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Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Study period:
From 2021-11-17 to 2023-06-27 (estimated)
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
Based on an ECHA decision (decision number TPE-D-2114449866-32-01/F), an extended one-generation reproductive toxicity study (test method OECD TG 443) in rats by the oral route has been requested by ECHA. A dose-range finding study similar to OECD TG 421 has been conducted to determine the doses for the main study (see section 7.8.1). The requested OECD TG 443 study has been started on 17 November 2021 and follows the study design specifications made by ECHA:

- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.

Due to the high workload of the laboratories, the deadline of 31 May 2021 specified in ECHA decision TPE-D-2114449866-32-01/F cannot be met.The in-life phase of the study was completed in August 2022 and the final report is planned to be issued on 23 June 2023 according to Study Plan Amendment 7, p. 11 (see "Attached justification").

This section will be updated with the results of the EOGRTS as soon as the study is finalized (presumably end of 2023).
Cross-reference
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
screening for reproductive / developmental toxicity
Study period:
2021-02-24 to 2021-12-17 (estimated)
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
Based on an ECHA decision (decision number TPE-D-2114449866-32-01/F), an extended one-generation reproductive toxicity study (test method OECD TG 443) in rats by the oral route has been requested by ECHA. A dose-range finding study similar to OECD TG 421 has been conducted to determine the doses for the main study. The in-life phase of the study has been completed and the results will be included as soon as the final report ist available.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Not all parameters measured as the study was conducted as a range finding study for the main OECD 443 study.
GLP compliance:
no
Remarks:
No claim of compliance with Good Laboratory Practice (GLP) will be made for this study; however, the work performed will be followed good scientific practices and adhere to the study plan, any amendments and applicable SOPs.
Limit test:
no
Justification for study design:
The study design was chosen on the basis of OECD guideline 421.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
The Sprague-Dawley rat (sexually mature and virgin) is an accepted species for reproductive toxicity studies by regulatory agencies and historical control data are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 10-12 weeks; (F1) 4 weeks
- Weight at study initiation: (P) Males: 351-400 g; Females: 227-254 g; (F1) Males: will be added when the study is finished; Females: will be added when the study is finished
- Fasting period before study: Not indicated
- Housing: Polycarbonate caging with stainless steel grid (pairing period) or with solid polycarbonate floor (rest of the study) with softwood based bark-free fiber, sterilized by autoclaving
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes: Air will be filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to approx. day 60 (F0 females), from day 0 to approx. day 37 (F0 males), from approx. day 37 to approx. day 91 (F1 offspring)
Route of administration:
oral: feed
Vehicle:
other: Will be documented in the study data and included in the final report.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Unless advised otherwise, the procedure for production of a premix includes grinding the test item/diet admixture.

DIET PREPARATION
- Rate of preparation of diet: Weekly, and up to one week in advance of first feeding.
- Mixing appropriate amounts with: SDS VRF1
- Storage temperature of food: Prepared diets will be stored frozen (-10 to -30 °C). Formulated diets may be stored at ambient temperature (15 to 25ºC) (once defrosted), within the confirmed stability period of up to 8 days, pending feeding and accounting for the time in the food hoppers.
Details on mating procedure:
- M/F ratio per cage: 1.1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.
- Further matings after two unsuccessful attempts: A female showing no evidence of mating after completion of the pairing period will be separated from the male and vaginal smearing will be 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 cannot be pregnant and will be dispatched to necropsy as soon as practically possible. If an estrus smear is not seen the female will be dispatched to necropsy on or after day 25 after the last day of pairing.
- After successful mating each pregnant female was caged: Singly to permit collection of food consumption data individually for pregnant females and during lactation.
Analytical verification of doses or concentrations:
no
Remarks:
No formulation analysis will be performed on this study but was tested as part of another study (see below).
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test item in the diet during storage were confirmed as part of another study. In that study, prepared diets in the range 1500 to 15000 ppm were determined to be stable for:
- 8 days at ambient temperature (15 to 25 °C)
- 15 days when stored frozen (-10 to -30 °C)
Prepared diets will be stored frozen (-10 to -30 °C) until required for feeding.
Duration of treatment / exposure:
F0 females: Two weeks before pairing until Day 21 of lactation (approx. 60 days); F0 males: two weeks before pairing until after successful littering by females (approx. 37 days); F1 litters: From late lactation when offspring start to consume diet (approx. in the age of 28 days) to approximately week 7 of age after sexual maturity has been attained (approx. 26 days)
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
Remarks:
control
Dose / conc.:
3 750 ppm (nominal)
Remarks:
Expected achieved doses: Approx. 225 mg/kg/day (males) and 235 mg/kg/day (females)
Dose / conc.:
7 500 ppm (nominal)
Remarks:
Expected achieved doses: Approx. 456 mg/kg/day (males) and 485 mg/kg/day (females)
Dose / conc.:
15 000 ppm (nominal)
Remarks:
Expected achieved doses: Approx. 886 mg/kg/day (males) and 842 mg/kg/day (females)
No. of animals per sex per dose:
8 animals per sex per dose (F0 generation), 10 animals per sex per dose (F1 generation)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary levels for this study have been selected in consultation with the sponsor, based on the results of a 21-Day palatability study in Sprague Dawley Rats by dietary administration (see section 7.5.1). In this study, no premature decedents or treatment-related changes in clinical conditions were observed and only some slight effects on body weight and liver weight were observed. Thus the highest dose used in the palatibility study (15000 ppm, resulting in mean achieved doses of 886 mg/kg/day in males and 842 mg/kg/day in females) was chosen also for this dose-range finding test.
- Fasting period before blood sampling for clinical biochemistry: Animals were not fasted before blood sampling and clinical biochemistry.
Positive control:
Not included
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations included: Evidence of reaction to treatment or ill-health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice weekly; days 0, 3, 7, 10, 14, 18 and 20 after mating and days 1, 4, 7, 10, 14, 18 and 21 of lactation for F0 females.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly and at the day of necropsy (F0 males), twice weekly until mating detected; days 0, 3, 7, 10, 14, 17 and 20 after mating and on days 1, 4, 7, 11, 14, 18 and 21 of lactation (F0 females); days 21, 23, 25, 27 and 28 of age and twice weekly from nominal 4 weeks of age to termination at approximately 7 weeks of age (F1 animals)

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

HEMATOLOGICAL ANALYSIS
- Time schedule for examinations: At termination (no overnight deprivation of food)
- Anaesthetic: Isoflurane
- Parameters examined: Thyroid hormones, hematocrit, hemoglobin concentration, erythrocyte count, total leucocyte count, differential leucocyte count, platelet count, mean cell hemoglobin, mean cell volume, mean cell hemoglobin concentration, red cell distribution width, prothrombin time, activated partial thromboplastin time
Oestrous cyclicity (parental animals):
The oestrus cycle will be determined by dry smears (for 15 days before pairing, using cotton swabs) and by wet smears (after pairing until evidence of mating confirmed, using pipette lavage). Percentages of females showing the following cycle types will be calculated:
- Regular (all observed cycles are of 4 or 5 days; may be divided into cycles of 4, 4 and 5, and 5 days, if inter-group differences are apparent),
- Irregular: At least one cycle of 2, 3 or 6 to 10 days.
- Acyclic: At least 10 days without estrus (beginning before pairing).
Sperm parameters (parental animals):
Sperm parameters will not be examined.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- A maximum of 5 pups/sex/litter were selected. Excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical and behaviour abnormalities, anogenital distance (AGD), gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities with assessment of stomach for presence of milk, where possible. Abnormal tissues retained in an appropriate fixative.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after successful littering by females
- Maternal animals: All surviving animals on day 21 of lactation. If females will fail to produce viable litter on day 25 after mating, if litters die before weaning, on day last offspring dies. If females fail to mate, on or after day 25 after last day of pairing OR day of confirmed estrus after failure to mate.

GROSS NECROPSY
- Gross necropsy consists of external examinations (see table 1).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in table 1 will be prepared for microscopic examination and weighed, respectively. Any abnormal tissues retained and may be weighed at the discretion of necropsy staff. In addition, the following will be recorded for all F0 females (including those prematurely sacrificed, where possible): number of implantation sites, mammary tissue appearance for females whose litter dies before day 21 of lactation.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected are sacrificed at 4 days of age.
- These animals are subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Culled offspring with clinical signs on day 4 of age will be subject to complete macroscopic examination with assessment of stomach for presence of milk, where possible. Abnormal tissues retained in an appropriate fixative. Culled offspring with no clinical sign on Day 4 of age will be killed and discarded without necropsy examination.

GROSS NECROPSY
- Gross necropsy consisted of complete external examinations. Organs are weighted (see table 2).

HISTOPATHOLOGY / ORGAN WEIGTHS
Abnormal tissues will be retained in an appropriate fixative and checked histopathologically (see table 2).
Statistics:
The following data types will be 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; organ weights, both absolute and relative to body weight estrous cycles and vaginal opening to first estrous; pre-coital interval; mating performance and fertility; gestation length and gestation index; litter data; thyroid hormones.

For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group. For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Reproductive indices:
Percentage mating (Number animals mating x 100 / Animals paired), conception rate (Number animals achieving pregnancy x 100 / Animals mated), fertility index (number animals achieving pregnancy x 100 / Animals paired)
Offspring viability indices:
Post-implantation survival index (Total number offspring born x 100 / Total number uterine implantation sites), Live birth index (Number live offspring on Day 1 after littering x 100 / Total number of offspring born), Viability index (Number live off spring on Day 4 before culling x 100 / Number live off spring on Day 1 after littering), Lactation index (Number live off spring on Day 21 after littering x 100 / Number live off spring on Day 4 (after culling)
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Will be updated after completion of the study.
Key result
Critical effects observed:
not specified
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Will be updated after completion of the study.
Key result
Critical effects observed:
not specified
Key result
Reproductive effects observed:
not specified
Executive summary:

An oral feeding study with the test item in male and female Sprague Dawley rats similar to OECD 421 is currently running as a preliminary study to a extended one-generation reproductive toxicity study (basic design) according to OECD guideline 443. The nominal dose range was chosen between  0 and 15000 ppm, which is expected to result in actual achieved doses of approx. 225 to 886 mg/kg bw/day (as determined in a 21-day palatability study, please refer to section 7.5.1). Clinical signs, body weight, food consumption, hematology, gross pathology estrus cyclicity and number of implantation sites + mammary tissue appearance (for females whose litter die before day 21 of lactation) will be examined in the P0 generation. Litters will be evaluated for number and sex of pups, sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical and behaviour abnormalities, anogenital distance (AGD) and gross evaluation of external genitalia. Reproductive indices and offspring viability indices will be calculated. This section will be updated with the results of the OECD 421 study as soon as the study is finalized. 

Data source

Reference
Reference Type:
study report
Title:
Unnamed

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Adopted 2018-06-25
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: According to a dose-range study according to OECD guideline 421. The dose level setting aims to induce systemic toxicity at the highest dose level and/or some reproductive toxicity effects.
- Inclusion/exclusion of extension of Cohort 1B: Cohort 1B is not planned to be extended according to ECHA decision number TPE-D-2114449866-32-01/F as no triggers for extension were identified.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Cohorts 2A and 2B are not planned to be included according to ECHA decision number TPE-D-2114449866-32-01/F as no triggers for extension were identified.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Cohort 3 is not planned to be included according to ECHA decision number TPE-D-2114449866-32-01/F as no triggers for extension were identified.
- Route of administration: Oral

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-dimethyloct-7-en-2-ol
EC Number:
242-362-4
EC Name:
2,6-dimethyloct-7-en-2-ol
Cas Number:
18479-58-8
Molecular formula:
C10H20O
IUPAC Name:
2,6-dimethyloct-7-en-2-ol

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague-Dawley rat is an accepted strain by regulatory agencies
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 28-35 days (P0 generation), 26-30 days (F1 generation)
- Weight at study initiation: Within a 30 g weight range (P0 generation)
- Fasting period before study: No
- Housing: Up to 4 animals/cage (except during mating and from gestation to weaning (females)) in polycarbonate cages

- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): Air is filtered but not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to approx. day 120 (P0 males and females), approx. from day 101 to day 189 (cohort 1A), approx. from day 101 to day 196 (cohort 1B), approx. from day 101 to day 161 (cohort 1C)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Will be included in the final report.

DIET PREPARATION
- Rate of preparation of diet: Will be included in the final report
- Mixing appropriate amounts with: Will be included in the final report
- Storage temperature of food: Will be included in the final report
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Ejected copulation plugs and/or sperm within vaginal smear referred to as day 0 of gestation
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Singly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At specified intervals during treatment, the test diets will be analyzed for achieved concentration of the test item. Analysis for achieved concentration will be performed and reported according to GLP regulations as part of this current study. The method of analysis will be documented in the study data and a summary included in the final report. The analytical method will involve the extraction of the test formulations with a suitable solvent followed by chromatographic assay. The formulated samples will be analyzed using a method validated with respect to the determination of the specificity of analysis, limits of detection, linearity of detector response, repeatability, method accuracy and precision.
Duration of treatment / exposure:
F0 animals: For 10 weeks before pairing until termination after litters are weaned.
Unselected F1 offspring: No direct treatment - killed on Day 22 of age
Cohort 1A: From weaning until approx. 13 weeks of age (Day 90)
Cohort 1B: From weaning to approximately 14 weeks of age
Cohort 1C: From weaning to completion of sexual maturation (approximately 6-8 weeks of age)
Frequency of treatment:
Continuously through feed
Details on study schedule:
- If mating of the F1 generation is triggered, animals paired at no less than 14 weeks of age.
- Selection of parents from F1 generation when pups are 18-20 days of age.
Doses / concentrationsopen allclose all
Dose / conc.:
11 000 ppm (nominal)
Remarks:
Selected F1 generation animals for nominal weeks 5 and 6 of age. Dietary levels will be closely monitored and may be increased during the progress of the selected F1 generation should the overall mean achieved dose fall close to or below 900 mg/kg/day for Group 4.
Dose / conc.:
5 500 ppm (nominal)
Remarks:
Selected F1 generation animals for nominal weeks 5 and 6 of age. Dietary levels will be closely monitored and may be increased during the progress of the selected F1 generation should the overall mean achieved dose fall close to or below 900 mg/kg/day for Group 4.
Dose / conc.:
2 750 ppm (nominal)
Remarks:
Selected F1 generation animals for nominal weeks 5 and 6 of age. Dietary levels will be closely monitored and may be increased during the progress of the selected F1 generation should the overall mean achieved dose fall close to or below 900 mg/kg/day for Group 4.
Dose / conc.:
7 500 ppm (nominal)
Remarks:
F0 females in lactation and selected F1 generation animals to the end of nominal Week 4 of age
Dose / conc.:
3 750 ppm (nominal)
Remarks:
F0 females in lactation and selected F1 generation animals to the end of nominal Week 4 of age
Dose / conc.:
1 875 ppm (nominal)
Remarks:
F0 females in lactation and selected F1 generation animals to the end of nominal Week 4 of age
Dose / conc.:
15 000 ppm (nominal)
Remarks:
F0 animals (except for females during lactation) and selected F1 generation animals from nominal Week 7 of age
Dose / conc.:
7 500 ppm (nominal)
Remarks:
F0 animals (except for females during lactation) and selected F1 generation animals from nominal Week 7 of age
Dose / conc.:
3 750 ppm (nominal)
Remarks:
F0 animals (except for females during lactation) and selected F1 generation animals from nominal Week 7 of age
Dose / conc.:
0 ppm (nominal)
Remarks:
Control group
No. of animals per sex per dose:
24 animals per sex per dose (P0), 20 animals per sex per cohort per dose (F1)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dietary levels were selected based on the findings of the Preliminary Reproductive Performance Study (Study No. 8446771, see IUCLID section 7.8.1). In that study, dietary administration of the test item at inclusion levels up to and including 15000 ppm was generally well tolerated for F0 males and F0 females in all phases with the exception of the lactation period. Three females given 15000 ppm were killed for welfare reasons on Day 20 of lactation, following signs of thin build and piloerection, these observations were first observed on Day 12 of lactation or later. With the exception of signs observed during lactation for females given 15000 ppm. there were no test item-related changes in general clinical condition among F0 generation animals.
- Fasting period before blood sampling for clinical biochemistry: Overnight
Positive control:
Not included

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations include: Evidence of reaction to treatment or ill-health


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week for all F0 and selected F1 generation animals. For F0 females on Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 females.
- Detailed clinical observations include: Physical condition and behavior during handling, signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: Day that treatment commences, afterwards each week and before necropsy (F0 males); day that treatment commences, afterwards each week until mating detected, days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating, days 1, 4, 7, 11, 14, 18, 21 and 28 post-partum and before necropsy (F0 females); days 21, 23, 24, 25, 26, 27, and 28 of age, from nominal 4 weeks of age, twice during Weeks 1, 2 and 3 of the F1 generation and weekly thereafter and before necropsy (F1 animals)


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

WATER CONSUMPTION AND COMPOUND INTAKE: No
Oestrous cyclicity (parental animals):
Dry smears: For 15 days before pairing
Wet smears: Daily after pairing until evidence of mating confirmed, using pipette lavage and on the day of scheduled necropsy
For females showing no evidence of mating: Following completion of the pairing period the females will be separated from the male and vaginal smearing will continue for up to five days or until the first estrus smear is seen. If a female shows an estrus smear during this period, the female will be killed as soon as practically possible and subject to macroscopic examination. If a female does not show an estrus smear, a wet smear will be taken on the morning of necropsy (Day 25 after removal from pairing where Day 0 = day of removal from pairing
Sperm parameters (parental animals):
Parameters examined in all P0 and F1 Cohort 1A males:
Testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups will be killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention is paid to the external reproductive genitals which will be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities. Where possible, decedent offspring ≤21 days of age (found dead or welfare kill) will be examined and carcass retained.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after at least 18 weeks of treatment and after weaning of the F1 animals, after confirmation that no further mating is required.
- Maternal animals: All surviving animals on day 28 postpartum

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 will be prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2-5 were prepared for microscopic examination and weighed, respectively.
Statistics:
For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group. For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary. Alternative or additional methods may be carried out if deemed appropriate following data review. Details will be included in the study report.
Reproductive indices:
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: (Calculated for each group as: Number of live litters born / Number pregnant) x 100
Offspring viability indices:
Post-implantation survival index: (Total number of offspring born / Total number uterine implantation sites) x 100;
Live birth index: (Number live offspring on Day 1 after littering / Total number of offspring born) x 100;
Viability index: (Number live offspring on Day 4 before culling / 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 culling)) x 100;
Sex ratio: Individual litter values tabulated for total at Day 1 and live at Days 1, 4 (before and after cull) and Day 21 of age;
Individual litter values tabulated for total at Day 1 and live at Days 1, 4 (before and after cull) and Day 21 of age;
Sexual maturation: Individual values tabulated for age and body weight at completion.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.

Reproductive function / performance (P0)

Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Will be updated after completion of the study.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.
Description (incidence and severity):
Will be updated after completion of the study.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined
Description (incidence and severity):
Will be updated after completion of the study.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined
Description (incidence and severity):
Will be updated after completion of the study.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Will be updated after completion of the study.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Applicant's summary and conclusion