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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-02-24 to 2021-12-17 (estimated)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
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.

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
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)

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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)

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.

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.

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

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 EOGRS soon as the study is finalized.