 days in the 11th and 12th weeks of exposure. Unsuccessful matings were repeated eight weeks later to determine if sexual maturity was delayed or if the mating pairs were infertile. Finally, during the 48thweek of exposure, mating trials were again conducted to evaluate the onset of reproductive senescence in the older animals. Litter data was collected on postnatal days 1, 2 and 21 in each of the four generations. Since there was no demonstrable effect of benzoic acid on reproduction and since the data from all four generations was similar, the data from all four generations was combined into a single table (Table 3). There was no difference between the groups with benzoic acid exposure and the control group for fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size.

The Kiekebusch and Lang paper also evaluated the effect of feeding 0.5 or 1.0% benzoic acid on the lifespan of rats. There was no difference between the 1.0% group and the control animals in terms of lifespan, while the incorporation of 0.5% benzoic acid in the diet appeared to lengthen the lifespan of the rats consuming this dietary concentration. The third generation of animals was necropsied after 16 weeks of exposure and organ weights of the brain, heart, liver, spleen kidneys and testes were collected. Tissues were saved and histopathological examination of the tissues did not reveal any difference between the control and two treated groups for either the organ weights or the histopathology findings.

While the design of this four-generation study for reproductive toxicity does not exactly match the endpoints included within the EOGRTS, it does include almost all of the endpoints associated with the OECD Guideline 416 – two generation reproductive toxicity that was previously considered adequate to satisfy the reproductive toxicity endpoint under the REACh regulation. The only missing reproductive endpoints within the Kiekebusch and Lang publication are collection of sperm parameters or estrous cyclicity data. However, the endpoints that are missing from the four-generation study with benzoic acid are available for benzyl acetate, a chemical that is metabolized completely to benzoic acid. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain). Therefore, it appears that all of the reproductive toxicity endpoints have been collected, albeit in two separate studies on benzoic acid and benzyl acetate. Based upon the recent information that has become available following receipt of the English translation of the Kiekebusch and Lang publication, the reproductive toxicity endpoint for benzoic acid should be satisfied based upon the available study data. Shortcomings of the Kiekebusch and Lang 1960 study can be addressed using the data from the 13-week feeding study with benzyl acetate (Morrissey et al., 1988). For these reasons, ECHA should consider the reproductive toxicity endpoint for benzoic acid to be satisfied with the available study data. Overall, taking into consideration both the Kieckebusch and Lang (1960), and Morrissey et al. (1988) studies, no effects on reproductive performance and off-spring were reported at 1% the test substance in feed (500 mg/kg bw). Therefore, the NOAEL for toxicity to reproduction is set at 500 mg/kg bw.

Benzaldehyde

A supporting dose range-finding toxicity (DRF) study (Labcorp Early Development Laboratories Ltd., 2022; draft report) was conducted to evaluate the general systemic toxic potential of the test material (Benzaldehyde; CAS# 100-52-7) in rats, including reproductive / developmental effects. This DRF was commissioned as a preliminary study to assist with setting dose levels for an Extended One-Generation Reproductive Toxicity Study (EOGRTS, OECD 443).

 

The test material was administered to Han Wistar (RccHan®;WIST) rats (10/sex/dose) once daily via oral gavage in a 0.5% methylcellulose with 0.5% Tween-80 in purified water at doses of 0, 100, 300, or 600 mg/kg/day. Male rats were treated for two weeks before pairing, up to necropsy after litters were weaned and females were treated for two weeks before pairing, throughout pairing until Day 20 of lactation. Due to a very high incidence of total litter loss amongst the high dose F0 females shortly after birth, they were prematurely terminated on animal welfare grounds since this dose level clearly exceeded the maximum tolerated dose in female rats for reproductive/developmental effects and would be unsuitable for use in future studies. The F0 males in the high dose group were subsequently prematurely terminated since the primary purpose of these males (to assess fertility) had been achieved.

 

The F1 generation (10 male and 10 female progeny from each surviving group, including controls) animals were treated from weaning (Day 21 of age) at 0, 100 or 300 mg/kg/day until Day 34 of age, with terminal procedures conducted on Day 35 of age.

 

The parameters evaluated in the study for the F0 generation included: clinical condition; post dosing observations; body weight; food consumption; estrous cycles; pre coital interval; mating performance; fertility; gestation length; organ weight; and macroscopic pathology. For all F1 offspring, clinical condition; litter size and survival; sex ratio; body weight; and macropathology were evaluated. Additionally, after weaning, the F1 generation animals were assessed for clinical condition, post dosing observations, body weight, food consumption and macroscopic pathology investigations.

 

There were no adverse treatment-related effects observed at 100 or 300 mg/kg/day either in the F0 generation, the F1 litters from birth to weaning, or in the selected F1 generation. No treatment-related premature deaths or changes in general clinical condition in the F0 or F1 generations were observed through the study period. Post-dosing observations were limited to a low incidence of transient piloerection in the F0 generation animals. Non-adverse reductions in body weight gain were apparent in F0 males at 100 or 300 mg/kg/day, accompanied by low food intake in Week 1 of treatment for F0 males given 300 mg/kg/day. Estrous cycle regularity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment at 100 or 300 mg/kg/day. 

 

Administration of the test material at 600 mg/kg/day was shown to exceed the maximum tolerated dose in pregnant female rats under the conditions of this dose-range finding study. Effects in the parental animals were limited to mean body weight loss accompanied by low food intake in F0 males in Week 1; reduced mean body weight gain in F0 females during Days 14-20 of gestation (the fetal growth phase); disruption to estrous cycle regularity in the 2-week pre-pairing period (in the absence of any effect on pre-coital interval or mating performance and fertility), and increased kidney and liver weights in the F0 males. Following parturition, however, of the first five females to give birth, only one female maintained a live litter, with all pups in the remaining four litters either cannibalised, found dead or killed for reasons of animal welfare due to poor clinical condition or poor survival prognosis. Consequently, the remaining five females were killed for reasons of animal welfare to avoid any further pup deaths (all of these five females were pregnant with live fetuses). There was no clear effect on litter size (number of offspring born, or number of uterine implantation sites/fetuses). All ten parental females showed inactive mammary tissue at macroscopic examination (This finding is preliminary and it is unclear whether it is an incidental finding or a treatment-related effect. The main OECD 443 study, currently on going, will provide a conclusive assessment on the same and the dossier will be updated upon completion of the main study).

 

Potential treatment-related effects were observed on the F1 litters but limited to slightly low post-implantation survival and marginally low birth weights in the 300 mg/kg/day group. There were no treatment-related macroscopic abnormalities detected in the F0 generation animals, the F1 offspring or the selected F1 generation animals, and differences in organ weights for the F0 generation animals were limited to slightly, but dose dependently, high liver weights for females in the 100 or 300 mg/kg/day groups.

 

Based on results observed in this preliminary dose range-finding study, oral gavage administration of benzaldehyde at 600 mg/kg/day to F0 generation animals was concluded to exceed the maximum tolerated dose due to the death of a majority of offspring shortly after birth. This dose level was therefore considered unsuitable for further investigation and the recommended high dose levels for the main Extended One-Generation Study (OECD 443) were 600 mg/kg/day for male and 450 mg/kg/day for female rats.

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. 

Effects on developmental toxicity

Description of key information

Rat (OECD 414): Developmental NOAEL = 300 mg/kg/day

Rabbit (OECD 414): Developmental NOAEL = 225 mg/kg/day

A key OECD Guideline 414 prenatal developmental toxicity study in rats was conducted to evaluate the effect/s of the test material (Benzaldehyde; CAS# 100-52-7) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Labcorp, 2022a; draft report).

 

The test material was administered via oral gavage to three groups of 22 time-mated female Han Wistar rats at doses of 100, 300 or 600 mg/kg/day once daily from Day 6 to 20 after mating. A similarly constituted Control group received the vehicle, 0.5% methylcellulose with 0.5% Tween-80 in purified water at the same volume dose as the treated groups. Animals were killed on Day 21 after mating for reproductive assessment and fetal examination/s.

 

Clinical observations, body weight, and food consumption were recorded and all adult females were examined macroscopically at necropsy on Day 21 after mating. Blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight was recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

No mortality or treatment-related signs of clinical toxicity were observed at routine examination or following dose administration through the study period. One female (4F 71) in the 600 mg/kg/day dose group was euthanized (for welfare reasons) on Day 11 of gestation, due to general poor clinical condition with clinical signs of decreased activity, piloerection, hunched posture and abnormal (swaying) gait. This female was also abnormally cold to touch and had a distended abdomen. Macroscopic examination revealed abnormal, pale contents in the stomach and a distended stomach. The female was found to be pregnant with 12 live fetuses. Since no other premature deaths occurred on study and no similar signs were observed among the remaining females, this premature death was not considered to be treatment-related.

 

Body weight gain, adjusted maternal body weight gain and food consumption were unaffected by treatment. At 600 mg/kg/day mean gravid uterine weight was slightly, but not statistically significantly, lower than Control.

 

Mean serum T3 or T4 concentration of maternal females in the 100 or 300 mg/kg/day remained unaffected post-treatment. Statistically significantly low mean serum T3 and T4 concentrations were observed at 600 mg/kg/day when compared to control animals (reduction of 24% and 26%, respectively). In the absence of any differences in thyroid weight or microscopic changes and no effect of treatment on TSH concentration at the highest dose level tested, it was considered that thyroid gland function was highly unaffected by treatment and these differences were adaptive not adverse. Thyroid weights were unaffected by treatment and gross necropsy did not reveal any remarkable treatment-related findings. There were no treatment-related microscopic findings observed either.

 

Litter data as assessed by the mean number of implantations, resorptions, extent of pre-and post-implantation losses, live young and sex ratio were unaffected by treatment with the test material. Mean total litter weights were slightly, but not statistically significantly, lower than Control at 600 mg/kg/day. Male, female and overall fetal weights were statistically significantly low at 600 mg/kg/day when compared to Controls (overall fetal weight was 16% lower than Controls). There was no effect of treatment on fetal weights at 100 or 300 mg/kg/day. Mean placental weights were unaffected by treatment and no effect of treatment on fetal ano-genital distance was observed.

 

At 600 mg/kg/day there were 8 fetuses in 4 litters affected with major eye abnormalities; anophthalmia and microphthalmia with absent optic nerve observed at fixed visceral examination and small orbital socket(s) observed at skeletal examination, all exceeding the Historical Control Data (HCD) range. In addition, at 600 mg/kg/day there was an increase in the incidence of minor skeletal abnormalities/variants compared to concurrent control which exceeded the HCD range; medially thickened/kinked ribs (the incidence was also increased to a lesser extent at 100 or 300 mg/kg/day), short supernumerary cervical ribs, supernumerary 14th rib(s), 20 thoracolumbar vertebrae, incompletely ossified cranial centres, sternebrae and cervical vertebrae These findings, although treatment-related, were not considered adverse.

 

At 100 and 600 mg/kg/day, there were 5 fetuses with apparent malrotation of the hindlimb(s) noted at necropsy external examination. At subsequent skeletal examination, although the limbs still appeared slightly malrotated, both the bones and cartilage of the hindlimbs were normal. This finding was therefore considered to be incidental and not treatment-related.

 

Based on the results observed in this prenatal developmental toxicity study in the rat, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 600 mg/kg/day and the NOAEL for embryo-foetal development was determined to be 300 mg/kg/day.

  

In another key OECD Guideline 414 pre-natal developmental toxicity study, the influence of the test material (Benzaldehyde; CAS# 100-52-7) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy was evaluated in the New Zealand White rabbit (Labcorp, 2022b; draft report).

 

The test material in a 0.5% methylcellulose with 0.5% Tween-80 vehicle was administered to female rabbits (24/dose) once daily via oral gavage at dose levels of 0, 100, 225, or 450 mg/kg bw/day from Day 6 to Day 28 after mating. Clinical observations, body weight, and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

Exposure to the test material at 100 or 225 mg/kg bw/day was well tolerated in maternal rabbits. No treatment-related changes in clinical condition orsigns were observed and there were no premature deaths recorded at these dose levels through the study period. Overall group mean body weight gainbetween GD 15-29 and GD 6-29 was observed to be low at 225 mg/kg bw/day. Group mean gravid uterine weight, adjusted weight loss, food consumption were unaffected by treatment and there were no treatment-related abnormalities recorded at necropsy at 100 or 225 mg/kg bw/day.

 

Exposure to the test material at 450 mg/kg bw/day was not tolerated in a majority of maternal rabbits and this group was prematurely dispatched to necropsy for welfare reasons with most females receiving 8 to 12 consecutive doses of benzaldehyde. Near to total food inappetence was observed in most females either immediately following the start of treatment or following an initial clear reduction in food consumption. This correlated with persistent body weight losses and manifested in clinical signs of decreased fecal pellets, reduced pellet size and thin build. A total of nine females were prematurely dispatched on welfare grounds prior to the subsequent termination of the group. A similar but slightly less severe pattern of reduced food consumption, low body weight gain and clinical signs were seen in the majority of the remaining females given 450 mg/kg bw/day, and no treatment-related abnormalities were found at macroscopicexamination with embryo-fetal survival unaffected by maternal treatment at this dose level.

 

There was no effect of maternal treatment of benzaldehyde on litter data. Mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss, placental weight, total litter or group mean male, female, or overall fetal weights were unaffected by treatment at 100 or 225 mg/kg bw/day.No adverse treatment-related external, major or minor, skeletal or visceral abnormalities were observed in fetuses following maternal exposure to the test material at 100 or 225 mg/kg bw/day.

 

Based on the effects observed, the systemic and developmental toxicity No Observed Adverse Effect Level (NOAEL) for benzaldehyde in rabbits was determined to be 225 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-AUG-19 to 2022-MAR-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

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.

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST.(Han Wistar)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 11 to 12 weeks old at arrival
- Weight at study initiation: 181 to 235 grams
- Fasting period before study: Not specified
- Housing: Individually in solid (polycarbonate) bottom cages during the acclimatization and gestation periods
- Diet (e.g. ad libitum): SDS VRF1 Certified, pelleted diet ad libitum
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum
- Acclimation period: 4 days (from arrival on Day 2 after mating to commencement of treatment on Day 6 after mating)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2021-AUG-26 to 29 To:2021-SEP-14 to 21

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:
Formulations were prepared weekly and in advance of the first day of dosing under yellow light. The required amount of test material was weighed into a suitable sized beaker to hold the whole formulation. Vehicle was added to achieve the required weight and the formulation was mixed using a high shear homogenizer until fully homogenous. Formulations were magnetically stirred for a minimum of 20 minutes then transferred to their final containers via syringe while being magnetically stirred. A series of formulations at the required concentrations were prepared by dilution of individual weighing’s of the test material and stored refrigerated at 2 to 8°C.

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: 0, 10, 30, or 60 mg/mL for the 0, 100, 300, and 600 mg/kg/day dose groups, respectively.
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity
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: One day at ambient temperature (15 to 25°C); 15 days when stored refrigerated (2 to 8°C).

Achieved concentration
Samples of each formulation prepared for administration in the first and last week of treatment were analyzed for achieved concentration of the test material.

Details on mating procedure:

- Impregnation procedure: 88 female rats purchased timed pregnant (arrived at CRO on Day 2 post mating)

Natural mating with Han Wistar of established fertility at the supplier’s facility. Males and females were not related and positive evidence of mating was designated as Day 0.

Duration of treatment / exposure:

Day 6 to Day 20 (inclusive) after mating.

Frequency of treatment:

Once daily

Duration of test:

Day 6 to Day 20 (inclusive) after mating.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses selected in this study (0, 100, 300 and 600 mg/kg/day) were were based on the results of the preliminary study (Labcorp Study Number 8462299). In that study doses of 0, 100, 300 and 600 mg/kg/day were investigated. There were no premature deaths and no treatment-related clinical signs. At 600 mg/kg/day, transient signs of decreased activity (in one female on Day 16 after mating and in two females on Day 17 of gestation), irregular breathing (in one female on Day 17 of gestation) and salivation (in one female on Day 18 of gestation) were observed post-dosing. Overall mean maternal body weight gain was slightly reduced at the 300 and 600 mg/kg/day dose levels, as was mean maternal body weight adjusted for the contribution of the gravid uterine weight at 600 mg/kg/day. Mean gravid uterine weight was slightly low at 600 mg/kg/day. Food intake was slightly low during Days 18-21 of gestation for females at 600 mg/kg/day.

One female that received 600 mg/kg/day was found not to be pregnant at macroscopic examination and one female that received 300 mg/kg/day had a total litter resorption. Post-implantation loss was higher than Control in all groups of treated females, however, no dose response was apparent. There was no clear effect of treatment on the number of implantations, resorptions (early, late and total), pre-implantation loss, or the number of live young or sex ratio at any dose level investigated. Mean placental weights were lower than Control for all groups of treated females, with a dose response apparent. Mean total litter weight was lower than Control at 600 mg/kg/day. Male, female, and overall fetal weights were statistically significantly lower than Control at 600 mg/kg/day. There were no findings at macroscopic examination of the adult females and no treatment related findings at external examination of the fetuses at any dose level investigated.

The high-dose level should produce some maternal and/or developmental toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity. Therefore, doses of 0, 100, 300 and 600 mg/kg/day were investigated in the current OECD 414 study in the rat.

- Rationale for animal assignment (if not random): Randomly to each group on the day of arrival
- Fasting period before blood sampling for (rat) dam thyroid hormones: No overnight deprivation of food
- Time of day for (rat) dam blood sampling: At termination
- Other:

Route of administration: The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Animal Model: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan®:WIST) strain was used because of the historical control data available at the CRO.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health and a viability check was performed near the start and end of each working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each adult was recorded on the day after arrival (Day 3 after mating) and on Days 6, 9, 12, 15, 18, and 21 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 after mating inclusive

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
Animals surviving until the end of the scheduled study period were killed by carbon dioxide asphyxiation on Day 21 after mating
- Organs examined: Gravid uterine weight (including cervix and ovaries); Thyroid

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
- Other: Fetuses (live and dead)

Blood sampling:

Blood samples (1.0 mL) were collected under Isoflurane anesthesia from the sublingual vein of all adult females (excluding premature deaths) at termination. Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation (At 2000g for ten minutes at 4°C). Two aliquots (Aliquot 1: 0.2 mL serum for T3/T4 and Aliquot 2: residual serum for TSH) were taken per animal.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
- Anogenital distance of all live rodent pups: Yes

Statistics:

For information on statistics please see 'Any other information on materials and methods incl. tables'.

Indices:

Reproductive Assessment
Prenatal losses were separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations) / Number of corpora lutea) x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations - Number of live fetuses) / Number of implantations) x 100

Historical control data:

July 2015 - Present Historical Control Data (HCD) presented in Tables 10, 11, 12, and 13 in 'Any other information on results incl. tables'.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related signs of clinical toxicity were observed at routine examination or following dose administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (4F 71) in the 600 mg/kg/day dose group was euthanized (for welfare reasons) on Day 11 of gestation, due to general poor clinical condition with clinical signs of decreased activity, piloerection, hunched posture and abnormal (swaying) gait. This female was also abnormally cold to touch and had a distended abdomen. Macroscopic examination revealed abnormal, pale contents in the stomach and a distended stomach. The female was found to be pregnant with 12 live fetuses. Since no other premature deaths occurred on study and no similar signs were observed among the remaining females, this premature death was not considered to be treatment-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no clear effect of treatment on body weight gain at any dose level investigated. No adverse effect of treatment on mean body weight gain adjusted for the contribution of the gravid uterine weight was observed at any dose level investigated. Mean adjusted body weight on Day 21 of gestation and mean adjusted body weight gain at 300 or 600 mg/kg/day were statistically significantly higher than Control. There was no clear effect of treatment on gravid uterine weight at 100 or 300 mg/kg/day. At 600 mg/kg/day, mean gravid uterine weight was slightly, but not statistically significantly, lower than Control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption remained unaffected through the study period.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Mean serum T3 or T4 concentration of maternal females in the 100 or 300 mg/kg/day remained unaffected post-treatment. Statistically significantly low mean serum T3 and T4 concentrations were observed at 600 mg/kg/day when compared to control animals (reduction of 24% and 26%, respectively). These differences were not considered to be adverse in the absence of any microscopic changes of the thyroid and with no effect of treatment on TSH concentration.

Mean serum TSH concentrations of animals in all treatment groups appeared unaffected by treatment with the test material. The mean serum TSH concentration in the 300 mg/Kg/day dose group was observed to be statistically significantly higher than control. However, one animal (3F 62) had a much higher TSH concentration than the other animals within the group, and therefore, the observed increased mean was more likely due to biological variation than exposure to the test material. No such effects were observed in animals in the 100 and 600 mg/kg/day dose groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid weights were unaffected by treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy did not reveal any remarkable treatment-related findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic findings in the thyroids

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Dead fetuses:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Three rats in the control group, two in the 100 mg/kg/day dose group, one in the 300 mg/kg/day dose group, and one female in the 600 mg/kg/day dose group were found to be not pregnant at macroscopic examination.

Other effects:

no effects observed

Description (incidence and severity):

Mean placental weights were unaffected by treatment

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 600 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Systemic Toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment on fetal weights at 100 or 300 mg/kg/day. However, male, female and overall fetal weights were statistically significantly low at 600 mg/kg/day when compared to Controls (overall fetal weight was 16% lower than Controls).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Number of live young was unaffected by treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by treatment.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Mean total litter weights were observed to be slightly, but not statistically significantly lower than Control at 600 mg/kg/day.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Fetal ano-genital distance was unaffected by treatment.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, treatment-related

Description (incidence and severity):

8 fetuses in 4 litters in the 600 mg/kg/day dose group were observed to have major eye abnormalities; anophthalmia and microphthalmia exceeding the Historical Control Data (HCD) range. These findings were considered to be adverse treatment-related effects.

At 100 and 600 mg/kg/day, there were 5 fetuses with apparent malrotation of the hindlimb(s) noted at necropsy external examination. At subsequent skeletal examination, although the limbs still appeared slightly malrotated, both the bones and cartilage of the hindlimbs were normal. This finding was therefore considered to be incidental and not treatment-related.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

8 fetuses in 4 litters in the 600 mg/kg/day dose group were observed to have small orbital socket(s) at skeletal examination, all exceeding the Historical Control Data (HCD) range. These findings were considered to be adverse treatment-related effects.

Additionally, at 600 mg/kg/day, there was an increase in the incidence of minor skeletal abnormalities/variants compared to concurrent control which exceeded the HCD range; medially thickened/kinked ribs (the incidence was also increased to a lesser extent at 100 or 300 mg/kg/day), short supernumerary cervical ribs, supernumerary 14th rib(s), 20 thoracolumbar vertebrae, incompletely ossified cranial centres, sternebrae and cervical vertebrae. These findings, although treatment-related, were not considered adverse.

At 100 and 600 mg/kg/day, there were 5 fetuses with apparent malrotation of the hindlimb(s) noted at necropsy external examination. At subsequent skeletal examination, although the limbs still appeared slightly malrotated, both the bones and cartilage of the hindlimbs were normal. This finding was therefore considered to be incidental and not treatment-related.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

8 fetuses in 4 litters in the 600 mg/kg/day dose group were observed to have major eye abnormalities; anophthalmia and microphthalmia with absent optic nerve at fixed visceral examination, all exceeding the Historical Control Data (HCD) range. These findings were considered to be adverse treatment-related effects.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

skeletal: skull

visceral/soft tissue: eye

Description (incidence and severity):

8 fetuses in 4 litters in the 600 mg/kg/day dose group were observed to have major eye abnormalities; anophthalmia and microphthalmia with absent optic nerve at fixed visceral examination. Small orbital socket(s) were also observed in these animals at skeletal examination, all exceeding the Historical Control Data (HCD) range.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

The mean concentrations of all test material formulations analyzed were within ± 20% of nominal concentrations.

 

Table 2. Results of Formulation Analysis
Sample ID	Nominal Concentration (mg/mL)	Concentration Found (mg/mL)	Expressed as % of Nominal
2021-AUGUST-28
F8046	0	ND	-
F8047	10	9.24	92
F8048	30	28.2	94
F8049	60	58.4	97
2021-SEPTEMBER-02
F8074	0	ND	-
F8073	10	8.32	83
F8072	30	26.7	89
F8071	60	55.9	93

Results are a mean of duplicate analysis

Table 3. Body weight and body weight change - group mean values (g) during gestation
Dose Group (mg/kg/day)		Day								Change
		3	6	9	12	15	18	21	6-9	9-12	6-12	12-15	15-18	18-21	6-21
Statistics test		Av	Av	Wi	Wi	Wi	Wi	Wi	Sh	Wi	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	203	211	218	230	243	268	294	7	12	19	12	25	26	82
	SD	11.2	10.4	11.6	13.4	14.8	17.7	22.0	2.9	4.2	4.8	4.5	5.9	6.5	15.0
	N	19	19	19	19	19	19	19	19	19	19	19	19	19	19
Group 2 (Control – 100 mg/kg/day)	Mean	201	210	217	229	241	266	294	7	12	19	12	25	28	84
	SD	9.5	9.4	9.9	12.8	15.1	18.0	22.4	2.6	4.2	4.7	6.1	7.6	7.3	18.2
	N	20	20	20	20	20	20	20	20	20	20	20	20	20	20
Group 3 (Control – 300 mg/kg/day)	Mean	208	217	226	238	249	275	303	9	12	21	11	26	27	86
	SD	17.8	15.8	17.2	17.9	17.7	18.8	22.9	3.4	3.7	4.9	4.1	3.8	6.2	11.9
	N	21	21	21	21	21	21	21	21	21	21	21	21	21	21
Group 4 (Control – 600 mg/kg/day)	Mean	205	214	221	234	246	269	296	7	12	19	12	23	27	82
	SD	13.1	13.1	16.2	16.5	16.9	21.5	29.2	5.6	4.6	5.0	3.4	6.9	9.9	20.1
	N	21	21	21	20	20	20	20	21	20	20	20	20	20	20

Av       Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Sh      Treated groups compared with Control using Shirley’s test

Wi      Treated groups compared with Control using Williams’ test

Table 4. Gravid Uterine Weight, Adjusted Body Weight and Adjusted Body Weight Change - Group Mean Values (g) on Day 21 of Gestation
Dose Group (mg/kg/day)		Body Weight Day 6	Terminal Body Weight Day 21	Body Weight Change Day 6-21	Gravid Uterine Weight	Adjusted Body Weight Day 21	Adjusted Body Weight Change Day 6-21
Statistics test		Av	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	211	293	82	67.3	226	15
	SD	10	22.0	15.0	17.22	16.9	10.5
	N	19	19	19	19	19	19
Group 2 (Control – 100 mg/kg/day)	Mean	210	294	83	63.5	230	20
	SD	9.4	22.4	18.3	18.86	9.3	6.6
	N	20	20	20	20	20	20
Group 3 (Control – 300 mg/kg/day)	Mean	217	303	86	64.7	238*	21*
	SD	15.8	23.2	12.1	15.39	16.8	8.2
	N	21	21	21	21	21	21
Group 4 (Control – 600 mg/kg/day)	Mean	214	296	81	57.7	238*	24**
	SD	13.4	29.3	20.2	21.28	16.7	10.1
	N	20	20	20	20	20	20

Av       Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi      Treated groups compared with Control using Williams’ test

*         p<0.05

**       p<0.01

Table 5. Litter Data – Group Mean Values on Day 21 of Gestation
Dose Group (mg/kg/day)		Corpora lutea	Implantations	Resorptions			Implantation loss (%)		Live young			Sex ratio (%M)
				Early	Late	Total	Pre-	Post-	Male	Female	Total
Statistics test		Wi	Wi				Wi	Wi			Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	11.6	10.4	0.7	0.1	0.7	11.4	9.0	5.3	4.4	9.6	56.4
	SD	1.61	2.75	0.82	0.23	0.81	19.28	12.89	2.47	2.06	2.87	22.17
	N	19	19	19	19	19	19	19	19	19	19	19
Group 2 (Control – 100 mg/kg/day)	Mean	12.2	9.7	0.7	0.1	0.7	18.1	6.5	4.4	4.7	9.0	46.4
	SD	2.53	3.16	0.93	0.22	0.98	24.13	8.62	2.16	2.21	2.97	18.06
	N	20	20	20	20	20	20	20	20	20	20	20
Group 3 (Control – 300 mg/kg/day)	Mean	12.0	10.0	0.5	0.0	0.5	15.4	4.8	5.0	4.5	9.5	53.5
	SD	2.06	2.46	1.12	0.00	1.12	19.19	10.28	2.09	2.04	2.50	17.88
	N	21	21	21	21	21	21	21	21	21	21	21
Group 4 (Control – 600 mg/kg/day)	Mean	12.0	9.9	0.7	0.0	0.7	18.9	9.0	4.5	4.8	9.3	46.5
	SD	1.45	3.39	0.81	0.00	0.81	23.72	16.70	2.06	2.35	3.63	16.84
	N	20	20	20	20	20	20	20	20	20	20	20

Wi      Treated groups compared with Control using Williams’ test

Table 6. Placental, Litter and Fetal Weights - Group Mean Values (g) on Day 21 of Gestation
Dose Group (mg/kg/day)		Placental weight	Total litter weight	Male fetal weight	Female fetal litter	Overall fetal weight	Fetal weight (g)	Ano-genital distance (mm)
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	0.50	49.73	5.31	5.04	5.20	5.3	3.6
	SD	0.185	14.541	0.232	0.231	0.225	0.23	0.16
	N	19	19	19	18	19	19	19
Group 2 (Control – 100 mg/kg/day)	Mean	0.48	47.20	5.39	5.17	5.25	5.4	3.8
	SD	0.101	15.063	0.347	0.270	0.306	0.35	0.19
	N	20	20	19	20	20	19	19
Group 3 (Control – 300 mg/kg/day)	Mean	0.46	48.15	5.21	4.97	5.09	5.2	3.6
	SD	0.063	12.231	0.408	0.375	0.383	0.41	0.23
	N	21	21	21	21	21	21	21
Group 4 (Control – 600 mg/kg/day)	Mean	0.50	40.25	4.49**	4.27**	4.36**	4.5**	3.6
	SD	0.099	15.818	0.448	0.416	0.419	0.45	0.19
	N	20	20	19	20	20	19	19

**       p<0.01

Wi      Treated groups compared with Control using Williams’ test

Table 7. Fetal Examinations - Major Abnormality Findings - Group Incidences
Group		Fetuses				Litters
		Group 1 (Control – 0 mg/kg/day)	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)	Group 1 (Control – 0 mg/kg/day)	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)
Number Examined		183	180	200	185	19	20	21	20
Total Number Affected		1	3	0	13	1	2	0	7
Head Skeletal Visceral	Small orbital socket (s) Brachygnathia Anophthalmia/absent optic nerve Microphthalmia/absent optic nerve	0 0 0 0	0 1 0 0	0 0 0 0	2 0 3 4	0 0 0 0	0 1 0 0	0 0 0 0	2 0 2 4
Cervical/Thoracic Skeletal Visceral	Multiple thoracic vertebral/rib abnormalities Complete situs inversus	0 1	0 0	0 0	1 0	0 1	0 0	0 0	1 0
Appendicular Skeletal External	Misshapen clavicle Bent scapula(e) Hyperflexion handlimbs(s) Malrotated handlimbs(s)	0 0 0 0	1 0 1 3	0 0 0 0	0 2 0 2	0 0 0 0	1 0 1 2	0 0 0 0	0 1 0 2

Table 8. Fetal examinations - minor skeletal abnormality and variants findings - group incidences
		Fetuses				Litters
Group		Group 1 (Control – 0 mg/kg/day)	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control - 600 mg/kg/day)	Group 1 (Control – 0 mg/kg/day)	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)
Number Examined		89	87	95	86	19	20	21	19
Minor skeletal abnormalities Vertebral element abnormality	thoracic scoliosis minimal	0 0	0 0	0 0	1 1	0 0	0 0	0 0	1 1
Ribs	Medially thickened/kinked absent	0 0	3 0	6 0	22 1	0 0	2 0	5 0	8 1
Sternebrae	Biparte ossified Misaligned ossification sites Misaligned hemicentres Supernumerary site Misshapen	0 0 0 1 0	0 0 1 0 0	0 1 0 0 1	1 0 4 0 0	0 0 0 1 0	0 0 1 0 0	0 1 0 0 1	1 0 3 0 0
Costal cartilage	2^ndnot connected to sternum Partially fused Misaligned Branched 7^thnot connected to sternum	1 0 0 0 0	0 0 1 0 0	0 0 0 1 0	0 1 4 0 2	1 0 0 0 0	0 0 1 0 0	0 0 0 1 0	0 1 3 0 1
Appendicular Total affected by one or more of the above	Misshapen cranial margin scapula(e)	0 2	1 5	0 8	0 27	0 2	1 4	0 5	0 10
Rib and vertebral configuration Cervical rib 13^thrib Number of 14^thribs	Short supernumerary short Short supernumerary full supernumerary total	4 0 11 1 11	0 0 21 0 21	4 0 21 0 21	12 1 36 5 37	4 0 7 1 7	0 0 12 0 12	2 0 13 0 13	9 1 13 3 13
Thoracolumbar vertebrae Pelvic girdle	20 unilateral cranial shift unilateral caudal shift	1 0 0	0 0 3	0 0 1	5 1 2	1 0 0	0 0 2	0 0 1	4 1 2
Delayed/ Incomplete ossification/unossified Cranial Sternebrae	cranial centres 5^thand/or 6^th 1^stto 4^th total	1 8 2 10	1 13 1 14	4 13 0 13	9 71 12 73	1 4 2 5	1 9 1 9	2 9 0 9	4 19 8 19
Vertebrae	Cervical thoracic	0 1	0 0	1 0	22 4	0 1	0 0	1 0	12 3
Increased ossification Cranial Talus	partially fused jugal to maxilla ossified	3 0	1 1	1 0	3 0	3 0	1 1	1   0	3 0

Table 9. Fetal Examinations - Minor Visceral Abnormality and Necropsy Findings - Group Incidences
		Fetuses				Litters
Group		Group 1 (Control – 0 mg/kg/day)	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)	Group 1 (Control – 0 mg/kg/day)	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)
Number Examined		94	93	105	99	18	19	21	20
Number of Heads Examined at Detailed Visceral Examination		94	93	105	99	18	19	21	20
Head abnormalities (fixed visceral) Eyes	folded retina dilated orbital sinus variation in lens shape	1   1   1	0   2   0	1   0   0	0   1   0	1 1   1	0 2 0	1 0 0	0 1   0
Brain Total affected by one or more of the above	Subdural haemorrhage	3 6	3 5	2 3	1 2	3 6	3   5	2 3	1   2
Necropsy observations (fresh visceral) Thymus     Abdominal cavity Kidney(s) Ureter(s) Umbilical Total affected by one or more of the above	partially undescended lobe blood in haemorrhage dilated left	0     0 0 1 10     11	1     1 1 0 5    7	0   0 0 0 6      6	0     2 0 0 10        12	0     0 0 1 7     7	1     1 1 0 3        4	0     0 0 0 6        6	0     1 0 0 7        7
Necropsy observations (external) Tail Skin Total affected by one or more of the above	tail tip kinked subcutaneous edema	1 0 1	0 0     0	0 0 0	1 1 2	1 0 1	0 0 0	0 0 0	1 1 2

Table 10. Fetal Examinations - Major Historical Control Data
Group		8461367								HCD Range
		Fetuses				Litters				Fetuses	Litters
		Group 1 (Control – 0 mg/kg/day)	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)	Group 1 (Control – 0 mg/kg/day)	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)
Number Examined		183	180	200	185	19	20	21	20	1213	119
Head Skeletal	small orbital socket(s) Brachygnathia	0 0	0 1	0 0	2 0	0 0	0 1	0 0	2 0	0-1 0-1	0-1 0-1
Visceral	Anophthalamia/absent optic nerve Microphthalmia/absent optic nerve	0   0	0   0	0   0	3   4	0 0	0   0	0 0	2 4	0-0   0-0	0-0   0-0
Cervical/Thoracic Skeletal	Multiple thoracic vertebral/rib abnormalities	0	0	0	1	0	0	0	1	0-0	0-0
Appendicular Skeletal	Misshapen clavicle Bent scapula(e)	0 0	1 0	0 0	0 2	0 0	1 0	0 0	0 1	0-0 0-0	0-0 0-0
External	Hyperflexion hindlimb(s) Malrotated hindlimb(s)	0   0	1   3	0   0	0   2	0   0	1   2	0   0	0   2	0-0   0-1	0-0   0-1

Table 11. Fetal Examinations - Minor Skeletal Historical Control Data
Group		8461367								HCD Range
		Fetuses				Litters				Fetuses	Litters
		Group 1 (Control – 0 mg/kg/day	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)	Group 1 (Control – 0 mg/kg/day	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)
Number Examined		89	87	95	86	19	20	21	19	635	119
Minor skeletal abnormalities
Vertebral element abnormality	thoracic	0	0	0	1	0	0	0	1	0-1	0-1
	scoliosis minimal	0	0	0	1	0	0	0	1	0-0	0-0
Ribs	Medially thickened/kinked	0	3	6	22	0	2	5	8	0-2	0-2
	absent	0	0	0	1	0	0	0	1	0-0	0-0
Sternebrae	Biparte ossified	0	0	0	1	0	0	0	1	0-0	0-0
	Misaligned ossification sites	0	0	1	0	0	0	1	0	0-1	0-1
	Misaligned hemicentres	0	1	0	4	0	1	0	3	0-3	0-3
	Misshapen	0	0	1	0	0	0	1	0	0-2	0-1
Costal cartilage	Partially fused	0	0	0	1	0	0	0	1	0-2	0-2
	Misaligned	0	1	0	4	0	1	0	3	0-4	0-3
	Branched	0	0	1	0	0	0	1	0	0-0	0-0
	7th not connected to sternum	0	0	0	2	0	0	0	1	0-1	0-1
Appendicular	Misshapen cranial margin scapula(e)	0	1	0	0	0	1	6	0	0-1	0-1

Table 12. Fetal Examinations - Minor Skeletal Historical Control Data
Group		8461367								HCD Range
		Fetuses				Litters				Fetuses	Litters
		Group 1 (Control – 0 mg/kg/day	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)	Group 1 (Control – 0 mg/kg/day	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)
Number Examined		89	87	95	86	19	20	21	19	635	119
Rib and vertebral configuration
Cervical rib	short supernumerary	4	0	4	12	4	0	2	9	0-3	0-3
13th rib	short	0	0	0	1	0	0	0	1	0-0	0-0
Number of 14th ribs	short supernumerary	11	21	21	36	7	12	13	13	4-33	3-16
	full supernumerary	1	0	0	5	1	0	0	3	0-3	0-2
	total	11	21	21	37	7	12	13	13	4-33	3-16
Thoracolumbar vertebrae	20	1	0	0	5	1	0	0	4	0-3	0-2
Pelvic girdle	unilateral cranial shift	0	0	0	1	0	0	0	1	0-0	0-0
	unilateral caudal shift	0	3	1	2	0	2	1	2	0-4	0-3
Delayed/Incomplete ossification / unossified
Cranial	cranial centres	1	1	4	9	1	1	2	4	0-4	0-3
Sternebrae	5th and/ or 6th	8	13	13	71	4	9	9	19	0-11	0-7
	1st to 4th	2	1	0	12	2	1	0	8	0-13	0-4
	total	10	14	13	73	5	9	9	19	1-15	1-9
Vertebrae	Cervical	0	0	1	22	0	0	1	12	0-0	0-0
	thoracic	1	0	0	4	1	0	0	3	0-0	0-0
Increased ossification
Talus	ossified	0	1	0	0	0	1	0	0	0-0	0-0

Table 13. Fetal Examinations - Visceral Historical Control Data
Group		8461367								HCD Range
		Fetuses				Litters				Fetuses	Litters
		Group 1 (Control – 0 mg/kg/day	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)	Group 1 (Control – 0 mg/kg/day	Group 2 (Control – 100 mg/kg/day)	Group 3 (Control – 300 mg/kg/day)	Group 4 (Control – 600 mg/kg/day)
Number Examined		94	93	105	99	18	19	21	20	992	119
Necropsy observations (fresh visceral)
Thymus	partially undescended lobe	0	1	0	0	0	1	0	0	0-5	0-5
Abdominal cavity	blood in	0	1	0	2	0	1	0	1	0-2	0-2
Kidney(s)	haemorrhage	0	1	0	0	0	1	0	0	0-0	0-0
Necropsy observations (external)
Skin	subcutaneous edema	0	0	0	1	0	0	0	1	0-0	0-0

Conclusions:

Based on the results observed in this prenatal developmental toxicity study in the rat, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 600 mg/kg/day and the NOAEL for embryo-foetal development was determined to be 300 mg/kg/day.

Executive summary:

A key OECD Guideline 414 prenatal developmental toxicity study in rats was conducted to evaluate the effect/s of the test material (Benzaldehyde; CAS# 100-52-7) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy.

 

The test material was administered via oral gavage to three groups of 22 time-mated female Han Wistar rats at doses of 100, 300 or 600 mg/kg/day once daily from Day 6 to 20 after mating. A similarly constituted Control group received the vehicle, 0.5% methylcellulose with 0.5% Tween-80 in purified water at the same volume dose as the treated groups. Animals were killed on Day 21 after mating for reproductive assessment and fetal examination/s.

 

Clinical observations, body weight, and food consumption were recorded and all adult females were examined macroscopically at necropsy on Day 21 after mating. Blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight was recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

No mortality or treatment-related signs of clinical toxicity were observed at routine examination or following dose administration through the study period. One female (4F 71) in the 600 mg/kg/day dose group was euthanized (for welfare reasons) on Day 11 of gestation, due to general poor clinical condition with clinical signs of decreased activity, piloerection, hunched posture and abnormal (swaying) gait. This female was also abnormally cold to touch and had a distended abdomen. Macroscopic examination revealed abnormal, pale contents in the stomach and a distended stomach. The female was found to be pregnant with 12 live fetuses. Since no other premature deaths occurred on study and no similar signs were observed among the remaining females, this premature death was not considered to be treatment-related.

 

Body weight gain, adjusted maternal body weight gain and food consumption were unaffected by treatment. At 600 mg/kg/day mean gravid uterine weight was slightly, but not statistically significantly, lower than Control.

 

Mean serum T3 or T4 concentration of maternal females in the 100 or 300 mg/kg/day remained unaffected post-treatment. Statistically significantly low mean serum T3 and T4 concentrations were observed at 600 mg/kg/day when compared to control animals (reduction of 24% and 26%, respectively). In the absence of any differences in thyroid weight or microscopic changes and no effect of treatment on TSH concentration at the highest dose level tested, it was considered that thyroid gland function was highly unaffected by treatment and these differences were adaptive not adverse. Thyroid weights were unaffected by treatment and gross necropsy did not reveal any remarkable treatment-related findings. There were no treatment-related microscopic findings observed either.

 

Litter data as assessed by the mean number of implantations, resorptions, extent of pre-and post-implantation losses, live young and sex ratio were unaffected by treatment with the test material. Mean total litter weights were slightly, but not statistically significantly, lower than Control at 600 mg/kg/day. Male, female and overall fetal weights were statistically significantly low at 600 mg/kg/day when compared to Controls (overall fetal weight was 16% lower than Controls). There was no effect of treatment on fetal weights at 100 or 300 mg/kg/day. Mean placental weights were unaffected by treatment and no effect of treatment on fetal ano-genital distance was observed.

 

At 600 mg/kg/day there were 8 fetuses in 4 litters affected with major eye abnormalities; anophthalmia and microphthalmia with absent optic nerve observed at fixed visceral examination and small orbital socket(s) observed at skeletal examination, all exceeding the Historical Control Data (HCD) range. In addition, at 600 mg/kg/day there was an increase in the incidence of minor skeletal abnormalities/variants compared to concurrent control which exceeded the HCD range; medially thickened/kinked ribs (the incidence was also increased to a lesser extent at 100 or 300 mg/kg/day), short supernumerary cervical ribs, supernumerary 14th rib(s), 20 thoracolumbar vertebrae, incompletely ossified cranial centres, sternebrae and cervical vertebrae These findings, although treatment-related, were not considered adverse.

 

At 100 and 600 mg/kg/day, there were 5 fetuses with apparent malrotation of the hindlimb(s) noted at necropsy external examination. At subsequent skeletal examination, although the limbs still appeared slightly malrotated, both the bones and cartilage of the hindlimbs were normal. This finding was therefore considered to be incidental and not treatment-related.

 

Based on the results observed in this prenatal developmental toxicity study in the rat, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 600 mg/kg/day and the NOAEL for embryo-foetal development was determined to be 300 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-FEB-14 to 2022-APR-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

yes

Remarks:

There was no impact on the validity or integrity of the study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

yes

Remarks:

There was no impact on the validity or integrity of the study.

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau

Version / remarks:

November 2000

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

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 (The Netherlands); Batch no. 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 (approx. 21°C) 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: Stable for one day at ambient temperature (15 to 25°C); 15 days when stored refrigerated (2 to 8°C)

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Colorless liquid

OTHER SPECIFICS
- Relative density: 1.042 (at 25°C)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Time-mated female rabbits

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) (Hillcrest, Dodgeford Lane, Loughborough LE12 9TE, UK)
- Age at study initiation: 18 to 22 weeks old (Day 1 after mating)
- Weight at study initiation: 2.69 to 4.28 kg (Day 1 after mating)
- Fasting period before study: Not specified
- Housing: Individually housed in suspended cages fitted with perforated floor panels and mounted in batteries.
- Diet (e.g. ad libitum): Teklad 2930, pelleted diet (200 g/animal/day); Additionally, a small supplement of autoclaved hay was given on a daily basis to
promote gastric motility. A small amount of chopped fresh vegetables were given twice weekly.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum
- Acclimation period: Five days before commencement of treatment (Days 1 to 5 of gestation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21°C
- Humidity (%): 45-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2022-MARCH-01 To: 2022-APRIL-01

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:

Formulations were formulated under yellow light with the required amount of test material weighed into a suitable sized beaker to hold the whole formulation.
Vehicle was added to achieve the required weight and mixed using a high shear homogenizer until fully homogenous. Formulations were magnetically stirred for a minimum of 20 minutes then transferred to their final containers via syringe while being magnetically stirred. Formulations were stored refrigerated (2 to 8°C) prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% methylcellulose with 0.5% Tween-80 in purified water.
- Concentration in vehicle: 0, 20, 45, or 90 mg/mL for the control, 100, 225, and 450 mg/kg/day dose groups, respectively.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of formulations during storage were determined as part of another study (Labcorp Study Number 8462384). In that study, formulations in the range 1 to 250 mg/mL were determined to be stable for: One day at ambient temperature (15 to 25°C); 15 days when stored refrigerated (2 to 8°C).

Concentration analysis: Samples of each formulation prepared for administration in the first and last week of treatment were analyzed for achieved concentration of the test material.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant (96 female rabbits)

Natural mating with New Zealand White bucks of established fertility at the supplier’s facility. Males and females were not closely related. Each female was injected intravenously with 25 i.u. luteinizing hormone. Day of mating was designated as Day 0 of gestation.

Duration of treatment / exposure:

Day 6 to 28 after mating

Frequency of treatment:

Once daily

Duration of test:

Day of mating (Day 0 of gestation) to Day 29 of mating.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control - vehicle)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low dose)

Dose / conc.:

225 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate dose)

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels selected in this study were selected based on the results of a pilot rabbit study (Labcorp Study Number 8461366) and a preliminary embryo-fetal rabbit study (Labcorp Study Number 8461368).

In the pilot rabbit study (Labcorp Study Number 8461366), a staggered dosing of 300, 600, and 450 mg/kg bw/day was used for the assessment of toxicity. The dose level of 600 mg/kg bw/day was not tolerated in rabbits with findings including total food inappetence and body weight loss from Day 6 of treatment in 2/3 females. One female treated with 600 mg/kg bw/day was found dead on Day 9, and the remaining two females in this dose group were prematurely terminated on Day 9. Based on the outcome of the pilot rabbit study, 450 mg/kg bw/day was selected as the top dose for the preliminary embryo-fetal rabbit study.

In the preliminary embryo-fetal rabbit study, dose levels of 150, 300, and 450 mg/kg bw/day were tolerated. There was no evidence of an effect of maternal treatment of benzaldehyde on litter data, as assessed by the mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss, and there was no effect of treatment on mean placental weights at 150, 300, or 450 mg/kg bw/day. Male, female and
overall fetal weights were low at 300 or 450 mg/kg bw/day, when compared to control animals, but there were no changes in male, female, or overall fetal weights at maternal treatment of 150 mg/kg bw/day. There were no findings at macroscopic examination of the adult females and no treatment-related findings at external examination of the fetuses at any dose level investigated.

The high-dose level produced some maternal and/or developmental toxic effects, but not death nor obvious suffering. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level produced no observable indications of toxicity. Therefore, doses of 0, 100, 225, and 450 mg/kg bw/day were selected for this current OECD 414 study.

- Rationale for animal assignment (if not random): Randomly assigned to group and cage position. Females mated on any one day were evenly distributed amongst the groups.

- Fasting period before blood sampling for (rat) dam thyroid hormones: Not examined

- Time of day for (rat) dam blood sampling: Not examined

- OTHER:

- Justification for route of exposure: The oral gavage route of administration was chosen to simulate the conditions of possible human exposure and as the route specified in the Final Decision Letter from the European Chemicals Agency (ECHA).

- Justification for species selected: The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available with the CRO.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on GD 1, 6, 12, 18, 23 and 29. Detailed observations were recorded daily during the treatment period at pre-dosing; 1-2 hours post-dosing; and as late as possible in the working day in relation to dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on GD 1, 3 and 6 to 29.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from GD 2 to termination.

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

POST-MORTEM EXAMINATIONS: Yes
All adult animals were sacrificed by intravenous injection of sodium pentobarbitone
- Sacrifice on gestation day # 29
- Organs examined: Uterus (gravid uterine weight)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses (live and dead)

Blood sampling:

- Plasma: Not examined
- Serum: Not examined

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Not examined

Statistics:

Please see 'Any other information on materials and methods incl. tables' for information on statistics.

Indices:

1) Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations / Number of corpora lutea) x 100

2) Post-implantation loss (%) = (Number of implantations – Number of live fetuses / Number of implantations) x 100

Historical control data:

Historical Control Data (HCD) is provided in Attachment 14.3 of the final study report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Daily exposure to the test material at 100 or 225 mg/kg bw/day was well tolerated and there were no treatment-related changes in clinical condition or signs observed.

Subsequent to exposure, 12 out of 24 females at 450 mg/kg bw/day displayed near to total food inappetence. Further clinical signs from Gestation Day 9 to dispatch were observed and consisted of decreased fecal pellets (15 out of 24 females), reduced pellet size (10 out of 24 females), and thin build (7 out of 24 females).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

No premature deaths were recorded subsequent to daily exposure to the test material at 100 or 225 mg/kg bw/day.

Daily exposure to the test material at 450 mg/kg bw/day was not tolerated by a majority of maternal rabbits and this group was prematurely dispatched to necropsy for welfare reasons with most females receiving 8 to 12 consecutive doses of the test material.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related effect on mean body weight or body weight gain was observed throughout the treatment period in females given 100 mg/kg bw/day.

Overall group mean body weight gain (GD 15-29 and GD 6-29) was statistically significantly low at 225 mg/kg bw/day when compared to the control animals.

In the 450 mg/kg bw/day dose group, 12 out of 24 females showed a response with near to total food inappetence, recorded either immediately or following an initial clear reduction in food consumption when compared to pre-dose food intake. Given the unexpected nature of this observation, extra dry and wet hay was offered. Although this was initially consumed, the performance of the females did not improve and deteriorated further and was associated with persistent body weight losses (16 out of 24 females showed a body weight loss prior to termination with one female (4F 86) losing up to 680 grams body weight between GD 6 to 13 with five more females losing 390 grams or more).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was unaffected by treatment up to 225 mg/kg bw/day. At the 450 mg/kg bw/day dose level, near to total food inappetence, recorded either immediately or following an initial clear reduction in food consumption when compared to pre-dose food intake was observed in half the animals.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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

Description (incidence and severity):

There was no effect of treatment on group mean gravid uterine weight for females dosed at 100 or 225 mg/kg bw/day on Day 29 of gestation. When overall mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight loss was recorded at 100 and 225 mg/kg bw/day, including Controls, but no clear effect of treatment was apparent.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross necropsy did not reveal any remarkable treatment-related findings in animals treated at 100 or 225 mg/kg bw/day. Macroscopic examination at termination of animals in the 450 mg/kg bw/day dose group did not reveal an increased incidence of treatment-related abnormalities. Findings were limited to one female (4F 88) that had abnormal colour of lung lobes with a thickened stomach, and one female (4F 89) that had abnormal contents and dark areas in the stomach with edema of the non-glandular and glandular mucosa.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre- and post-implantation loss were unaffected by treatment at dose levels up to 225 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Unaffected by treatment at dose levels up to 225 mg/kg bw/day.

Dead fetuses:

no effects observed

Description (incidence and severity):

Number of live young unaffected by treatment up to 225 mg/kg bw/day.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

At termination on Day 29 of gestation, one Control female (No. 4) and three females (No. 54, 58 and 72) at 225 mg/kg bw/day were found to be not pregnant. Therefore, the assessment was based on 23, 24, and 21 litters at 0, 100, and 225 mg/kg bw/day, respectively.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 225 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Systemic Toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effect on group mean male, female, or overall fetal weights was observed at 100 or 225 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live young was unaffected by treatment up to 225 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio was unaffected by treatment up to 225 mg/kg bw/day.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Litter size:
At termination on Day 29 of gestation, one Control female (No. 4) and three females (No. 54, 58 and 72) at 225 mg/kg bw/day were found to be not pregnant. Therefore, the assessment was based on 23, 24, and 21 litters at 0, 100, and 225 mg/kg bw/day, respectively.

Litter weights:
No treatment-related effect on total litterl weights was observed at 100 or 225 mg/kg bw/day.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Absent spleen was observed in one fetus in the 100 mg/kg bw/day. No such effects were observed in fetuses in the 225 mg/kg bw/day dose group.

At 100 mg/kg bw/day an increased incidence of major abnormalities (8 fetuses in 8 litters) predominantly affecting the heart/great vessels and vertebral column was observed. Most of these major abnormalities were within Historical Control Data (HCD) range. Similar findings at a lower incidence were observed in the Control group (3 fetuses in 3 litters) and at 225 mg/kg bw/day (two fetuses in one litter), all of these being within the HCD range.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Major skeletal abnormalities such as thoracic hemivertebra exceeding the HCD range were observed at 100 mg/kg bw/day (affected two fetuses in two litters). Additionally, an increase in the incidence of minor skeletal abnormalities exceeding the HCD range were observed at 225 mg/kg bw/day and included small/misshapen 1st cervical vertebral arch (two fetuses in two litters) and cervical transverse process cartilage fused to 1st (one fetus).

One incidental finding (slight increase in the incidence of bent cornua of hyoid compared to concurrent control) was reported at 100 mg/kg bw/day but this was within HCD range. Additionally, across treated groups there was an increased incidence of 20 thoracolumbar vertebrae (mostly within fetal HCD range and within litter HCD range at 225 mg/kg bw/day) and delayed/incomplete ossification of epiphyses (all within HCD range). Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity, and was therefore not considered adverse.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Major visceral abnormalities exceeding the HCD range were observed at 100 mg/kg bw/day and included truncus arteriosus, muscular ventricular septal defect and enlarged atrium (all affected two fetuses in two litters). At 225 mg/kg bw/day, one fetus was affected with one major visceral abnormality that exceeded HCD range; partially fused ascending aorta to pulmonary trunk.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 225 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental Toxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Formulation Analysis

Formulations administered in the first week were shown to be within +5%/-1% of nominal concentration, confirming accurate formulation for all groups. For the last week formulations, (45 mg/mL) was outside the acceptance criteria at +19% of nominal concentration. Prior to dose administration, this formulation was re-made, and further analysis of these samples showed satisfactory achieved concentration and was within specification at -5% of nominal concentration. Last week (20 mg/mL) formulation was within specification at -13%.

Table 2. Clinical Signs - Group Distribution of Observations after Mating
Category	Observation	Number of animals affected
		Group 1 0 mg/kg bw/day	Group 2 100 mg/kg bw/day	Group 3 225 mg/kg bw/day	Group 4 450 mg/kg bw/day
	Initial Number:	24	24	24	24
Build (Deformity)	Swollen area, Vaginal area	0	1	0	0
Build Conformation	Thin	0	0	0	7
Coat	Hair loss, Dorsal surface	0	1	0	0
	Hair loss, Forelimbs	1	0	0	0
	Hair loss, Ventral surface	0	3	1	0
	Hair loss, head	1	0	0	0
	Ungroomed	0	0	1	0
Excreta	Decreased fecal pellets	0	0	0	15
	Fecal pellets reduced in size	0	0	0	10
	Feces loose	0	0	0	1
	Reduced urine output	0	0	0	1
Reason for dispatch	General poor clinical condition	0	0	0	21
Skin	Encrustation, Forelimb - right	1	0	0	0
	Encrustation, Upper ventral surface	0	0	1	0
Skin abrasion	Dry, Upper ventral surface	0	0	1	0
Skin colour	Reddening, Forelimb - right	1	0	0	0
	Reddening, Upper ventral surface	0	1	1	0
	Reddening, head	1	0	0	0
Staining	Abnormal color, brown, Forelimbs	0	0	1	1
	Abnormal color, brown, Hindlimbs	2	6	5	4
	Abnormal color, brown, Lower dorsal surface	0	1	0	0
	Abnormal color, brown, tail	6	6	5	6
	Abnormal color, red, tail	1	1	0	0
	Abnormal color, yellow, Forelimbs	0	4	5	4
	Abnormal color, yellow, Hindlimbs	4	7	10	6
	Abnormal color, yellow, tail	4	4	6	2

Table 3. Body weight and body weight change - group mean values (kg) during gestation
Group		Change
		1-6	6-15	15-29	6-29
	Statistics test	Av	Wi	Wi	Wi
Group 1 0 mg/kg bw/day	Mean	0.02	0.14	0.28	0.41
	SD	0.077	0.088	0.108	0.138
	N	23	23	23	23
Group 2 100 mg/kg bw/day	Mean	0.03	0.12	0.25	0.37
	SD	0.093	0.095	0.111	0.148
	N	24	24	24	24
Group 3 225 mg/kg bw/day	Mean	0.02	0.12	0.18*	0.31*
	SD	0.109	0.092	0.132	0.164
	N	21	21	21	21
Group 4 450 mg/kg bw/day	Mean	0.02	-0.03
	SD	0.085	0.269
	N	22	12

Group 4 during gestation presented for information, not included in statistical analysis

Av Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi Treated groups compared with Control using Williams’ test

Table 4. Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (kg) on Day 29 of gestation
Dose Group (mg/kg/day)		Body Weight Day 6	Terminal Body Weight Day 29	Body Weight Change Day 6-29	Gravid Uterine Weight	Adjusted Body Weight Day 29	Adjusted Body Weight Change Day 6-29
Statistics test		Av	Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	3.40	3.80	0.41	0.523	3.28	-0.11
	SD	0.358	0.279	0.138	0.0776	0.263	0.146
	N	23	23	23	23	23	23
Group 2 (Control – 100 mg/kg/day)	Mean	3.40	3.78	0.37	0.468	3.31	-0.10
	SD	0.287	0.238	0.145	0.1020	0.232	0.131
	N	24	24	24	24	24	24
Group 3 (Control – 225 mg/kg/day)	Mean	3.44	3.75	0.30*	0.466	3.28	-0.16
	SD	0.378	0.305	0.162	0.0962	0.267	0.181
	N	21	21	21	21	21	21

Av Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi Treated groups compared with Control using Williams’ test

Table 5. Macropathology - Group Distribution of Findings on Day 29 of Gestation
Tissue/Organ and Findings	Number of Animals Affected
Number of Animals	23	24	21
Number of animals within normal limits	22	21	20
Kidneys
Cyst(s)	0	1	0
Liver
Abnormal Color	0	1	0
Lobular Pattern Accentuated	0	0	1
Pale Area(s)	0	0	1
Lungs and Bronchi
Dark area(s)	1	2	0

Table 6. Placental, litter and fetal weights - group mean values (g) on Day 29 of gestation
Dose Group (mg/kg/day)		Placental Weight	Total Litter Weight	Male Fetal Weight	Female Fetal Weight	Overall Fetal Weight
Statistics test		Wi	Wi	Wi	Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	4.9	341.7	41.6	40.6	41.1
	SD	0.54	59.18	2.94	3.43	2.64
	N	23	23	23	23	23
Group 2 (Control – 100 mg/kg/day)	Mean	5.0	318.0	41.7	42.4	42.0
	SD	0.67	76.15	4.46	4.69	3.87
	N	24	24	24	24	24
Group 3 (Control – 225 mg/kg/day)	Mean	4.8	315.9	39.4	38.9	39.1
	SD	0.66	66.08	3.68	4.01	3.54
	N	21	21	21	21	21

Wi Treated groups compared with Control using Williams’ test

Table 7. Litter data - Group Mean Values on Day 29 of Gestation
Group		Corpora Lutea	Implantations	Resorptions			Implantation Loss (%)		Live Young			Sex Ratio (% M)
				Early	Late	Total	Pre-	Post-	Male	Female	Total
	Statistics Test	Wi	Wi				sWi	Wi			Wi	Wi
Group 1 (Control – 0 mg/kg/day)	Mean	9.8	9.5	0.7	0.4	1.1	4.7	11.6	4.9	3.4	8.3	58.1
	SD	1.44	1.38	0.97	0.66	1.25	6.55	12.48	1.68	1.16	1.61	13.88
	N	23	23	23	23	23	23	23	23	23	23	23
Group 2 (Control – 100 mg/kg/day)	Mean	9.5	8.4	0.5	0.2	0.7	12.1	8.9	3.7	4.0	7.7	49.4
	SD	2.00	2.17	0.78	0.51	0.86	14.82	11.78	1.30	2.04	2.27	16.70
	N	24	24	24	24	24	24	24	24	24	24	24
Group 3 (Control – 225 mg/kg/day)	Mean	9.1	8.7	0.3	0.2	0.5	9.3	5.8	4.0	4.2	8.1	48.4
	SD	1.87	1.93	0.58	0.51	0.81	9.63	9.05	2.01	1.94	1.88	20.59
	N	21	21	21	21	21	21	21	21	21	21	21

Wi Treated groups compared with Control using Williams’ test

s Data were square root transformed for the statistical analysis

Table 8. Summary of Fetal examinations - Major Abnormalities and HCD Range
		Study Number 8461369						HCD Range
		Fetuses			Litters			Fetuses	Litters
Group		Group 1 0 mg/kg bw/day	Group 2 100 mg/kg bw/day	Group 3 225 mg/kg bw/day	Group 1 0 mg/kg bw/day	Group 2 100 mg/kg bw/day	Group 3 225 mg/kg bw/day
Number Examined		192	185	171	23	24	21	2776	367
Cervical/Thoracic	Thoracic hemivertebra	0	2	0	0	2	0	0-1	0-1
Visceral	Truncus arteriosus	0	2	0	0	2	0	0-1	0-1
	Muscular ventricular septal defect	0	2	0	0	2	0	0-1	0-1
	Enlarged atrium(a)	0	2	0	0	2	0	0-1	0-1
Lumbar (and abdominal) / Sacral / Caudal
External	Absent spleen	0	1	0	0	1	0	0-0	0-0

Conclusions:

Based on the effects observed, the systemic and developmental toxicity No Observed Adverse Effect Level (NOAEL) for benzaldehyde in rabbits was determined to be 225 mg/kg bw/day.

Executive summary:

In a key OECD Guideline 414 pre-natal developmental toxicity study, the influence of the test material (Benzaldehyde; CAS# 100-52-7) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy was evaluated in the New Zealand White rabbit.

 

The test material in a 0.5% methylcellulose with 0.5% Tween-80 vehicle was administered to female rabbits (24/dose) once daily via oral gavage at dose levels of 0, 100, 225, or 450 mg/kg bw/day from Day 6 to Day 28 after mating. Clinical observations, body weight, and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

Exposure to the test material at 100 or 225 mg/kg bw/day was well tolerated in maternal rabbits. No treatment-related changes in clinical condition or

signs were observed and there were no premature deaths recorded at these dose levels through the study period. Overall group mean body weight gain

between GD 15-29 and GD 6-29 was observed to be low at 225 mg/kg bw/day. Group mean gravid uterine weight, adjusted weight loss, food consumption were unaffected by treatment and there were no treatment-related abnormalities recorded at necropsy at 100 or 225 mg/kg bw/day.

 

Exposure to the test material at 450 mg/kg bw/day was not tolerated in a majority of maternal rabbits and this group was prematurely dispatched to necropsy for welfare reasons with most females receiving 8 to 12 consecutive doses of benzaldehyde. Near to total food inappetence was observed in most females either immediately following the start of treatment or following an initial clear reduction in food consumption. This correlated with persistent body weight losses and manifested in clinical signs of decreased fecal pellets, reduced pellet size and thin build. A total of nine females were prematurely dispatched on welfare grounds prior to the subsequent termination of the group. A similar but slightly less severe pattern of reduced food consumption, low body weight gain and clinical signs were seen in the majority of the remaining females given 450 mg/kg bw/day, and no treatment-related abnormalities were found at macroscopic examination with embryo-fetal survival unaffected by maternal treatment at this dose level.

 

There was no effect of maternal treatment of benzaldehyde on litter data. Mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss, placental weight, total litter or group mean male, female, or overall fetal weights were unaffected by treatment at 100 or 225 mg/kg bw/day.No adverse treatment-related external, major or minor, skeletal or visceral abnormalities were observed in fetuses following maternal exposure to the test material at 100 or 225 mg/kg bw/day.

 

Based on the effects observed, the systemic and developmental toxicity No Observed Adverse Effect Level (NOAEL) for benzaldehyde in rabbits was determined to be 225 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

225 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Two key OECD 414 Guideline studies (Rat and Rabbit) available for assessment.

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

Benzaldehyde

A key OECD Guideline 414 prenatal developmental toxicity study in rats was conducted to evaluate the effect/s of the test material (Benzaldehyde; CAS# 100-52-7) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Labcorp, 2022a; draft report).

 

The test material was administered via oral gavage to three groups of 22 time-mated female Han Wistar rats at doses of 100, 300 or 600 mg/kg/day once daily from Day 6 to 20 after mating. A similarly constituted Control group received the vehicle, 0.5% methylcellulose with 0.5% Tween-80 in purified water at the same volume dose as the treated groups. Animals were killed on Day 21 after mating for reproductive assessment and fetal examination/s.

 

Clinical observations, body weight, and food consumption were recorded and all adult females were examined macroscopically at necropsy on Day 21 after mating. Blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight was recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

No mortality or treatment-related signs of clinical toxicity were observed at routine examination or following dose administration through the study period. One female (4F 71) in the 600 mg/kg/day dose group was euthanized (for welfare reasons) on Day 11 of gestation, due to general poor clinical condition with clinical signs of decreased activity, piloerection, hunched posture and abnormal (swaying) gait. This female was also abnormally cold to touch and had a distended abdomen. Macroscopic examination revealed abnormal, pale contents in the stomach and a distended stomach. The female was found to be pregnant with 12 live fetuses. Since no other premature deaths occurred on study and no similar signs were observed among the remaining females, this premature death was not considered to be treatment-related.

 

Body weight gain, adjusted maternal body weight gain and food consumption were unaffected by treatment. At 600 mg/kg/day mean gravid uterine weight was slightly, but not statistically significantly, lower than Control.

 

Mean serum T3 or T4 concentration of maternal females in the 100 or 300 mg/kg/day remained unaffected post-treatment. Statistically significantly low mean serum T3 and T4 concentrations were observed at 600 mg/kg/day when compared to control animals (reduction of 24% and 26%, respectively). In the absence of any differences in thyroid weight or microscopic changes and no effect of treatment on TSH concentration at the highest dose level tested, it was considered that thyroid gland function was highly unaffected by treatment and these differences were adaptive not adverse. Thyroid weights were unaffected by treatment and gross necropsy did not reveal any remarkable treatment-related findings. There were no treatment-related microscopic findings observed either.

 

Litter data as assessed by the mean number of implantations, resorptions, extent of pre-and post-implantation losses, live young and sex ratio were unaffected by treatment with the test material. Mean total litter weights were slightly, but not statistically significantly, lower than Control at 600 mg/kg/day. Male, female and overall fetal weights were statistically significantly low at 600 mg/kg/day when compared to Controls (overall fetal weight was 16% lower than Controls). There was no effect of treatment on fetal weights at 100 or 300 mg/kg/day. Mean placental weights were unaffected by treatment and no effect of treatment on fetal ano-genital distance was observed.

 

At 600 mg/kg/day there were 8 fetuses in 4 litters affected with major eye abnormalities; anophthalmia and microphthalmia with absent optic nerve observed at fixed visceral examination and small orbital socket(s) observed at skeletal examination, all exceeding the Historical Control Data (HCD) range. In addition, at 600 mg/kg/day there was an increase in the incidence of minor skeletal abnormalities/variants compared to concurrent control which exceeded the HCD range; medially thickened/kinked ribs (the incidence was also increased to a lesser extent at 100 or 300 mg/kg/day), short supernumerary cervical ribs, supernumerary 14th rib(s), 20 thoracolumbar vertebrae, incompletely ossified cranial centres, sternebrae and cervical vertebrae These findings, although treatment-related, were not considered adverse.

 

At 100 and 600 mg/kg/day, there were 5 fetuses with apparent malrotation of the hindlimb(s) noted at necropsy external examination. At subsequent skeletal examination, although the limbs still appeared slightly malrotated, both the bones and cartilage of the hindlimbs were normal. This finding was therefore considered to be incidental and not treatment-related.

 

Based on the results observed in this prenatal developmental toxicity study in the rat, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 600 mg/kg/day and the NOAEL for embryo-foetal development was determined to be 300 mg/kg/day.

  

In another key OECD Guideline 414 pre-natal developmental toxicity study, the influence of the test material (Benzaldehyde; CAS# 100-52-7) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy was evaluated in the New Zealand White rabbit (Labcorp, 2022b;draft report).

 

The test material in a 0.5% methylcellulose with 0.5% Tween-80 vehicle was administered to female rabbits (24/dose) once daily via oral gavage at dose levels of 0, 100, 225, or 450 mg/kg bw/day from Day 6 to Day 28 after mating. Clinical observations, body weight, and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

Exposure to the test material at 100 or 225 mg/kg bw/day was well tolerated in maternal rabbits. No treatment-related changes in clinical condition or signs were observed and there were no premature deaths recorded at these dose levels through the study period. Overall group mean body weight gain between GD 15-29 and GD 6-29 was observed to be low at 225 mg/kg bw/day. Group mean gravid uterine weight, adjusted weight loss, food consumption were unaffected by treatment and there were no treatment-related abnormalities recorded at necropsy at 100 or 225 mg/kg bw/day.

 

Exposure to the test material at 450 mg/kg bw/day was not tolerated in a majority of maternal rabbits and this group was prematurely dispatched to necropsy for welfare reasons with most females receiving 8 to 12 consecutive doses of benzaldehyde. Near to total food inappetence was observed in most females either immediately following the start of treatment or following an initial clear reduction in food consumption. This correlated with persistent body weight losses and manifested in clinical signs of decreased fecal pellets, reduced pellet size and thin build. A total of nine females were prematurely dispatched on welfare grounds prior to the subsequent termination of the group. A similar but slightly less severe pattern of reduced food consumption, low body weight gain and clinical signs were seen in the majority of the remaining females given 450 mg/kg bw/day, and no treatment-related abnormalities were found at macroscopic examination with embryo-fetal survival unaffected by maternal treatment at this dose level.

 

There was no effect of maternal treatment of benzaldehyde on litter data. Mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss, placental weight, total litter or group mean male, female, or overall fetal weights were unaffected by treatment at 100 or 225 mg/kg bw/day.No adverse treatment-related external, major or minor, skeletal or visceral abnormalities were observed in fetuses following maternal exposure to the test material at 100 or 225 mg/kg bw/day.

 

Based on the effects observed, the systemic and developmental toxicity No Observed Adverse Effect Level (NOAEL) for benzaldehyde in rabbits was determined to be 225 mg/kg bw/day.

Sodium Benzoate

Studies with sodium benzoate are available in rats, mice, hamsters and rabbits. Dose levels applied showed no evidence of maternal toxicity. No effects on foetal development were reported. The NOAEL is based on the rat and mouse studies and set at >= 175 mg/kg bw. This level is considered to be very conservative and rats and mice seem to be the most sensitive species.

From the toxicokinetic assessment, it was concluded that the test substance after oral dosing will be metabolised to benzoic acid (see also above). Therefore it is considered acceptable to use the data from developmental study in rats and mice with the structural analogue of benzoic acid, sodium benzoate, to set the NOAEL. The NOAEL for toxicity to reproduction is set at 175 mg/kg bw.

Justification for classification or non-classification

Taking into account the available data on reprotoxicity and developmental toxicity, benzaldehyde meets the criteria for classification as a reproductive toxicant and is classified Cat 1B ( H360D: May damage the unborn child) under CLP EU Regulation 1272/2008.