Isopentyl acetate

Toxicity to reproduction

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2018 - December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Extended One-Generation Reproductive Toxicity Study, June 2018

Deviations:

yes

Remarks:

Incidental recording interruptions of the validated system for temp and rel. hum. recording occurred. The non-validated back- up recording system was used instead.This system is verified and calibrated yearly.This did not impact the validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151, July 2013

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 range finding study
- Exclusion of extension of Cohort 1B
- Termination time for F2 : not applicable
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Exclusion of developmental immunotoxicity Cohort 3
- Route of administration : oral gavage
- Other considerations: vehicle was corn oil (based on solubility).

Test material

Test material information

Constituent 1

Reference substance name:

Isopentyl acetate

EC Number:

204-662-3

EC Name:

Isopentyl acetate

Cas Number:

123-92-2

Molecular formula:

C7H14O2

IUPAC Name:

3-methylbutan-1-ol

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: OXITENO S.A. Industria e Comercio. Avenida Ayrton Senna Da Silva 3001 Maua SP Brasil.
Batch no. 180225M035041 used for test item preparation from 09 November 2018 to 22 February 2019 and 180525M035403 used for test item preparation from 01 March 2019 to 17 Mai 2019.
- Expiration date of the lot/batch: 25 February 2019 for batch No. 180225M035041 and 25 May 2019 for batch No. 180525M035403.
- Purity test date: 26 February 2018 for Batch no. 180225M035041; 10 July 2018 for Batch no. 180525M035403

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area set to maintain 15°C to 25°C.
- Stability under test conditions: Stability of the test item in the control item: Stability analyses performed previously in validation Study No. AB22399 demonstrated that the test item is stable at least 16 days in the vehicle when prepared and stored in a refrigerator set to maintain +2 to +8°C, 24 hours at room temperature (+15 to +25°C) and 3 weeks frozen (-20°C) for concentrations of 5 mg/mL to 200 mg/mL and 24 hours at room temperature (+15 to +25°C), 9 days in a refrigerator set to maintain +2 to +8°C and at least 3 weeks frozen (-15 to -25°C) for a concentration of 250 mg/mL.
- Solubility and stability of the test substance in the solvent/vehicle: Stability analyses performed previously in validation Study No. AB22399 demonstrated that the test item is stable at least 16 days in the vehicle when prepared and stored in a refrigerator set to maintain +2 to +8°C, 24 hours at room temperature (+15 to +25°C) and 3 weeks frozen (-20°C) for concentrations of 5 mg/mL to 200 mg/mL and 24 hours at room temperature (+15 to +25°C), 9 days in a refrigerator set to maintain +2 to +8°C and at least 3 weeks frozen (-15 to -25°C) for a concentration of 250 mg/mL

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel:The test item was prepared as a solution in the control item at concentrations of 25, 75 and 250 mg/mL according to Standard Operating Procedures of the Test Facility.
Formulations were prepared at least once weekly (up to 7 days) and dispensed into daily aliquots, which were stored in a refrigerator set to maintain +2 to +8°C until use. Control formulations were placed under the same storage conditions. The aliquots were allowed to warm to room temperature before dosing. After use, any residual volumes were discarded.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI. Outbred, SPF-Quality

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: F0 yes
- Age at at the initiation of dosing: (P Approximately 6 weeks.; (F1) 3 weeks (from PND21)
- Weight at study initiation:
(P) Males: 136.1 to 169.6 g. Females: 107.6 to 170.9 g;
(F1) Males: mean 62.75-66.10 g; Females: mean 60.77-63.17 g
- Fasting period before study: Adult animals were fasted overnight before sampling for clinical laboratory determinations.
- Housing: One air-conditioned room in a barrier protected unit (building K2).
Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages meeting European directive 2010/63/EU requirements with following numbers of animals per cage:
Premating: Males: 5; Females:5
Mating: Males: 1 + 1 female (housed together)
Gestation: Males: 5; Females: 1
Lactation: Females 1+ litter
Post-weaning: Males: 5; Females: 5
Each female was temporarily placed in the cage of the male for mating. Cellulose bedding (analysed twice a year for chemical and bacterial contaminants) or dust-free sawdust made from spruce tree wood (each batch is analysed for chemical and bacterial contaminants). Shredded paper was provided.
- Diet (e.g. ad libitum): Rat pelleted commercial complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analyzed. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water (e.g. ad libitum): Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system or via bottles). Water is analysed twice a year for bacterial and chemical contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Acclimation period: F0 males and females: 7 days between arrival and the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C (target range). Temperature was within the target range throughout the study.
- Humidity (%): 35 to 70% (target). Relative humidity was outside the target (minimum of 26%) on several occasion(s) during the study. These deviations were of short duration and did not noticeably affect the clinical condition of the animals or the outcome of the study.
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark.

IN-LIFE DATES: 05 November 2018 - 24 May 2019.
-F0 animals
From: Animal arrival of males and females: 05 November 2018.
To: Necropsy F0 males incl. blood sampling/urinalysis and sperm examinations: 04 to 07 February 2019.
Necropsy F0 females incl. blood sampling/urinalysis (L22-24): from 07 March 2019.
-F1 animals
From: Allocation into cohorts (PND 21): from 06 March 2019.
To:
Cohort 1A: Necropsy (PND 85-93): from 14 to 17 May 2019.
Cohort 1B: Necropsy (PND 92 to PND 100): from 21 to 24 May 2019.
Cohort 1C: Necropsy of females: 04 and 05 April 2019; Necropsy of males: 16 to 19 April 2019.
Surplus: Blood sampling for thyroid hormones and necropsy (PND 21): from 06 March 2019.
Experimental completion date (last necropsy of Cohort 1B animals): 24 May 2019.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a solution in the control item at concentrations of 25, 75 and 250 mg/mL according to Standard Operating Procedures of the Test Facility.
Formulations were prepared at least once weekly (up to 7 days) and dispensed into daily aliquots, which were stored in a refrigerator set to maintain +2 to +8°C until use. Control formulations were placed under the same storage conditions. The aliquots were allowed to warm to room temperature before dosing. After use, any residual volumes were discarded.
Formulations were maintained under continuous stirring for at least 30 minutes before and throughout the dosing procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Volume of administration: 4 mL/kg/day Individual dose volumes were calculated using the latest body weight.
- Lot/batch no. (if required): MKCF8882, MKCG3257 and MKCH1635

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: The mating period consisted of a maximum of 14 consecutive days. Mated females were separated from the males once mating was confirmed and smearing ceased.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility (was not needed).
- Further matings after two unsuccessful attempts: [no (was not applicable)]
- After successful mating each pregnant female was caged: 1 Female/cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample for analysis: Analyses were conducted on formulations used on Day 1, Week 11 and Week 22 of treatment. Samples of formulations were analysed for homogeneity (highest and lowest concentrations) and stability (highest concentration) only on Day 1 and accuracy of preparation (all concentrations) was analysed for all groups on all occasions. The homogeneity results obtained from the top, middle and bottom for the Groups 2 and 4 preparations were averaged and utilized as the
concentration results. All samples analyzed were transferred at ambient temperature to the analytical laboratory at the Test Facility.
All analyzed samples for formulations prepared at nominal concentrations of 25, 75 and 250 mg/mL of Isoamyl Acetate in vehicle (corn oil), taken from each preparation, including the vehicle, on day 1, week 11 and week 22 of treatment were in agreement with acceptance criteria (± 15%).
The deviations from the nominal concentrations ranged from -5.1 % to 2.0%.
Furthermore, on day 1 for the concentration at 250 mg/mL, the stability was checked and all results were in the acceptance criteria with deviation from the nominal concentrations between 0.5 and 0.6%.
Furthermore, the test item prepared as a suspension in the vehicle was homogenous as the RSD on the six aliquots (Top, Middle, Bottom) was ≤ 0.8 % (acceptance criteria ≤ 10%).
Finally, no test item was present in the vehicle sample.

Duration of treatment / exposure:

F0 animals:
- For both sexes: 10 weeks before mating, throughout the mating period and up to the day before necropsy.
- For females:
- during gestation (the first day of gestation is designated as G0) and at least 21 days after parturition, up to and including the day before scheduled necropsy (the first day of birth is designated as L0).
- apparently unmated females for 24 days after the last day of the mating period.
On 14 November 2018, Group 4 Female No. 181 was overdosed (unknown quantity).
F1 animals:
During lactation (up to PND 21), pups were not treated directly but could potentially have been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
After weaning (PND 21), F1-animals were treated up to and including the day before scheduled necropsy (PND 85 to 93 for cohort 1A, PND 92 to PND 100 for cohort 1B or after sexual maturation for cohort 1C). The F1 surplus animals were not dosed.

Frequency of treatment:

Once daily

Details on study schedule:

F1 animals were not mated.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

F0: 25 animals per sex per dose
F1: 20 animals per sex per cohort (1A,1B,1C) per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Rationale for the dose selection: the dose levels were selected based on the results of a preliminary 4 weeks dose range-finding study in the wistar rat by oral administration (gavage) with the test item, Isoamyl Acetate, at concentrations of 100, 300 and 1000 mg/kg/day. In this study, preliminary test item-related effects were limited to transient hypersalivation associated with abnormal foraging and a slightly higher mean body weight gain in all treated groups for both sexes and a slight increase in mean food consumption for all treated males compared with the control group during the overall dosing period. There was no clear test item-related effect on clinical chemistry parameters or organ weights.
- Rationale for animal assignment (if not random): Allocation to treatment groups: Performed during the acclimatization period, using a computer-generated randomisation. The mean body weights of each group at allocation was not statistically significantly different from each other (analysis of variance), each sex being considered separately.
Culling in PND4: To reduce variability among the litters, eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily at the beginning and at the end of each working day (including weekends and public holidays).
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed daily during the study. During treatment, animals were observed at least twice daily, up to the day prior to necropsy. These clinical observations were at least conducted prior to dosing and after dosing. A specific individual out-of-cage examination for post-dosing hypersalivation and any associated clinical changes was performed once weekly just after administration (within 5 minutes) and 1 to 3 hours later. A full clinical examination was performed on each weighing day. Towards the end of the gestation, females were examined daily for signs of parturition.
Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded if applicable.

BODY WEIGHT: Yes
- Time schedule for examinations: Each male was weighed at least weekly. Each female was weighed as follows:
-At least weekly during pre-mating and mating periods (only pre-mating data are reported).
-On Days 0, 4, 7, 11, 14, 17 and 20 of gestation.
-On Days 1, 4, 7, 10, 14, 17 and 21 of lactation.
Animals were individually weighed.

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
Food consumption of males was recorded weekly during the pre-mating period. Food consumption was quantitatively measured per cage except for the day of scheduled euthanasia.
Food consumption of females was recorded for the following periods:
-weekly during the pre-mating period
-gestation: Days 0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17, 17 to 20,
-lactation: Days 1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 21.
-Food consumption during mating was not recorded for practical reasons.

HAEMATOLOGY: Yes, in tubes containing EDTA-K2 for hematology parameters, both F0 and F1A
- Time schedule for collection of blood: on the day of necropsy. In addition, a blood smear was prepared for each animal on each occasion but not further analysed.
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes, overnight.
- How many animals: 10/sex/group (randomly selected), (ten surviving females with litter for F0 females).
- Parameters examined:
Red blood cell count
Hemoglobin concentration
Hematocrit (Packed cell volume in the study plan)
Mean corpuscular volume
Mean corpuscular hemoglobin concentration
Mean corpuscular hemoglobin
Reticulocyte count (absolute
Platelet count
White blood cell count
Neutrophil count (absolute and percentage)
Lymphocyte count (absolute and percentage)
Monocyte count (absolute and percentage)
Eosinophil count (absolute and percentage)
Basophil count (absolute and percentage)
Large unstained cells (absolute and percentage)

COAGULATION: Yes, in tubes containing trisodium citrate for coagulation parameters, both F0 and F1A
Blood samples were processed for plasma, and plasma was analyzed for the following parameters:
Activated partial thromboplastin time
Prothrombin time

CLINICAL CHEMISTRY: Yes, in tubes without anticoagulant for clinical chemistry parameters, both F0 and F1A
- Time schedule for collection of blood: on the day of necropsy.
- Animals fasted: Yes, overnight
- How many animals: 10/sex/group (randomly selected), (ten surviving females with litter for F0 females).
- Blood samples were processed for serum, and the serum was analyzed for the following parameters:
Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
Total bilirubin
Urea
Chloride
Creatinine
Calcium
Inorganic phosphorus
Total protein
Albumin
Globulin
Albumin/globulin ratio
Glucose
Cholesterol
Triglycerides
Sodium
Potassium
Bile acids

URINALYSIS: Yes, both F0 and F1A.
- Time schedule for collection of urine: Urine was collected in individual metabolism cages for approximately 16 hours from animals deprived of food and water but receiving 20 mL/kg of tap water by gavage before the beginning of the collection period.
- Metabolism cages used for collection of urine: Yes, individual metabolism cages were used.
- Animals fasted: Yes, but receiving 20 mL/kg of tap water by gavage before the beginning of the collection period.
- Parameters examined:
Color
Appearance/turbidity
Specific gravity
Volume
pH
Protein
Glucose
Urobilinogen
Bilirubin
Ketones
Blood
Sediments:
White blood cells
Red blood cells
Casts
Epithelial cells
Crystals
Bacteria

HORMONE ANALYSIS (F0- Animals, F1- Animals Cohort 1A and Surplus, PND 4 Pups):
- F0-male & females (10/sex/group): On the day of scheduled necropsy T4 & TSH
- F1 (2 pups/litter): PND 4 T4
- F1-cohort 1A (10/sex/group) On the day of scheduled necropsy T4 & TSH
- F1 cohort Surplus (All surplus pups): 20/sex/group PND 21T4 & TSH.
Blood samples of approximately 0.9 mL for F1 surplus and 1 mL for F0 and F1 cohort 1A were withdrawn from a retro-orbital sinus under isoflurane anaesthesia for F0 an F1 cohort 1A and by aorta puncture under anaesthesia using isoflurane for surplus. F0 and F1 cohort 1A animals were fasted overnight before sampling and F1-surplus animals were not fasted before sampling.
The samples were collected in tubes without anticoagulant between 7.00 and 10.30 am because of diurnal variation of hormone concentrations.
The samples were centrifuged at 1800 g, at approximately +4°C for 10 minutes and the serum was separated in two aliquots (at least 225 µL for the first aliquot and the remainder for the second aliquot).
Serum samples were stored deep-frozen (between -70 and -90°C) prior to dispatch packed in dry ice to Test Site for analysis.
The first set of samples analysed was dispatched to the Test Site after the last sampling occasion.
Study samples were analyzed for Total T4 and/or TSH at the Test Site in accordance with Standard Operating Procedure HAE-B-268 using the Immulite 1000®.

Oestrous cyclicity (parental animals):

F0: Daily vaginal lavage was performed beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal smears continued for those females with no evidence of copulation until termination of the mating period. On the day of terminal necropsy a vaginal lavage was also taken to determine the stage of estrus. Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.
F1 Cohort 1A: On the day of scheduled necropsy, a vaginal lavage was taken. Stage of estrus was determined by examining the vaginal
cytology of samples obtained by vaginal lavage procedure.

Sperm parameters (parental animals):

Parameters examined in [F0/F1 cohort 1A] male parental generations:
Method:
Immediately after sacrifice, the left (unless specified) cauda epididymis was removed and a fragment of the cauda epididymis was immersed in approximately 5 mL of 199 medium with 250 μL of 1% BSA at 37 ± 3°C for 8 to 30 minutes (the analysis was performed within 30 minutes). This allowed the spermatozoa to diffuse into the medium. A Leja slide was then filled by capillary action. The slide has a 100 μm deep cavity. The slide was placed on the stage of the Hamilton Thorne Research (HTR) IVOS computer-assisted sperm analyser. The operator checked the settings and adjusted the focus for the x10 UV objective and selected five appropriate fields (excluding those containing debris), for analysis by the system. Sufficient fields were analysed to include at least 200 spermatozoa. If this was not achieved in the first five fields selected, successive additional fields were selected, until at least 200 spermatozoa or a maximum of 10 fields had been analysed.

F0-generation:
Sperm analysis was performed for all surviving males using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive motility. In the case of an abnormality of the left epididymis, the right organ was used for sperm analysis. This occurred for the following animals: Male Nos. 51, 56, 57, 104 and 153.
Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The numbers of sperm with each type of abnormality was recorded.

F1-cohort 1A
For all surviving males of Cohort 1A, the following assessments was performed:
Sperm analysis was performed using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive motility.
Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The numbers of sperm with each type of abnormality was recorded.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
-The in-life procedures, observations, and measurements listed below were performed for the pups until weaning (PND 21).

MORTALITY/MORIBUNDITY CHECKS:
Pups were observed twice daily at the beginning and at the end of each working day (including weekends and public holidays) with the mortality/moribundity check of the dam for general health/mortality and moribundity. Pups were not removed from cage during observation, unless necessary for confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
Pups were observed twice daily at the beginning and at the end of each working day (including weekends and public holidays) with the mortality/moribundity check of the dam for general health/mortality and moribundity. Pups were not removed from cage during observation, unless necessary for confirmation of possible findings. Detailed clinical observations were made for all pups.

BODY WEIGHTS:
Live pups were individually weighed on PND 1, 4, 7, 14, 21.

SEX:
Sex was externally determined for all pups on PND 1 and 4.

ANOGENITAL DISTANCE:
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

AREOLA/NIPPLE RETENTION:
All males in each litter were examined for the number of areola/nipples on PND 13.

-The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards.

MORTALITY/MORIBUNDITY CHECKS (COHORTS 1A, 1B, 1C):
All animals were observed twice daily at the beginning and at the end of each working day (including weekends and public holidays) to detect any which were dead or moribund.
Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

CLINICAL OBSERVATIONS (COHORTS 1A, 1B, 1C):
The animals were observed daily during the study.
During treatment, animals were observed at least twice daily, up to the day prior to necropsy. These clinical observations were at least conducted prior to dosing and after dosing. A specific individual out-of-cage examination for post-dosing hypersalivation and any associated clinical changes were performed once weekly just after administration (within 5 minutes) and at approximately the same time than the standard post-dosing clinical observation.
A full clinical examination out of cage was performed weekly.
Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded if applicable.

BODY WEIGHTS (COHORTS 1A, 1B, 1C):
Animals were individually weighed at least weekly from weaning onwards.
In addition, the body weight was recorded of each female on the day of acquisition of vaginal opening and of each male on the day of acquisition of balano preputial skinfold cleavage.

FOOD CONSUMPTION (COHORTS 1A, 1B, 1C):
Food consumption was quantitatively measured by cage weekly from weaning onwards up to the day prior to scheduled necropsy.

VAGINAL OPENING (COHORTS 1A, 1B, 1C):
Vaginal opening was monitored by visual inspection of the vaginal area daily for all females from PND 28 onwards. Examinations was continued until vaginal opening is detected. Body weight was recorded on the day of acquisition of vaginal patency.

BALANOPREPUTIAL SEPARATION (COHORTS 1A, 1B, 1C):
Balanopreputial skinfold cleavage were monitored by visual inspection of the genital area daily for all males from PND 38 onwards. Examinations were continued until balanopreputial skinfold cleavage is detected. Body weight was recorded on the day of acquisition of balanopreputial skinfold cleavage.

STAGE OF ESTRUS DETERMINATION (COHORT 1A):
Stage of estrus was determined by examining the vaginal cytology of samples obtained by vaginal lavage procedure on the day of scheduled necropsy, a vaginal lavage was taken.

- The in-life procedures, observations, and measurements listed below was performed for the F1-females of Cohort 1A only.

ESTROUS CYCLE DETERMINATION (COHORT 1A):
Daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events.
Daily vaginal lavage was performed at least 2 weeks before autopsy.
Estrous stages and cycles were evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.
The day on which the first cornified smear (i.e. vaginal smear on which only cornified epithelial cells are observed is observed was recorded) in order to determine the time interval between these two events.
If a vaginal thread was observed, it was recorded daily until no longer present.
On 16 March 2019 at the time of the vaginal lavage, a pinworm was noted in the anal area for Female No. 437. This finding was observed only once for one animal in the control group. Furthermore, all rats from the F1 generation were in good general condition and none of the rats showed any clinical sign that could be related to a pinworm infection. Analysis were performed on feces samples collected from all cages including control and all results were positives for syphacia muris. This pinworm was part of the background of findings at Charles River Laboratories and is considered without impact on the health of the animals and the study outcome.

GROSS EXAMINATION OF DEAD PUPS:
Pups (extra pups on PND 4 or any moribund pups) were killed by intraperitoneal injection of sodium pentobarbitone.
Recognizable foetuses of females that died spontaneously or were euthanized in extremis were euthanized by oral administration of sodium pentobarbitone (if necessary) and examined externally, sexed.
Pups (including any found dead or killed moribund) were necropsied. For any pups found dead or killed moribund, the stomach was examined for the presence of milk and defects or cause of death was evaluated, if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Splenic lymphocyte subpopulation analysis has been performed by Flow cytometry at termination, from 10 selected animals/sex/group in Cohort 1A.
The study design was the following: F1 animals (including Cohort 1A) where not treated during lactation (up to PND 21) but could have been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces. After weaning (PND 21), F1-animals have been treated up to and including the day before scheduled necropsy (PND 85 to 93 for cohort 1A). Groups 2 to 4 have been treated by oral route once daily with Isoamyl Acetate and Group 1 treated likewise with vehicle.
Spleens were harvested from 10 selected animals/sex/group of cohort 1A at necropsy. Half of the spleen was collected and placed in a sterile tube containing approximately 5mL of sterile RPMI medium supplemented with 20% (v/v) of Serum (RPMI-20%) and maintained on molten ice until immunophenotyping processing. Single cell suspensions were prepared into RPMI-20%, by mechanical dissociation of the spleen through cell strainers (70 and 40 μm mesh) attached to a 50-mL conical tube. After washes with RPMI-20%, the cell pellets were re-suspended in RPMI- 20% Serum, enumerated with an automatic hematology analyzer (ADVIA) and adjusted at 10x10E6 cells/mL in RPMI-20% serum. Depletion of red blood cells was performed using a red blood cell lysing solution.
After washing steps, cells were stained for specific markers.
Briefly, a fixed analysis mask was applied for the identification of the cell populations: based on size and granulosity the lymphoid cells are defined. Within this cell population, gating for B-cells, T-cells, T-helper and T-cytotoxic lymphocytes and NK cells was defined. This fixed analysis mask for populations was not adjusted between animals unless in case of atypical profile at the discretion of scientific judgment. Consequently, any modification of the analysis mask was justified.
Compensation matrix was revised for each experiment before editing analyses.
Data are reported as the percentage of each population of interest within the lymphoid population Immunophenotyping data were analyzed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogeneous variances and normal distribution in all groups were analyzed using ANOVA followed by Dunnett’s test. Data showing non homogeneous variances or a non-normal distribution in at least one group were analyzed using Kruskal-Wallis test followed by Wilcoxon's rank sum test.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after a minimum of 12 weeks of treatment
- Maternal animals: All surviving animals.
Females which deliver: L22 - 24.
Females which fail to deliver:
With evidence of mating: G26 or G27.
Without evidence of mating: Approximately 26 days after the last day of the mating period.
Females with total litter loss: Dams with no surviving pups were euthanized within 24 hours after the last pup is found dead or missing.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
All animals (including total litter death) were submitted to necropsy procedures including an examination of following:
–external surface,
–all orifices,
–cranial cavity,
–thoracic and abdominal cavities and organs and their contents,
–the carcass.
Special attention was paid to the organs of the reproductive system.
Any abnormalities observed were recorded and preserved in an appropriate fixative.
The numbers of former implantation sites were recorded for all paired females.
For females sacrificed before parturition, the ovaries and uterus were removed and examined including examination of the placentae. The following data were recorded:
–pregnancy status,
–number of corpora lutea,
–number of intrauterine implantations,
–number of live embryos,
–number of intrauterine deaths (resorption sites).
The uterus of all adult females were placed in ammonium sulphide solution in order to stain any previously undetected implantation sites. The number of corpora lutea was counted.
Any foetuses were examined externally where possible and discarded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs listed below were weighed at scheduled necropsy for all animals:
Adrenal glands
Brain
Cervix (1)
Epididymides
Heart
Kidneys
Liver
Ovaries (3)
Oviducts (3)
Parathyroid glands (2)
Prostate
Seminal vesicles with coagulating gland
Spleen
Testes
Thymus
Thyroid glands (2)
Uterus (1)
Pituitary gland
(1) Cervix and uterus were weighed together.
(2) Thyroid and parathyroid glands were weighed together.
(3) Ovaries and oviducts will be weighed together.
Paired organs were weighed together with the exception epididymis and if a difference in size is observed, the abnormally-sized organ(s) will also be weighed separately.
Organs were weighed after dissection of fat and other contiguous tissues.
Organ weights were expressed as absolute values (g) and relative values (g per 100 g of body weight).

The organ/ tissues below were sampled for all animals. All organs/tissues sampled were fixed and preserved in 10% neutral formalin with the following exceptions: testes, epididymides, eyes and optic nerves were fixed in modified Davidson's fluid.
Animal identification
Macroscopic lesions
Adrenal glands
Bone (sternum) with bone marrow
Brain
Caecum
Cervix*
Coagulating gland*
Colon
Duodenum
Epididymides*
Eyes
Heart
Ileum
Jejunum
Kidneys
Larynx
Liver
Lungs (infused with formalin)
Lymph nodes (mandibular)
Lymph node (mesenteric)
Mammary gland (females andmales, inguinal area)
Nasopharynx
Oesophagus
Optic nerves
Ovaries*
Oviducts*
Parathyroid glands
Pituitary gland
Prostate*
Rectum
Salivary glands (mandibular, sublingual)
Seminal vesicles*
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach
Testes*
Thymus
Thyroid glands
Tongue
Trachea
Urinary bladder
Uterus*
Vagina*
Vas deferens*

Tissue preparation and microscopic examination for all animals was performed on the organs/tissues listed below:
Macroscopic lesions
Adrenal glands
Bone (sternum) with bone marrow
Brain
Caecum
Cervix*
Coagulating gland*
Colon
Duodenum
Epididymides*
Eyes
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (infused with formalin)
Lymph nodes (mandibular) (left only)
Lymph node (mesenteric)
Mammary gland (females and males, inguinal area)
Oesophagus
Optic nerves
Ovaries*
Oviducts*
Parathyroid glands
Pituitary gland
Prostate*
Rectum
Seminal vesicles*
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach
Testes*
Thymus
Thyroid glands
Trachea
Urinary bladder
Uterus*
Vagina*
Vas deferens*
Histopathological examinations was performed as follows:
- for all organs/tissues from all adult animals found dead or killed moribund during the study
- for all macroscopic lesions from all dose group animals
- for all organs/tissues from all adult animals of groups 1 (control) and 4 (high dose)
- for the reproductive organs of all males that fail to sire, females which fail to deliver and females with total litter loss.
All organs/tissues listed in the list above were examined histologically from groups 1 and 4, where possible. Any exceptions are recorded in the individual animal data.
All gross lesions were examined histologically where possible. The bone marrow smears were not examined in view of the lack of treatment-related findings in the other organs or parameters.
All sections were stained with haematoxylin and eosin (HE) (except bone marrow smears, which were stained using the May Grünwald Giemsa Method).

Tissue preparation and microscopic examination performed for males that fail to sire, females which fail to deliver and females with total litter loss were:
Macroscopic lesions
Cervix*
Coagulating gland*
Epididymides*
Mammary gland (females and males, inguinal area)
Ovaries*
Prostate*
Seminal vesicles*
Testes*
Uterus*
Vagina*
Vas deferens*

Legend: *: Reproductive organs.

Postmortem examinations (offspring):

SACRIFICE
-Terminal procedures-F1-generation until weaning
Unscheduled Deaths – F1 -Generation:
Pups (extra pups on PND 4 or any moribund pups) were killed by intraperitoneal injection of sodium pentobarbitone.
Recognizable foetuses of females that died spontaneously or were euthanized in extremis were euthanized by oral administration of sodium pentobarbitone (if necessary) and examined externally, sexed.
Pups (including any found dead or killed moribund) were necropsied. For any pups found dead or killed moribund, the stomach was examined for the presence of milk and defects or cause of death was evaluated, if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs.

-Terminal procedures-F1-generation from weaning onwards
For animal euthanized moribund, a necropsy was conducted within 24 hours.
Spare F1-animals that were not assigned to one of the Cohorts were sacrificed on PND 21 by intraperitoneal injection of sodium pentobarbital. Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined. Descriptions of all external abnormalities were recorded
*Cohort 1A
Scheduled necropsy of Cohort 1A was conducted on PND 85 to PND 93. Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available.
The animals had a terminal body weight recorded and were killed by carbon dioxide inhalation and exsanguination. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
*Cohort 1B
Scheduled necropsy of Cohort 1B was conducted on PND 92 to PND 100. Cohort 1B animals were not deprived of food overnight before necropsy. These animals had a terminal body weight recorded and were killed by carbon dioxide inhalation and exsanguination.
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
*Cohort 1C
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal opening or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy. Terminal body weight was not recorded. The animals were killed by carbon dioxide inhalation and exsanguination.
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. In case of macroscopic abnormalities, gross lesions were preserved in the most appropriate fixative together with the identification marks.
*Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 21. Cohort Surplus animals were not be deprived of food overnight before necropsy. Terminal body weight was recorded. On PND 21, blood samples were collected between 7.00 and 10.30 a.m. from all animals by aorta puncture under anaesthesia using isoflurane as part of the necropsy for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4).
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

GROSS NECROPSY
- Gross necropsy consisted of:
spare pups and any moribund or found dead pups until weaning (PND21): external examinations with special attention to the external reproductive organs
Cohort 1A: full post mortem examination, with special attention to the reproductive organs
Cohort 1B and C: limited examination with special attention to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
The organs listed below were weighed at scheduled necropsy for all Cohort 1A and Cohort 1B animals:
Adrenal glands
Brain
Cervix (1)
Epididymides
Heart
Kidneys
Liver
Ovaries (3)
Oviducts (3)
Parathyroid glands (2)
Prostate
Seminal vesicles with coagulating gland
Spleen
Testes
Thymus
Thyroid glands (2)
Uterus (1)
Pituitary gland
Lymph nodes (mesenteric)** (Cohort 1A only)
Lymph nodes (mandibular)** (Cohort 1A only)
(1) Cervix and uterus were weighed together.
(2) Thyroid and parathyroid glands were weighed together.
(3) Ovaries and oviducts will be weighed together.
** Lymph nodes will be weighed for 10 animals/sex/group from all treated groups.
Paired organs were weighed together with the exception epididymis and if a difference in size is observed, the abnormally-sized organ(s) will also be weighed separately.
Organs were weighed after dissection of fat and other contiguous tissues.
Organ weights were expressed as absolute values (g) and relative values (g per 100 g of body weight).

All organs/tissues sampled were fixed and preserved in 10% neutral formalin with the following exceptions: testes, epididymides, eyes and optic nerves were fixed in modified Davidson's fluid.
The Organ Processing Table for the F1 Generation is given under “Any other information on material and methods incl. tables”.
Histopathological examinations was performed as follows:
• Cohort 1A and the unscheduled deaths
- Histopathological examination was performed on tissues of animals in the control and high dose groups (Groups 1 and 4), unless otherwise indicated (see below).
- For all animals of Groups 1 and 4, and from the 10 first animals/sex of Groups 2 and 3, the bone marrow was evaluated histopathologically.
- For the 10 first animals/sex of all groups, the lymph nodes were weighed and evaluated histopathologically.
- For all females, HE-stained step sections of ovaries at a thickness of 6 micrometers (4 step sections in total, including the routine section) were prepared for quantitative evaluation of follicles.
- For all females of groups 1 and 4 of all groups (20/group), ovarian follicle counts, quantitative evaluation of primordial and small growing follicles, as well as corpora lutea, was performed.
- For the right testis of all males, detailed qualitative examination was made taking into account the tubular stages of the spermatogenic cycle. In addition to the haematoxylin and eosin-stained slide, an additional slide was prepared and examined, stained with PAS. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
• Cohort 1B
The reproductive organs of all Cohort 1B animals were processed to block stage. Further processing and histopathological examination were not required based on the results of Cohort 1A.

The study plan-defined tissues identified as not examined have been annotated in the individual animal data macroscopic and microscopic observations. The number of missing tissues was inconsequential and did not negatively impact the interpretation of this study.
Severity grades were assigned to non-neoplastic histologic diagnoses, as follows:
Present: Finding present, severity not graded.
Grade 1: Minimal/very few/very small.
Grade 2: Slight/few/small.
Grade 3: Moderate/moderate number/moderate size.
Grade 4: Marked/many/large.
Grade 5: Severe/massive/very many/very large.
A pathology peer review was conducted by David Ravelo, DVM.
Provantis version 9 was used for acquisition, storage and reporting of histopathological diagnoses. The individual animal Provantis records contain all of the recorded data and served as the basis for this report.

Statistics:

Statistical analysis was performed, where appropriate, by the data acquisition software, as follows:
The best transformation for the data (none, log or rank) was determined depending upon:
- the kurtosis of the data
- the probability of the Bartlett's test for homogeneity of the variances and
- an assessment of whether the size of the groups are approximately equal or not. Non- or log-transformed data were analyzed by parametric methods.
Rank transformed data were analyzed using non-parametric methods.
Data were then analyzed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analyzed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
All litter-based percentages were analyzed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis was significant.
Selected incidence data were analyzed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 is significant.
See further under "Any other information on material and methods incl. tables".

Reproductive indices:

The following reproductive indices were calculated:
Pre-coital interval (in days): Sum of days until successful insemination / Number of inseminated females
Copulation index (in %): (Number of inseminated females / Number of paired females) x 100
Fertility index (in %): (Number of pregnant females / Number of inseminated females) x 100
Pre-birth loss (%): [(number of implantations - number of offspring born) / number of implantations] x 100
Live birth index (in %): (number of pups born alive / number of pups born) x 100
Viability index (in %): (number of pups alive on PND / number of pups alive at birth) x 100
Lactation index (%): (number of pups alive on PND 21 / number of pups alive on PND 4 (after culling)) x 100
Sex ratio (proportion of male pups in %): (number of males / number of pups) x 100
Irregularity index: Mean standard deviation of length of the oestrous cycle / √ (length of the oestrous cycle)
Days in estrus: (number of estrus days / number of smears) x 100

Offspring viability indices:

Live birth index (in %): (number of pups born alive / number of pups born) x 100
Viability index (in %): (number of pups alive on PND / number of pups alive at birth) x 100
Lactation index (%): (number of pups alive on PND 21 / number of pups alive on PND 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of hypersalivation with or without abnormal foraging and/or pedalling during pre-mating period in all treated groups and at 300 and 1000 mg/kg/day for females during gestation and lactation. This sign was considered to be a physiological response probably related to the irritancy/taste of the test item and/or vehicle rather than a sign of systemic toxicity and was not considered as adverse.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no test item-related effect in any group on mean F0 body weight (gain) for either sexes.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no test item-related effect on food consumption for females throughout the treatment period, including during gestation and lactation for females. There was a slight increase in mean food consumption for males in all treated groups during the pre-mating period considered non adverse in view of the minor change witout impact on the terminal mean body weight.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on hematology and coagulation parameters for F0 animals at any dose for either sex.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on serum clinical chemistry parameters for F0 animals at any dose for either sex.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on urinary parameters for F0 animals at any dose for either sex.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effects on histology examinations for F0 animals in any group for either sex. In the heart, males treated at 1000 mg/kg/day had a slightly higher incidence of myocardial
degeneration (8 treated males versus 3 control males). This finding is frequently observed as part of the background changes in the Wistar rat. It was of minimal severity in males and absent in females. This difference of incidence was considered incidental.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no test item-related effect in any group on mean estrous cycle lengths.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect in any group on sperm parameters. There was a statistically significant higher mean number of combined abnormalities at 1000 mg/kg/day but in view of the low magnitude of the change and in the absence of similar findings for the F1 generation, this was considered incidental. At 1000 mg/kg/day, the mean percent of progressive sperm was slightly below the historical control data range (55% of progressively motile sperm at 1000 mg/kg/day compared with 56 to 68% in the HCD). In view of the low magnitude of the change, this was considered incidental.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no test item-related effect in any group on F0 male and female mating and fertility, male copulation, mean number of implantations, mean estrous cycle lengths, mean precoital intervals, gestation lengths and sperm parameters.

Details on results (P0)

-Mortality:
There were no unscheduled deaths in any group.
-Clinical Signs:
Males:
There was a dose-related increased incidence of transient hypersalivation with or without abnormal foraging and/or pedaling, noted just after dosing (+1 minute) and no longer present at +1h30, from Day 4 and throughout the dosing period.
Females:
Before mating, there was a dose-related increased incidence of hypersalivation with or without abnormal foraging and/or pedalling in all treated groups from Day 4. Hypersalivation and associated clinical signs were transient as they were noted just after dosing (+1 minute) and were no longer present at +1h30 for all occasions (except for female no. 144 given 300 mg/kg/day on Day 32).
During gestation and lactation, transient hypersalivation associated or not with abnormal foraging and/or pedalling was still observed with a dose-related increased incidence for all treated female groups when compared with the control group. However, signs of recovery were noted as the occurrence of hypersalivation and associated clinical signs was lower during gestation and lactation when compared with the pre-mating period.
The hypersalivation effect was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect, the signs of recovery noted after repeated administration for females and its time of occurrence (i.e. just after dosing). This sign may be related to irritancy/taste of the test item and/or vehicle and was not considered as adverse in the absence of effects on the general health status of rats.
Straub tail was noted on a single occasion (Day 32) for two females at 1000 mg/kg/day
(Nos. 187 and 190) and was therefore considered incidental.
Sporadic clinical signs including, scabs, sores, induration, noisy/laboured breathing, localised hairloss or red vaginal discharge were noted among control and treated groups and considered incidental or related to the pregnancy status of the females.
-Body Weight:
There was no statistically significant differences in mean body weight for males at the end of the dosing period or for females at the end of the pre-mating, gestation and lactation periods.
There was no test item-related effect on mean body weight gain during the pre-mating period for either sex or during gestation and lactation periods for females in any group.
Despite some transient statistically significant changes noted for both sexes, the overall mean body weight gains during the pre-mating period were comparable with, or higher than that of the control group in all treated groups for both sexes.
There was a slightly but not statistically significant lower mean body weight gain during gestation (G 0-20) at 300 mg/kg/day (approximately -9%) and during lactation at 100 and 300 mg/kg/day (-11 and -7%, respectively) when compared with the concurrent control. In the absence of similar findings at 1000 mg/kg/day and of any impact on terminal body weight, this effect was considered incidental and not test item-related and toxicologically relevant.
-Food Consumption:
There was a slight increase in mean food consumption for males (mostly statistically significant) throughout the pre-mating period that led to a statistically significant dose-related higher mean food consumption during the overall pre-mating period (+4.8%, +6.4% and +6.8% at 100, 300 and 1000 mg/kg/day, respectively, from Day 1 to Day 71) when compared with the concurrent control. This minor change had no impact on the terminal mean body weight and therefore considered no adverse.
There was a slight decrease in mean food consumption for females at 300 mg/kg/day during pre-mating, gestation and lactation periods (-6.2%, -6.5% and -8.2%, respectively, statistically significant during premating and lactation periods) and at 1000 mg/kg/day during the lactation period (-8.7%, not statistically significant) when compared with concurrent control. In the absence of clear dose-relationship and/or without associated effects on mean body weight gain, this was considered not adverse.
-Hematology:
There were no test item-related changes in hematological parameters in any group for either sex.
Minor statistically significant differences arising between control and treated animals, such as decreased neutrophil count and increase in percentage of lymphocytes at 1000 mg/kd/day for males or increased monocytes count at 1000 mg/kg/day for females were considered not toxicologically relevant as they were of low magnitude, observed in one sex only and remained within the historical control data range.
-Clinical Chemistry:
There were no test item-related changes in serum clinical chemistry parameters in any group for either sex.
Minor statistically significant differences arising between control and treated animals such as increased glucose in all treated groups and triglyceride concentration at 1000 mg/kg/day for males and increased albumin concentration at 100 mg/kg/day for females were considered not toxicologically relevant as they were of low magnitude, observed in one sex only, not dose-related and remained within the historical control data range.
-Thyroid Hormone Analysis:
Males: T4 and TSH levels in F0-males were considered to be unaffected by treatment.
Females: Total T4 and TSH levels in F0-females were considered to be unaffected by treatment.
-Urinalysis:
There was no test item-related effect on urine analysis parameters in any group for either sex.
-Organ Weights:
There were no organ weights differences that were considered to be associated with administration of the test item.
All organ weight differences including those that were statistically significant were considered to be incidental and unrelated to the test item administration.
-Macroscopic Observations:
There were no macroscopic observations that were considered to be associated with administration of the test item.
-Microscopic Observations:
There were no microscopic observations that were considered to be associated with administration of the test item.
In the heart, males treated at 1000 mg/kg/day had a slightly higher incidence of myocardial degeneration (8 treated males versus 3 control males). This finding is frequently observed as part of the background changes in the Wistar rat. It was of minimal severity in males and absent in females. This difference of incidence was considered incidental.
Any other microscopic observations were considered to be incidental and unrelated to the test item administration.
Of note, there were no relevant histopathologic findings in males that failed to sire, females that failed to deliver and or had total litter loss.
-Estrous Cycle Data:
Compared with controls, the mean length and regularity of estrous cycles in the treated groups were not affected by treatment with test item in any group.
-Sperm Analysis:
There was no test item-related effect on mean sperm count, sperm morphology or motility, including the percentage of progressively motile sperm, in any group. Mean sperm count and motility were comparable with the concurrent control and/or within the historical control data range (see Appendix 38). There was a statistically significant higher mean number of combined abnormalities at 1000 mg/kg/day but in view of the low magnitude of the change and in the absence of similar findings for the F1 generation, this was considered incidental. At 1000 mg/kg/day, the mean percent of progressive sperm was slightly below the historical control data range (55% of progressively motile sperm at 1000 mg/kg/day compared with 56 to 68% in the HCD). In view of the low magnitude of the change, this was considered incidental.
Similarly, mean cauda epididymis weights were comparable in all groups.
-Mating Index:
There was no test item-related effect on mating performance in any group.
After two weeks of mating, there were 24, 23, 25 and 25 pairs of animals that mated in the control, 100, 300 and 1000 mg/kg/day groups, respectively.
-Precoital Time:
The majority of mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle).The mean pre-coital interval was therefore normal in all groups (i.e. 2.6, 2.5, 2.9 and 2.7 days in the control, 100, 300 and 1000 mg/kg/day groups, respectively).
-Number of Implantation Sites:
Number of implantation sites was considered not to be affected by treatment.
-Fertility Index:
There was no test item-related effect on fertility. Most mated females became pregnant, with the exception of one female in each of the control, 100 and 1000 mg/kg/day groups (Female Nos. 46, 82 and 181, respectively). This was considered incidental.
-Gestation Index and Duration:
There was no test item-related effect on the gestation index and duration of gestation in any group.
There were 23, 22, 25 and 24 pregnant females in each the control, 100, 300 and 1000 mg/kg/day groups, respectively, all of which successfully completed delivery of liveborn pups with the exception of one female in each the control and 300 mg/kg/day group (Nos. 38 and 136 ) that lost their entire litter on PND 0 and one female at 300 mg/kg/day with a total litter resorption (No. 140).
The mean duration of gestation was approximately 22 (22.2-22.6) days in each group and all values remained within the historical control data range (22.0 to 22.8 days).
-Parturition/ Maternal Care:
There were no difficult littering-related clinical signs.
-Pre-Birth Loss:
There was no test item-related effect on pre-birth loss in any group.
The mean percentage of pre-birth loss were lower in all treated groups (7.4%, 6.8% and 6.8% at 100, 300 and 1000 mg/kg/day, respectively) when compared with the control group (11.0%).
-Litter Size:
The mean numbers of pups delivered were comparable with or higher than that of the control group and the historical control data mean in all treated groups
-Sex Ratio:
There was no test item-related effect on sex ration in any group.
-Live birth index:
The number of live offspring at birth compared to the total number of offspring born was considered not to be affected by treatment.
There were 6, 3, 2 and 20 stillborn pups from 4, 3, 2 and 4 litters in the control, 100, 300 and 1000 mg/kg/day groups, respectively. The live birth index was slightly lower in the 1000 mg/kg/day group (92.5%) compared with the concurrent control (97.8%). This was due to two females (Nos.179 and 198) that delivered 9 dead pups each and that disproportionately influenced the mean value. Subsequently, Female No.179 lost her entire litter on PND 1. The dose of 1000 mg/kg/day was not associated with any effect on pup viability, pre-birth loss or pup body weight and a female with total litter death was already observed in the historical control data. This finding was therefore considered incidental.
-Viability index:
The number of live offspring on Day 4 before culling compared to the number of offspring on PND 0 (viability index) was considered not to be affected by treatment.
There were 3, 2, 3 and 4 dead, missing or cannibalized pups from 3, 2, 3 and 3 litters, respectively in the control, 100, 300 and 1000 mg/kg/day, between PND 0 and PND 4. Viability index (PND 0 to 4) was consequently comparable in all groups.
-Weaning Index:
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.
The number of dead pups after culling and therefore the weaning index (PND 4 to 21) were comparable in all groups. Female No. 184 in the 1000 mg/kg/day group lost and/or cannibalized 1 pup between PND 4 and PND 21. This isolated finding was considered incidental.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No parental systemic toxicity was observed up to the highest dose level tested (1000 mg/kg).

2

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg).

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of hypersalivation with or without abnormal foraging and/or pedalling for both sexes in all treated groups. This sign was considered to be a physiological response probably related to the irritancy/taste of the test item and/or vehicle rather than a sign of systemic toxicity and was not considered as adverse.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were one (No. 462, on Day 63) and two females (Nos. 358 on Day 62 and 541 on Day 55) at 100 and 1000 mg/kg/day, respectively, euthanized moribund after swallowing the dosing cannula. Before death, marked piloerection was observed for Female No. 462 and body weight loss for Female No. 541 (-0.2g from Day 44 to Day 50).
At necropsy, a segment of the cannula was found on the esophagus of all three females. These findings were therefore clearly related to the procedure of administration. Dark foci on the liver and dark thymus were also noted for Female No. 541 given 1000 mg/kg/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no test item-related effect on mean body weight gain throughout the treatment period for F1 animals in any group for either sex.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no test item-related effect on mean food consumption throughout the treatment period for F1 animals in any group for either sex.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There was no test item-related effect on hematology and coagulation parameters for the F1 males and females after approximately 12 weeks of dosing (cohort 1A) at any dose.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was no test item-related effect on serum clinical chemistry parameters for the F1 males and females after approximately 12 weeks of dosing (cohort 1A) at any dose.
Total T4 and TSH levels in F1 cohort 1A animals were considered to be unaffected by treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no test item-related effect on urinary parameters for the F1 males and females after approximately 12 weeks of dosing (cohort 1A) at any dose.

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the splenic lymphocyte population for the F1 males and females after approximately 12 weeks of dosing (cohort 1A) at any dose.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related effects on organs weights for F1 animals in any group for either sex.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on macroscopic examination for F1 animals in any group for either sex.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related effects on histology examination for F1 animals in any group for either sex.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related effects on T4 and TSH in F1 cohort 1A animals after approximately 12 weeks of dosing in any group.

Details on results:

General toxicity F1-Generation Data (Cohort 1A, 1B and 1C) from Weaning onwards (considered "P1" under the results section)

-Mortality:
There were one (No. 462, on Day 63) and two females (Nos. 358 on Day 62 and 541 on Day 55) at 100 and 1000 mg/kg/day, respectively, euthanized moribund after swallowing the dosing cannula. Before death, marked piloerection was observed for Female No. 462 and body weight loss for Female No. 541 (-0.2g from Day 44 to Day 50).
At necropsy, a segment of the cannula was found on the esophagus of all three females. These findings were therefore clearly related to the procedure of administration. Dark foci on the liver and dark thymus were also noted for Female No. 541 given 1000 mg/kg/day.

-Clinical Signs:
There was a dose-related increased incidence of transient hypersalivation with or without abnormal foraging and/or pedalling noted mostly just after dosing (+1 minute) for both sexes in all treated groups throughout the dosing period. Males appeared more affected than females, including control ones. This sign was also observed sporadically for more than half control males and for approximately one third of females. However, signs of recovery were noted as the occurrence of hypersalivation and associated clinical signs was lower toward the end of the dosing period for both sexes. As for the F0-generation, this effect was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect, the signs of recovery after repeated administration and its time of occurrence (i.e. just after dosing). This sign may be related to irritancy/taste of the test item and/or vehicle and was not considered as adverse in the absence of effects on the general health status of rats.
Sporadic clinical signs including, bent tail, bleeding, chromodaccryorrhea, extensive/localised hairloss, decreased activity, lying down, incomplete hair growth, lacrimation, partly closed eye(s), piloerection or scab(s)/sore(s) were noted among control and treated groups and considered incidental.

-Body Weight:
There was no statistically significant differences in absolute mean body weight at the end of the dosing period for either sexes.
There was no test item-related effect on mean body gain in any group for either sex.
Despite some transient statistically significant changes noted for both sexes, the overall mean bodyweight gain was comparable or slightly higher in all groups for both sexes (+2.3%, +1.9% and +1.5% at 100, 300 and 1000 mg/kg/day, respectively, for males and +6.4%, +4.6% and +7.7% at 100, 300 and 1000 mg/kg/day, respectively, for females) when compared with the concurrent controls.

-Food Consumption:
There was no test-item related effect on food consumption in any group for either sex.
Despite some transient statistically significant changes noted for both sexes, the overall mean food consumptions during the dosing period Day 1 to 71 were comparable with, or higher than that of the control group in all treated groups for both sexes (-1.1%, -2.1% and -0.9% at 100, 300 and 1000 mg/kg/day, respectively, for males and +2.9%, +1.2% and +2.8% at 100, 300 and 1000 mg/kg/day, respectively, for females, versus control groups).

-Hematology:
There were no test item-related changes in hematological parameters in any group for either sex.
Minor statistically significant changes arising between control and treated animals were noted including slight decrease of red blood cells in all treated groups and a slight increase of LUC and monocytes (absolute and relative) at 1000 mg/kg/day for males, slightly increased hemoglobin concentration at 300 and 1000 mg/kg/day, mean corpuscular hemoglobin concentration at 300 and 1000 mg/kg/day, decreased total white blood cells at 300 and 1000 mg/kg/day, monocytes at 300 and 1000 mg/kg/day and lymphocytes in all dose groups and increased percentage of eosinophiles in all dose group for females.
These changes were considered of no toxicological significance as they were of low magnitude, observed in one sex only and/or without dose-relationship and remained within the historical control data range.

-Clinical Chemistry:
There were no test item-related changes in serum clinical chemistry parameters in any group for either sex.
Minor statistically significant changes arising between control and treated animals were noted only for females including increased chloride concentration at all dose levels, protein concentration at 300 and 1000 mg/kg/day and globulin concentration at all dose levels and decreased phosphorus concentration at all dose levels, total bilirubin concentration at all dose levels and albumin/globulin ratio at 300 and 1000 mg/kg/day.
These changes were considered of no toxicological significance as they were of low magnitude, observed in one sex only and/or without dose-relationship and remained within the historical control data range.

-Thyroid Hormone Analysis:
Total T4 and TSH levels in F1 cohort 1A animals were considered to be unaffected by treatment.

-Urinalysis:
There was no test item-related effect on urine analysis parameters in any group for either sex.

-Splenic Lymphocyte Subpopulation Analysis:
Analysis of the splenic lymphocyte population by flow cytometry did not reveal any significant modification of the immune system of the cohort 1A induced by the test item, at doses of 100, 300 and 1000 mg/kg/day. This included the following subsets: total CD45+ lymphoid cells, CD3+CD45RA- T lymphocytes, CD3-CD45RA+ B lymphocytes, CD3+CD4+ T helper lymphocytes, CD3+CD8+ cytotoxic T lymphocytes and CD3-CD161a+ natural killer cells.

-Organ Weights:
There were no organ weights differences that were considered to be associated with administration of the test item.
The mean absolute and relative testes weights were statistically significantly lower in all treated groups from Cohort 1A than in corresponding controls. Male No. 321 (treated at
1000 mg/kg/day) had individual low value of the testes weight that correlated with unilateral atrophy/hypoplasia of moderate grade, and that contributed to the lower mean weight in males treated at 1000 mg/kg/day. In addition, there were no histopathologic testicular findings that could correlate with this lower weight. Finally, there were also no relevant differences between treated and control groups from Cohort 1B males , as well as between treated groups and historical control data.
Any other organ weight differences including those that were statistically significant were considered to be incidental and unrelated to the test item administration.

-Macroscopic Observations:
In pups killed and examined on PND 21, there were no macroscopic abnormalities that were considered to be associated with administration of the test item.
In all three Cohorts 1A, 1B and 1C, there were no macroscopic observations that were considered to be associated with administration of the test item.

-Microscopic Observations:
There were no microscopic observations that were considered to be associated with administration of the test item.
In particular, the histopathologic evaluation of the testes and the quantitative evaluation of ovarian follicles and corpora lutea did not show any difference between animals treated at 1000 mg/kg/day and controls.
Any other microscopic observations were considered to be incidental and unrelated to the test item administration.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item-related effects on mean F1 estrous cycle lengths noted in any group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item-related effects on mean F1 spermatogenic parameters noted in any group.

Reproductive performance:

not examined

Description (incidence and severity):

This is not part of the basic design of the EOGRTS as requested by ECHA.

Details on results (P1)

Reproductive function/performance F1-Generation Data (Cohort 1A, 1B and 1C) from Weaning onwards (considered "P1" under the results section)
-Estrous Cycle Data:
There was no test item-related effect on estrous cycle data in any group.
The duration from vaginal opening to first estrus in the treated groups ((3.0, 3.5 and 3.5 days at 100, 300 and 1000 mg/kg/day, respectively) was comparable to the control group (2.9 days). None of the differences was statistically significant.
The mean lengths of estrous cycles for F1 females in the treated groups (3.9, 4.0 and 4.0 at 100, 300 and 1000 mg/kg/day, respectively) during the last two weeks of dosing (PND71/75 to PND89/93) were similar to the control group value (4.0). In addition, the percentage of females cycling in the treated groups (30, 35 and 25% at 100, 300 and 1000 mg/kg/day, respectively) were similar to the concurrent control group (30%).

-Sexual Maturation:
There was no test item-related effect on sexual maturation in any group for either sex.
The mean occurrence of balanopreputial cleavage for treated males (51.3, 52.6, and 50.7 days in the 100, 300, and 1000 mg/kg/day groups, respectively) was comparable to the concurrent control (50.3 days). Mean body weights at the age of attainment of treated groups (233.8, 239.5 and 231.9g in the 100, 300, and 1000 mg/kg/day groups, respectively) were comparable to the concurrent control (226.7g).
The mean occurrence of vaginal opening for treated females (34.4, 33.7, and 33.9 days in the 100, 300, and 1000 mg/kg/day groups, respectively) was comparable to the concurrent control (33.8). Mean body weights at the age of attainment of treated groups (114.8, 109.7, and 111.4g in the 100, 300, and 1000 mg/kg/day groups, respectively) were comparable to the concurrent control (110.1g)

-Sperm Analysis:
There was no test item-related effect on mean sperm counts, sperm morphology or motility, including the percentage of progressively motile sperm, in any group. All values were comparable with the concurrent control and/or within the historical control data range.
Similarly, mean cauda epididymis weights were comparable in all groups.

Effect levels (P1)

1

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity F1 after weaning

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No parental systemic toxicity was observed up to the highest dose level tested (1000 mg/kg).

2

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg).

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There was no test item-related effect on F1 pup clinical condition at any dose level.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item-related effect on F1 postnatal survival in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup birth weights as well as mean pup weights on PND21 were comparable across all groups.
There was no test item-related effect on mean pup body weight during lactation in any group. Mean values in all treated groups for both sexes were comparable with the concurrent control and/or within the historical control data range from PND 1 and throughout the preweaning period.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Not applicable for pups until weaning ( considered "F1" in the results section).

Food efficiency:

not examined

Description (incidence and severity):

Not applicable for pups until weaning ( considered "F1" in the results section).

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

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on F1 pup anogenital distance at any dose level.
There was a slight but statistically significant increased anogenital distance (normalized to the cube root of body weight) for males at 300 (2.4 mm.g3) and 1000 mg/kg/day (2.3 mm.g3) when compared with the control group (2.2 mm.g3). In view of their low magnitude and in the absence of a dose-related trend, these differences were considered incidental

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There was no test item-related effect on F1 pup areolae/nipple at any dose level.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Description (incidence and severity):

There were no test item-related effects on T4 and TSH in F1 pups on PND4 and PND21 in any group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

F1-GENERATION UNTIL WEANING (considered "F1" under results)

-Clinical Signs during Lactation (F1-pups):
No clinical signs occurred among pups that were considered to be test item-related.
Incidental clinical signs of pups consisted of occasional pale/cyanotic/cold pup, hematoma, , closed eyes, complete or partial absence of tail, weakness, thinness, incomplete hair growth, scabs or marks on the teeth. In view of their nature and/or sporadic occurrence including in controls, these findings were considered of no toxicological relevance.

-Pup Body Weight during Lactation (F1-pups)
There was no test item-related effect on mean pup body weight in any group. Mean values in all treated groups for both sexes were comparable with the concurrent control and/or within the historical control data range from PND1 and throughout the preweaning period.

-Pup Anogenital Distance:
There was not test item-related effect on pup anogenital distance in any group for either sex.
There was a slight but statistically significant increased anogenital distance (normalized to the cube root of body weight) for males at 300 (2.4 mm.g3) and 1000 mg/kg/day (2.3 mm.g3) when compared with the control group (2.2 mm.g3). In view of their low magnitude and in the absence of a dose-related trend, these differences were considered incidental.

-Clinical Biochemistry (T4 and TSH levels) on PND 4 and 21:
Total T4 levels in F1-PND 4 pups were considered to be unaffected by treatment.
Total T4 and TSH levels in F1 PND 21 pups were considered to be unaffected by treatment.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No parental systemic toxicity was observed up to the highest dose level tested (1000 mg/kg).

2

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Validation of an Ultra Performance Liquid Chromatographic Method for the Determination of Isopentyl Acetate in Dose Formulations
Charles River Test Facility Study No. No. AB22399

An analytical procedure for the determination of Isopentyl Acetate (also named Isoamyl Acetate) in corn oil dosing formulations has been validated. In addition, this procedure has been used to validate the formulation procedure for Isoamyl Acetate in corn oil dosing formulations at Charles River.

In the validated analytical procedure samples of the dosing formulations are diluted in Isopropanol/Acetone (60:40, v/v) as necessary and analysed by UPLC with UV detection at 210 nm. Internal standardisation was employed.

Formulation of Isoamyl Acetate at 5 and 200 mg/mL were stable after storage for 24 hours at room temperature (+15 to +25°C) and after 16 days under refrigerated conditions (+2 to +8°C) and 22 days under frozen conditions (-15 to -25°C).

Formulation of Isoamyl Acetate at 250 mg/mL was stable after storage for 24 hours at room temperature (+15 to +25°C) and after 9 and 22 days under refrigerated conditions (+2 to +8°C) and 22 days under frozen conditions (-15 to -25°C).

Satisfactory results were obtained for homogeneity of formulations at 5, 200 and 250 mg/mL of Isoamyl Acetate in vehicle.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the study, Isoamyl Acetate administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg induced no parental toxicity for F0-males exposed for 10 weeks prior to mating, during mating, and up to termination (at least 84 days) or for F0-females that delivered and exposed for 10 weeks prior to mating, during mating, during post-coitum, and during at least 21 days of lactation (for at least 115 days). For F1-males and F1-females exposed at the same dose levels up to sexual maturation for cohort 1C, during approximately 12 weeks for cohort 1A and during approximately 13 weeks for cohort 1B, no developmental toxicity was observed.
In conclusion, based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg in male and female Wistar Han rats was established.

Executive summary:

A key Extended One-Generation Reproductive Toxicity Study (EOGRTS) was performed with Isoamyl acetate to evaluate reproductive toxicity, pre- and postnatal effects on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and in young and adult offspring (Charles River, 2019b). Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, were expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the test item, Isoamyl Acetate, on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

The test item, Isoamyl Acetate, was administered by oral gavage at dose levels of 100, 300 and 1000 mg/kg/day at a dose volume of 4 mL/kg/day to groups of 25 males and 25 females Wistar rats (F0-generation). The males and females were treated for ten weeks before pairing. Treatment then continued throughout mating and up to the day before necropsy. A fourth group received the control item (Corn oil) at the same dose volume. Clinical condition, body weights and food consumption of the males and females were monitored throughout the study. The inseminated females were allowed to litter and development of the offspring was observed up to weaning on postnatal day (PND) 21.

Pups were then allocated to 3 cohorts (1A, 1B and 1C) of 20 males and 20 females each (F1 generation). F1 animals were administered test item by oral gavage at dose levels of 100, 300 and 1000 mg/kg/day and at a dose volume of 4 mL/kg/day from PND21 up to the day before necropsy. A fourth group received the control item (Corn oil) at the same dose volume.

The following parameters and end points were evaluated in all F0 animals: survival, clinical signs, body weights, body weight gains, food consumption, clinical pathology, hormone analysis, estrous cycle data, mating performance and fertility, delivery and litter data, sperm analysis, macroscopic observations, organ weights and histology examination. F1 offspring were observed for survival, clinical signs, body weights, body weight gains, food consumption, clinical pathology, hormone analysis, pup anogenital distance, pup number of aerolae/nipples, estrous cycle data, sexual maturation, sperm analysis, macroscopic observations, organ weights and histology examination. Additionally, spleen samples were taken for splenic lymphocyte subpopulation analysis on 10 rats/sex/group from F1 cohort 1A animals.

F0 males were sacrificed after a minimum of 12 weeks of treatment and F0 females between Days 22 (L 22) and (L 24) of lactation. F1 animals were sacrificed after sexual maturation for cohort 1C or between PND 85 and PND 100 for cohort 1A and 1B.

Results

F0 generation

There were no unscheduled deaths in any group for F0 animals.

There was an increased incidence of hypersalivation with or without abnormal foraging and/or pedalling during pre-mating period in all treated groups and at 300 and 1000 mg/kg/day for females during gestation and lactation. This sign was considered to be a physiological response probably related to the irritancy/taste of the test item and/or vehicle rather than a sign of systemic toxicity and was not considered as adverse.

There was no test item-related effect in any group on mean F0 body weight (gain) for either sexes and food consumption for females throughout the treatment period, including during gestation and lactation for females.

There was a slight increase in mean food consumption for males in all treated groups during the pre-mating period considered non adverse in view of the minor change without impact on the terminal mean body weight.

There was no test item-related effect in any group on F0 male and female mating and fertility, male copulation, mean number of implantations, mean estrous cycle lengths, mean precoital intervals, gestation lengths and sperm parameters.

There were no test item-related effects on mean T4 and TSH levels, on hematology, coagulation, serum clinical chemistry and urinary parameters for F0 animals at any dose for either sex.

There were no test item-related effects on organs weights, on macroscopic and histology examinations for F0 animals in any group for either sex.

F1 generation

There was no test item-related effect on F1 postnatal survival in any group. The mean number of pups delivered was comparable with or higher than that of the control group and the historical control data mean in all treated groups.  

There were no test item-related effects on F1 pup clinical condition, anogenital distance, or areolae/nipple at any dose level. Mean male and female pup birth weights as well as mean pup weights on PND21 were comparable across all groups. 

There was no test item-related effects on T4 and TSH in F1 pups on PND4 and PND21, and in F1 cohort 1A animals after approximately 12 weeks of dosing in any group. 

There was no test item-related effect on the mean ages and body weights at attainment of balanopreputial cleavage and vaginal opening for F1 animals in any group. The duration from vaginal opening to first estrous in the treated groups was comparable to the control group.

There was an increased incidence of hypersalivation with or without abnormal foraging and/or pedalling for both sexes in all treated groups. This sign was considered to be a physiological response probably related to the irritancy/taste of the test item and/or vehicle rather than a sign of systemic toxicity and was not considered as adverse.

There was no test item-related effect on mean body weight gain or mean food consumption throughout the treatment period for F1 animals in any group for either sex.

There were no test item-related effects on mean F1 estrous cycle lengths or spermatogenic parameters noted in any group.

There were no test item-related effects on hematology, coagulation, serum clinical chemistry and urinary parameters for the F1 males and females after approximately 12 weeks of dosing (cohort 1A) at any dose.

There was no test item-related effect on the splenic lymphocyte population for the F1 males and females after approximately 12 weeks of dosing (cohort 1A) at any dose.

There was no test item-related effects on organs weights and on macroscopic or histology examination for F1 animals in any group for either sex.

Conclusion

Under the experimental conditions of the study, Isoamyl Acetate administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg induced no parental toxicity for F0-males exposed for 10 weeks prior to mating, during mating, and up to termination (at least 84 days) or for F0-females that delivered and exposed for 10 weeks prior to mating, during mating, during post-coitum, and during at least 21 days of lactation (for at least 115 days). For F1-males and F1-females exposed at the same dose levels up to sexual maturation for cohort 1C, during approximately 12 weeks for cohort 1A and during approximately 13 weeks for cohort 1B, no developmental toxicity was observed.

Parental NOAEL (F0-generation and F1-generation): No parental systemic toxicity was observed up to the highest dose level tested (1000 mg/kg).

Reproductive NOAEL (F0-generation): No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg).

Developmental NOAEL (F1-generation): No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

In conclusion, based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg in male and female Wistar Han rats was established.