(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

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:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 443 Guideline without deviations.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

other: Draft Study Plan

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS: as requested by ECHA in the final decision CCH-D-21 14366659-31-01/F

Test material

Test material information

Constituent 1

Reference substance name:

(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

EC Number:

207-856-6

EC Name:

(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

Cas Number:

498-15-7

Molecular formula:

C10H16

IUPAC Name:

3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

impurity 1

Reference substance name:

(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

Cas Number:

20296-50-8

Molecular formula:

C10H16

IUPAC Name:

(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

impurity 2

Reference substance name:

7-methyl-3-methyleneocta-1,6-diene

EC Number:

204-622-5

EC Name:

7-methyl-3-methyleneocta-1,6-diene

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-methyl-3-methyleneocta-1,6-diene

impurity 3

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-isopropenyl-1-methylcyclohexene

impurity 4

Reference substance name:

(S)-p-mentha-1,8-diene

EC Number:

227-815-6

EC Name:

(S)-p-mentha-1,8-diene

Cas Number:

5989-54-8

Molecular formula:

C10H16

IUPAC Name:

(4S)-isopropenyl-1-methylcyclohexene

impurity 5

Reference substance name:

(-)-pin-2(10)-ene

EC Number:

242-060-2

EC Name:

(-)-pin-2(10)-ene

Cas Number:

18172-67-3

Molecular formula:

C10H16

IUPAC Name:

(1S,5S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

impurity 6

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

impurity 7

Reference substance name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

2243-33-6

Molecular formula:

C10H16

IUPAC Name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 8

Reference substance name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

EC Number:

224-167-6

EC Name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

4221-98-1

Molecular formula:

C10H16

IUPAC Name:

(5R)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 9

Reference substance name:

3-isopropyl-(6S)-methylenecyclohexene

Cas Number:

6153-16-8

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-(6S)-methylenecyclohexene

impurity 10

Reference substance name:

3-isopropyl-(6R)-methylenecyclohexene

Cas Number:

6153-17-9

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-(6R)-methylenecyclohexene

impurity 11

Reference substance name:

p-mentha-1,3-diene

EC Number:

202-795-1

EC Name:

p-mentha-1,3-diene

Cas Number:

99-86-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,3-diene

impurity 12

Reference substance name:

(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Cas Number:

19902-08-0

Molecular formula:

C10H16

IUPAC Name:

(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Test material form:

liquid

Details on test material:

Batch No. : 1000117557
Purity : 82.5%
Name of test material (as cited in study report): delta-3-carene
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 10 March 2022

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by
regulatory agencies. The Crl:CD(SD) strain, sexually mature and virgin, was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited.
- Age at study initiation: 22 to 28 days old (approximately 14-15 weeks of age at pairing).
- Weight at study initiation: Males 51 to 80g. Females 40 to 70g.
- Housing (see Table 1 below): Grid bottomed cages were suspended above absorbent paper which were changed daily during pairing. Solid bottomed cages have bedding (softwood based bark-free fiber, sterilized by autoclaving) changed at appropriate intervals. Cages, cage-trays, food hoppers and water bottles were changed at appropriate intervals.
Covance has a policy of improvement of animal welfare which includes permitting social interaction through multiple housing of rats whenever possible and this policy were followed for this study, except after mating when females were housed singly to permit collection of food consumption data individually for pregnant females and during lactation, when adult females are singly housed with their litter for good husbandry practice.
- Diet: SDS VRF1 Certified, powdered diet; Non-restricted (except when diet removed overnight for blood sampling for hematology or blood chemistry or during urine collection); Before delivery each batch of diet is analyzed by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier's analytical certificates are scrutinized and approved before any batch of diet is released for use. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Potable water from the public supply. Non-restricted via polycarbonate bottles with sipper tubes and changed at appropriate intervals. Certificates of analysis are routinely received from the supplier.
- Acclimation period: at least 5 days.
- Environmental enrichment: During the acclimatization and appropriate study periods environmental enrichment in the form of Aspen wood based products (soft white untreated wood product) and a plastic shelter were available in each home cage.
From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material were changed at the same frequency as the cage bedding. See Table 2 below.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered, not recirculated
- Photoperiod: 12 h light : 12 h dark

IN-LIFE DATES: FROM: 02 June 2021 TO: [TBC]

Administration / exposure

Route of administration:

oral: feed

Vehicle:

corn oil

Remarks:

Corn oil at a ratio of test item to corn oil of 5 to 1

Details on exposure:

RATIONALE FOR ROUTE OF ADMINISTRATION
Dietary, to simulate the conditions of possible human exposure.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly, subdivided into aliquots, and may be prepared in advance of the first day of dosing; retained frozen (-10 to -30°C) following preparation.
- Mixing appropriate amounts with (Type of food): VRF1 diet with corn oil stabilizer at a ratio of test item to corn oil of 5 to 1.
- Storage and stability of formulations: The homogeneity and stability of the test material in the diet at inclusion levels of 500 to 15000 ppm were confirmed for 15 days following frozen storage (-10 to -30 ºC) and for 8 days following ambient storage (15 to 25˚C) as part of a separate study Covance Study Number MJ98TB).
- Method: [The formulation procedure will be documented in the study data and included in the final report] = TBC

Details on mating procedure:

The F0 animals were paired on a 1:1 basis within each treatment group after 10 weeks of treatment and up to 2 weeks. The cohort 1B animals were paired on the same basis, approximately 10 weeks after selection if mating is triggered. Sibling pairing was not permitted.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm within vaginal smear.
- Day 0 of gestation: When positive evidence of mating detected.
- Male/female separation: Day when mating evidence detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Females showing no evidence of mating - following completion of the pairing period females were separated from the male and vaginal smearing continued for up to five days or until the first estrus smear is seen. If an estrus smear is seen during this period, the female was killed as soon as practically possible and subject to macroscopic examination. If necropsy is not possible on the day of estrus, smears was continued until the morning of necropsy.
If a female does not show an estrus smear, wet smears were re-commence on Day 22 after separation from pairing (where day of separation = Day 0) for a period of four days with the last smear on the morning of necropsy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before commencement of treatment, the suitability of the proposed mixing procedures and the homogeneity and stability of the test material in the carrier diet was determined as part of a separate study, Covance Study Number MJ98TB.
At specified intervals during treatment, the diet formulations was analyzed for achieved concentration of the test item.
- Method: [The method of analysis will be documented in the study data and a summary included in the final report: TBC]

Duration of treatment / exposure:

F0 ANIMALS: For 10 weeks before pairing until termination after litters are weaned.

F1 ANIMALS: From weaning until termination of respective cohort*.

UNSELECTED F1 offspring: Retention of brain, spleen, thymus and mammary tissue and organ weights - no direct treatment, killed on Day 22 of age.

COHORT 1A : Primary assessment of effects upon reproductive systems and of general toxicity – treated from weaning to approximately 13 weeks of age.

COHORT 1B : Reproductive /developmental toxicity testing - treated from weaning to approximately 13 weeks of age.
* Although direct treatment starts at or soon after weaning (or when the offspring start to consume some treated diet whilst still with the dam), all offspring have potential indirect exposure in-utero, and/or through the milk during lactation.

Frequency of treatment:

Continuous via the diet; to be replaced at least twice weekly.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

Group 1 (basal diet plus corn oil)

Doses / concentrations 2

Dose / conc.:

2 000 ppm

Remarks:

Group 2 (test item)

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Remarks:

Group 3 (test item)

Doses / concentrations 4

Dose / conc.:

12 000 ppm

Remarks:

Group 4 (test item)

No. of animals per sex per dose:

F0 generation: 24 animals/sex/dose + 8 animals spares per sex
F1 generation: 20 animals/sex/dose + 8 animals spares per sex
See Table 3 below.

Control animals:

yes, plain diet

Details on study design:

DOSE SELECTION
Dietary concentrations are selected, in conjunction with the Sponsor, based on the findings of the preliminary Reproductive/Development Toxicity Screening Study (Covance Study Number: 8434961).
In the preliminary study the F0 generation, three groups of eight male and eight female Sprague Dawley rats received (+)-delta-3-carene orally, via the diet, at concentrations of 3000, 6000 or 12000 ppm. The F1 generation, eight males and eight females, were treated from weaning until completion of sexual maturation of the females (5-6 weeks of age) at the same dietary concentrations as the F0 generation.
Dietary administration of (+)-delta-3-carene to Sprague Dawley rats at dietary concentrations up to 12000 ppm were considered suitable for investigation in this main extended one-generation reproductive performance study. Effects on body weight gain and food consumption were observed for F0 parental animals, comprising slight group mean body weight losses following the commencement of treatment for males that received 12000 or 6000 ppm and for all groups of treated females, and a reduction in mean food intake for all groups of treated animals, compared to Controls, also apparent following the commencement of treatment. Reduced body weight gain from Days 1 to 21 of age was observed for offspring derived from parents that received 12000 or 6000 ppm. In addition, slightly reduced overall group mean body weight gain for the selected F1 males and females at 12000 ppm was observed with a marginal reduction in group mean food intake for selected F1 males from Day 21 to Day 25 of age. Female sexual maturation was marginally delayed at 12000 ppm but was considered to reflect the lower mean body weight gain of these females. A slight increase in mean kidney weights in all groups of treated F1 males was apparent. An increase in liver weights was apparent for F1 males that received 6000 ppm as well as in F1 males and females that received 12000 ppm. Overall, the extent of the reduced body weight gain, reduced food intake, marginal delay in sexual maturation and organ weight changes were considered to be insufficient to preclude a dietary concentration of 12000 ppm from further investigation.
Based on the results obtained in the preliminary extended one-generation reproductive toxicity study, the high dose for the current main study is set at 12000 ppm with intermediate and low dietary concentrations set at 5000 and 2000 ppm, respectively, in order to fulfill the 2-fold to 4-fold dosing interval as specified in the test guideline.

ANIMAL GROUP SELECTION
- Allocation to treatment groups (F0 generation): Each F0 animals were allocated by sex, after a period of at least 5 days of acclimatization. Before or on Day 1 of study (prior to diet administration), animals were weighed. Body weights were reviewed by Study Management and allocation may be adjusted, if appropriate, to reduce inter-/intra-group variation.
At commencement of the study the weight variation should, if possible, not exceed 20% of the mean weight of each sex.

- Selection of Offspring to form F1 Generation: The selection of F1 animals was performed nominally at Day 28 of age. Selected animals were microchipped on Day 18-21 of age and separated from littermates on Day 21 of age.

ANIMAL REPLACEMENT
8 male and 8 female spare F0 animals were ordered to replace any individuals rejected, before the start of treatment. Up to 2 male and 2 female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortalities. These spares have body weights and clinical signs monitored weekly and were terminated after commencement of the F1 generation. The rejection before treatment may relate to ill-health or/and body weight range extremes. On Day 1 (before first diet administration) variations in body weight of animals should not exceed ±20% of the mean for each sex.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

MORTALITY: Yes
- Animals may be killed for reasons of animal welfare in a premature sacrifrice. In animals found dead, or killed for reasons of animal welfare, a necropsy is performed as soon as possible.

CLINICAL OBSERVATIONS & DETAILED CLINICAL OBSERVATIONS: Yes
F0 and F1 animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Only signs which are indicative of ill-health were routinely recorded as part of this twice daily health check. Those signs which are not indicative of ill-health were routinely recorded, as appropriate, as part of the detailed physical examination check.
A detailed physical examination was performed at nominally the same time of day on each occasion by an observer. After removal from the home cage, animals were assessed for physical condition and behavior during handling. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviations from normal were recorded with respect to nature, and, where appropriate, degree of severity.
- Time schedule: Once each week for all F0 and selected F1 generation animals.
Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 females (and F1 females if mating triggered).

BODY WEIGHT: Yes
F0 Males: Day that treatment commences, each week and before necropsy.
F0 Females: Day that treatment commences, each week until mating detected, days 0, 6, 13 and 20 after mating, days 1, 4, 7, 14 and 21 post-partum and before necropsy.
F1 selected animals: Days 21, 22, 25 and 28 of age, then each week from nominal 4 weeks of age (at the formal start of the F1 generation) and before necropsy.

FOOD CONSUMPTION: Yes
- F0 Animals: Twice weekly. Food consumption was not recorded for males and females during the period when paired for mating but was recommence for males once pairing of all the animals is completed. For females after mating food consumption schedule was match body weight schedule: on days 0-7, 7-14, 14-20 after mating and on days 1-4, 4-7, 7-14 and 14-21 of lactation.

- Selected F1 generation: Each week from nominal 4 weeks of age.

HEMATOLOGY, PERIPHERAL BLOOD (F0 and F1 Cohort 1A): Yes
After overnight deprivation of food, blood samples were collected under light general anesthesia (isoflurane) from sublingual vein for 10 animals/sex/group. Where blood sampling coincides with urine collection animals were also deprived of water overnight but have access to water for a minimum period of one hour prior to blood sampling. A blood film were prepared and may be examined at the discretion of the analyst, for samples showing abnormalities in the automated analysis.
All samples (0.5 mL) were examined for the following characteristics:
1) Using EDTA as anticoagulant: Hematocrit*, Hemoglobin concentration, Erythrocyte count, Total leucocyte count, Differential leucocyte count, Platelet count, Mean cell hemoglobin*, Mean cell volume, Mean cell hemoglobin concentration* and red cell distribution width.
2) Using citrate as anticoagulant: Prothrombin time and activated partial thromboplastin time
* Derived values calculated in ClinAxys.

BLOOD CHEMISTRY (F0 and F1 Cohort 1A): Yes
After overnight deprivation of food, blood samples were collected under light general anesthesia (isoflurane) from sublingual vein for 10 animals/sex/group. Where blood sampling coincides with urine collection animals were also deprived of water overnight but have access to water for a minimum period of one hour prior to blood sampling.
All samples (0.7 mL) were examined for the following characteristics, using lithium heparin as anticoagulant: Alkaline phosphatase, Alanine amino-transferase, Aspartate amino-transferase, Gamma glutamyl transpeptidase, Glucose, Total bilirubin, Total cholesterol, Creatinine, Urea, Total protein, Albumin (by chemical assay), Albumin/globulin ratio, Sodium, Potassium, Chloride, Calcium, Phosphorus

BIOMARKERS - TSH AND T4 (F0 and F1 generation): Yes
F0 adults: 10 animals/sex/group
F1 offspring: 10 litters/group on Day 22 of age
F1 adults (1A): 10 animals/sex/group (approx 13 weeks of age)
- Conditions: Adults: Following overnight deprivation of food; Offspring Day 22 of age: no overnight deprivation of food.
- Sample site/Volume: Adults and Offspring Day 22 of age: Sublingual vein/1.0 mL;
- Anesthesic: Adults and offspring on Day 22 of age: Isoflurane.
- Maximum no of samples per occasion: F0 adults, F1 offspring Day 22 of age and F1A adults: 80 per analyte; F1 offspring Day 4 of age: 40 for T4 only.
- Overall maximum no. of samples for analysis: T4: 240 and TSH: 240

URINALYSIS (F1 Cohort 1A): Yes
After overnight deprivation of food and water in individual metabolsim cage, urine samples were collected for 10 animals/sex/group.
Using manual methods: Clarity/colour (appearance), Volume, pH and Specific gravity.
Using semi-quantitative methods (Clinitek): Glucose, Ketone, Bilirubin (bile pigments), Blood pigments.
Using quantitative automated methods (Roche Cobas 6000): Protein (total and concentration), Sodium (total and concentration), Potassium (total and concentration) and Chloride (total and concentration)
The deposit, obtained from centrifugation, were examined microscopically for epithelial cell, leucocyte, erythrocytes, crystals, casts, spermatozoa and other abnormal components.

SPARE ANIMALS
Spare animals were also examined for clinical observations and their body weights were recorded as for F0 and F1 generation, until they are terminated.
Spare animals dying or killed for health reasons before removal from the study were subject to macroscopic investigations as detailed for F0 generation and histopathological examinations were only performed following discussion with the Sponsor and documented by study plan amendment.

Oestrous cyclicity (parental animals):

Dry smears - F0 only: Daily for 15 days before pairing, using cotton swabs.
Wet smears - F0 only: Daily after pairing until evidence of mating confirmed, using
pipette lavage, for four days before scheduled termination (nominally Days 25 to 28 post partum) and for F0 females that fail to produce a viable litter or with litter death, was smeared and terminated with first cohort of females with live litters.

The duration of gestation correspond to the time elapsing between mating and commencement of parturition, and parturition observations were made from Day 20 after mating, three times daily for evidence of parturition. If difficulties observed, noted progress of parturition process was monitored. Numbers of live and dead offspring was recorded.

Sperm parameters (parental animals):

Cohort 1A:
Vas deferens (from left side): Sperm sample assessed for motility using a computer assisted sperm analyzer (CASA). Each animal in each group. A manual assessment of sperm morphology were performed. Each animal in Groups 1 and 4#.

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

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

# If treatment-related changes are suspected, or at the request of the Sponsor, the examinations may be extended to all animals of all groups and documented.

Litter observations:

CLINICAL OBSERVATIONS: Yes
Animals were observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to maternal treatment. On Day 1 of age, all offspring receive a qualitative assessment of body temperature, state of activity and reaction to handling.

LITTER SIZE: Yes
- Schedule: Daily records were maintained of mortality and consequent changes in litter size on Days 1-21 of lactation. On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving where possible, five male and five female offspring in each litter. Culled offspring were examined.

SEXE RATIO: Yes
Recorded Days 1, 4 (before and after culling) and Day 21 of age.

INDIVIDUAL OFFSPRING BODY WEIGHT: Yes
Recorded on Days 1, 4, 7, 14 and 21 of age (day of necropsy) for all offspring, on day 22 af age for unselected F1 and on days 25 and 28* of age for selected F1.
*only applicable before formal commencement of the F1 generation at nominal Week 4 of age (Day 28 of age ± 2 days).

ANO-GENITAL DISTANCE: Offspring on Day 1 of age.

NIPPLE COUNT: Male offspring on Day 13 of age.

SEXUAL MATURATION: Yes
Weaning of offspring takes place on day 21 of age.
- Males: Examined daily from Day 38 of age for the completion of balano-preputial separation.
Body weight recorded on day of completion of separation.
- Females: Examined daily from Day 28 of age until vaginal opening occurs. Body weight recorded on the day of vaginal opening. For females in Cohort 1A, a wet smear were taken daily from the day of vaginal opening until first estrus.

COHORT SPECIFIC IN-LIFE INVESTIGATIONS: Yes
- Estrous cycle monitoring for F1 Cohort 1A:
Wet smears (using pipette lavage): Following onset of vaginal patency until the first cornified (estrus) smear is recorded. For at least 3 days prior to the start of the necropsy phase and on the day of termination.
Dry smears (using cotton swabs): For 2 weeks from approximately Day 75 of age.

- F1 Cohort 1B: These animals may be used for follow up assessment of reproductive performance by mating of the F1 animals and for obtaining additional histopathology data in cases of suspected reproductive or endocrine toxicants or when results from F1 Cohort 1A are equivocal.

Postmortem examinations (parental animals):

SACRIFICE
- F0 adult animals: Females were killed on day 28 post-partum. Males were killed after at least 10 weeks of treatment and after weaning of the F1 animals, after confirmation that no further mating is required.
For F0 females failing to mate, if an estrus smear is seen following completion of the pairing period, animals were terminated as soon as logistically possible. If no estrus smear is seen, animals were terminated on Day 25 after the last day of pairing (where Day 0 is the day of pairing separation).
For F0 females failing to produce a viable litter and those with total litter loss, they are terminated with first cohort of females with live litters.

- Method: Animals 14 days and older: Carbon dioxide. Each animal were subsequently exsanguinated.

MACROSCOPIC PATHOLOGY
A complete gross necropsy were performed on all animals, including surplus offspring culled on Day 4 of age, and unselected F1 offspring. Where possible, decedent offspring ≤ 21 days of age (found dead or welfare kill) were examined and carcass retained.
For F0 females, implantation site count was recorded.
Retained tissues were checked.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together unless otherwise specified. Organ weights are not routinely recorded for animals killed or dying prematurely; organ weights were recorded for groups terminated prematurely.

FIXATION
For F0 animals:
- Standard: 10% Neutral Buffered Formalin. Where possible, carcass retained for decedent offspring ≤ 21 days of age.
- Others: Testes (F0 and F1 adults only): modified Davidson’s fluid.
Eyes: Davidson’s fluid.

HISTOLOGY
Processing to slide:
- Full List for all F0 animals killed or dying prematurely and all F0 terminal animals of Groups 1 and 4.
- Reproductive Organs (testes, epididymides, seminal vesicles, prostate, ovaries, uterus -with cervix and oviducts, vagina and pituitary) for F0 suspect fertility animals Groups 2 and 3*.
- Abnormalities only for all F0 terminal animals of Groups 2 and 3.

Staining:
- Routine staining: 4-5 µm sections stained with haematoxylin and eosin.
- Special staining: None scheduled.

* Includes animals not pregnant, failed to mate, failed to sire a pregnancy, sired 2 or less live young, failed to litter, females with litter death, females with abnormal estrous cycles or males with abnormal seminology.

The pathology procedures was detailed in the Tables 4, 6 and 7 below.

Postmortem examinations (offspring):

SACRIFICE
- Unselected offspring: F1 animals were killed on Day 4 and Day 22 of age.
- Cohort 1A adult animals: Scheduled kill on approximately Week 13 of age.
- Cohort 1B adult animals: Scheduled kill on approximately Week 14 of age.

- Method: Animals 14 days and older: Carbon dioxide. Each animal were subsequently exsanguinated; Animals less than 14 days of age: intraperitoneal injection of sodium pentobarbitone.

MACROSCOPIC PATHOLOGY
A complete gross necropsy were performed on all animals, including surplus offspring culled on Day 4 of age, and unselected F1 offspring. Where possible, decedent offspring ≤ 21 days of age (found dead or welfare kill) were examined and carcass retained. Retained tissues were checked.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together unless otherwise specified. Organ weights are not routinely recorded for animals killed or dying prematurely; organ weights were recorded for groups terminated prematurely.

FIXATION
For unselected F1 animals (including decedent offspring), and F1 Cohorts 1A and 1B:
- Standard: 10% Neutral Buffered Formalin. Where possible, carcass retained for decedent offspring ≤ 21 days of age.
- Others: Testes from unselected F1 offspring on Day 22 of age retained in 10% Neutral Buffered Formalin; Eyes: Davidson’s fluid.

HISTOLOGY
Processing to slide:
- Full List: F1 Cohort 1A/1B: All animals killed or dying prematurely; F1 Cohort 1A: All terminal animals of Groups 1 and 4.
- Abnormalities only for F1 Cohort 1A: All terminal animals of Groups 2 and 3; F1 Cohort 1B: All animals.

Processing to block:
- For all F1 Cohort 1B animals, Reproductive Organs (testes, epididymides, seminal vesicles, prostate, ovaries, uterus -with cervix and oviducts, vagina and pituitary).

Staining:
- Routine staining: 4-5 µm sections stained with haematoxylin and eosin.
- Special staining: None scheduled.

IMMUNOPHENOTYPING OF SPLEEN LEUCOCYTES (F1 Cohort 1A)
- Necropsy procedures: Ten males and ten females per group from Cohort 1A, from as many litters as possible, were selected for immunophenotyping. Where possible, one male or one female were assigned from each selected litter (i.e. all surviving litters should be represented by at least one offspring). After the spleen has been weighed, a 3-5 mm mid transverse section will be removed and retained for histopathological examination.
The remaining spleen were weighed and then placed into a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held on wet ice until processing for analysis.

- Each sample were analysed for the following cell types: T cells, B cells, NK cells, CD4+ et CD8+ T cells, Monocytes and Neutrophils.

The pathology procedures was detailed in the Table 5 below.

Statistics:

The relative proportions (%) and cell numbers/spleen of each cell population were reported. The proportions of the T, B and NK cells were expressed as a percentage of lymphocytes, monocytes and granulocytes as a percentage of the total leukocytes. CD4+ and CD8+ T lymphocyte subsets were reported as a percentage of total T lymphocytes. The absolute cell numbers/spleen were obtained by performing a back calculation using the percentage proportions of the cell populations obtained from the cytometer and the total cell count of each spleen sample.
Percentage data were reported to two decimal places whilst absolute cell numbers (cells/spleen) were reported as whole integers.

DATA-TYPES
The following data types were analyzed at each timepoint separately, where required, in support of interpretation:
-body weight, using absolute weights and gains over appropriate study periods.
-food consumption, over appropriate study periods.
-estrous cycles and pre-coital interval.
-mating performance and fertility.
-gestation length.
-litter size and survival indices.
-pre-weaning examination (ano-genital distance).
-sexual maturation, age and body weight at completion.
-clinical pathology (hematology, blood chemistry, urinalysis).
-thyroid hormones (TSH and T4)
- spleen immunophenotyping.
- organ weights, both absolute and adjusted for terminal body weight.
- sperm analysis, motility, morphology and counts.
- corpora lutea and ovarian primordial follicle counts.


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

Reproductive indices:

Duration of gestation being the time elapsing between mating and commencement of
parturition was recorded. From Day 20 after mating animals checked 3 times daily for evidence of parturition. If difficulties observed, noted progress of parturition process monitored. Numbers of live and dead offspring recorded.

Percentage mating: (Number animals mating / Animals paired) x 100
Conception rate: (Number animals achieving pregnancy / Animals mated) x 100
Fertility index: (Number animals achieving pregnancy / Animals paired) x 100
Gestation index: (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

Survival indices (%):
- Post implantation survival index: (Total number of offspring born / Total number uterine implantation sites) x 100
- Live birth index: (Number live off spring on Day 1 after littering / Total number of offspring born) x 100
- Viability index: (Number live off spring on Day 4 before cull / Number live offspring on Day 1 after littering) x 100
- Lactation index: (Number live offspring on Day 21 after littering / Number live offspring on Day 4 (after cull)) x 100

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Remarks on result:

other: Study on-going

Results: F1 generation

Effect levels (F1)

Remarks on result:

other: Study on-going

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Study on-going