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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
other: Study started in November 2019
Adequacy of study:
key study
Study period:
Study started in November 2019. Draft report will be available in December 2020.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
other: Study plan
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The study design is based on final decision on acompliance check from ECHA dated 5th of April 2017. An Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows, was requested:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some 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;

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
3-aminomethyl-3,5,5-trimethylcyclohexylamine from Evonik, Batch: 19111847

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rat / CD® / Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Body weight (at start of dosing): *
Age (at start of mating): Approximately 20 weeks* (young adults; sexually mature)

Selection of species: The rat is a commonly used rodent species for such studies and required by the guideline.

Number of parental animals:
Pre-exposure period: At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
Main study: 192 animals (96 males and 96 females)

A sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.

Adaptation period: At least 5 days


ENVIRONMENTAL CONDITIONS

DIET Commercial diet ssniff R/M-Z V1324, offered ad libitum
Drinking water Tap water is offered daily ad libitum
Housing Singly in MAKROLON cages (type III plus)
Temperature 22°C ± 2 °C (maximum range)
Humidity 55% ± 10% (maximum range)
The rooms are alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified water
Details on exposure:
Route of administration: Oral, via gavage
Frequency of administration: Once daily; see section 2.9 for the treatment periods
Vehicle: Purified water
Administration volume: 10 mL/kg b.w.
Dosages: 0, 25, 80, 240 (160) mg/kg bw/d
Selection of route of administration: According to international guidelines.

Dose reduction since 9th of January 2020
Duration of treatment / exposure:
F0 ANIMALS
Males: 10 weeks prior to mating, during the mating period, and at least until weaning of the F1 generation (up to and including the day before sacrifice).
Females: 10 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).

F1 ANIMALS
Until weaning, F1 animals are indirectly exposed to the test item through the breast milk. After weaning, dosing will continue in the same way as for the parental generation
Cohort 1A: Until a dosing period of 10 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 91)
Cohort 1B: Until a dosing period of 11 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 98)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
240 mg/kg bw/day (nominal)
Remarks:
high dose group; dose reduced on 9th of November to 160 mg/kg bw/d
No. of animals per sex per dose:
20 males and females in P generation and 20 males and females in Cohort 1A and Cohort 1B
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CLINICAL SIGNS
All animals
Throughout the test period, each animal will be observed for clinical signs at least once daily. The frequency will be increased when signs of toxicitiy are observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity are recorded.

FOOD CONSUMPTION
Food consumption is recorded daily during pre-mating period in males and females and during gestation period on GD 7, 14, 21 and during lactation period on PND 1, 7, 14, 21 in females. In males food consumption ois further recorded during post mating period on a weekly basis.

Individual animals will be observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness.
Cageside observations will include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed will be recorded.
In addition, animals will be checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals will be checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change, and disappearance of clinical signs will be maintained on clinical history sheets for individual animals.
F0 animals and F1 animals after weaning
Additionally, a more detailed examination of all F0 and F1 Cohort 1B animals is conducted on a weekly basis. F0 animals will be examined once before the first exposure (to allow for within-subject comparisons) and weekly thereafter until termination. F1 animals will be examined weekly after weaning until termination.
Detailed clinical observations will be made in all animals outside the home cage in a standard arena, approximately at the same time of day, each time preferably by observers unaware of the treatment. Signs noted will include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) will also be recorded.

MORTALITY
Further checks will be made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This will allow post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure will be followed except that the final check will be carried out at approximately 3:30 p.m.

BODY WEIGHT
The animals will be weighed and the weights recorded as scheduled daily during pre-mating, mating period. Females will be weighted daily during gestation (reported for GD 0, 7, 14, 21), lactation period (reported for PND 4, 7, 14, 21) and males during post mating period.

Water Water consumption is monitored by visual appraisal daily throughout the study.

HAEMATOLOGY
10 males and 10 females randomly selected from each F0 group.

Differential blood count (relative)
Differential blood count (absolute)
Erythrocytes (RBC)
Leucocytes (WBC)
Haematocrit value (HCT)
Haemoglobin content (HGB)
Platelets (PLT)
Reticulocytes (RET)
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY
Sodium
Potassium
Calcium
Chloride
Albumin
Total bilirubin
Total cholesterol
Glucose
Total protein
Blood urea (BUN)
Creatinine
Alanine amino-transferase (ALAT/GPT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT/GOT)
Bile acids
Lactate dehydrogenase (LDH)
Sodium/Potassium ratio
Globulin
Albumin/globulin ratio
BUN/creatinine ratio

T4 and TSH Determination
From 10 males and females of F0 at sacrifice

Urinanalysis
At the end of the F0 dosing period

Parameters: Volume, pH and specific gravity
Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Haemoglobin, Nitrite
The deposit will be examined for the presence of the following parameters:
- Epithelial cells
- Leucocytes
- Erythrocytes
- Organisms
- Further constituents (i.e. sperm, casts)
- Crystalluria
Oestrous cyclicity (parental animals):
Vaginal smears will be taken and the oestrus cycle stages will be determined at the following time points:

F0 animals 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days) and during 10 weeks of premating until evidence of mating.
F1 animals, cohort 1A Start after onset of vaginal patency until first appearance of cornified cells and two weeks starting around PND 75.
F0 and F1 animals On the day of sacrifice, Shortly before necropsy.
Sperm parameters (parental animals):
Sperm viability and morphology (spermiogram)
• All F0 males
• All F1 Cohort 1A males
One epididymis and one testicle will be used for the sperm count. The sperm viability is determined and the sperm morphology is examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Litter observations:
Littering
Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts4 and the presence of gross abnormalities. Based on these parameters, the reproductive performance of the dams will be evaluated.
The pups will be examined as described below. Any abnormal behavior will be recorded.

Counting, sexing and weighing
Live pups will be counted and sexed. Live pups are weighed on PND 1 and regularly thereafter.

Ano-genital distance
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups will be determined using a scale.

Litter adjustment
After counting on PND 4, the litters will be adjusted to 10 pups per litter (5 pups/sex/per litter) by eliminating surplus pups following a randomisation scheme. Selective elimination of pups, e.g. based upon body weight is not appropriate. In case of unequal gender distribution, partial litter size adjustment is acceptable (e.g. 6 male and 4 female pups).

Nipples/areolae counting
Nipples/areolae will be counted in all male pups on PND 13.

Sexual maturation
Animals (all selected) are evaluated daily for balano-preputial separation or vaginal patency before expected achievement of these endpoints to detect if sexual maturation occurs early. Any abnormalities of the genitals are recorded. Sexual maturity of the animals is compared to physical development (i.e. body weight and age at balano-preputial separation or vaginal opening).

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

not yet available

Effect levels (P0)

Dose descriptor:
other: no Dose descriptor derived yet
Remarks on result:
other: study not yet completed

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: P1 (second parental generation)

Details on results (P1)

not yet avialable

Results: F1 generation

Effect levels (F1)

Dose descriptor:
other: no dose descriptor derived yet
Remarks on result:
other: study not completed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Up to now, there are no results of the study available.
Executive summary:

The OECD 443 study started in November 2019 and the first draft report is expected to be available in December 2020.