Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
The study was extended by additional examinations (estrous cycle, sperm parameters, Organ weight determinations, extended histopathology, signs of sexual maturation in selected pups and/or parental animals) that are required in the following test guidelines:
OECD Guideline for Testing of Chemicals, No. 416 (Jan. 2001 )
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test substance: 625-Triketon
- Test substance No.: 99/273-1
- Batch No.: LJ.292007118
- Date of production: June 21-30,1999
- Content: 97,3 g/100g
- Stability: Proven by reanalysis
- Homogeneity: Homogenous
- Physical state/appearance: Powder / yellow
- Storage conditions: Refrigerator

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Sulzfeld, Germany
- Strain/Quality: (CrlGlxBr1Han:Wi) former name: (CRL:WI (GWBRUHAN) IGS BR)
- Age at study initiation: 28 (± 1) days, females were nulliparous and non-pregnant at the beginning of the study
- Weight: male animals: 98.7 - 100.5 g; female animals: 85.3 - 86.1 g
- Fasting period before study: none
- Housing: individually; from day 18 of gestation until day 21 after birth, the pregnant animals and their litters were cohoused
- Diet: ground Kliba maintenance diet rat mouse/hamster meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Tap water ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Food analyses
The food used in the study was assayed for chemical as well as for microbiological contaminants.

Drinking water analyses
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF AG as well as for the presence of microorganisms by a contract laboratory.

Bedding analyses
The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

To prepare the suspensions, the appropriate amount of test substance was weighed, depending on the dose group, in calibrated beakers. Then the olive oil was filled up to the desired volume and mixed thoroughly with a high speed dispersing and emulsifying apparatus (Ultra Turrax, JAHNKE & KUNKEL AG, Germany). In respect to the proven stability the test substance preparations were prepared once weekly before dosing. After preparation the suspensions were split up in portions and deep frozen (-20°C). Before administration the required portions were defrosted.
Details on mating procedure:
Matings of F0 generation parental animals
In general, each of the male and female animals was mated overnight at a 1:1 ratio for a maximum of 2 weeks (exception in few matings) due to premature mortalities of single females). Generally, throughout the mating period, each male animal was mated with a predetermined female animal from the same dose group. Matings occurred by placing the female in the cage of the male mating partner from about 4.00 p.m. until 17.00 - 9.00 a.m. of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" p.c. (post coitum).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis
Analytical verifications of the stability of the test substance in olive oil (storage in freezer) over a period of up to 7 days was verified during this study. Homogeneity and concentration analyses were performed at the start of the administration period in samples of the high and low concentration. Samples of the oily test substance suspensions were sent to the analytical laboratory three times during the study period for verification of the concentrations. Three additional samples of the low concentration were sent towards the beginning of the application period due to an analytical problem.
Duration of treatment / exposure:
10 weeks prior to mating, during mating, gestation, lactation until weaning of F1 pups
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 6, 36 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Standardisation of litter:
- Day of standardization: 4 days p.p.
- Standard litter size: in total 8 pups, 4 female and 4 male if possible

Examinations

Parental animals: Observations and examinations:
Parental animals
Mortality
At least once daily a check was made for dead or moribund animals.

Clinical observations
Parameters recorded once daily: clinical signs of toxicity in the parental animals; nesting and lactation behaviour of the dams
Parameters recorded twice daily (except holidays): littering behaviour of the dams

Food consumption
Food consumption determined once weekly: F0 parental animals during premating period (until 10th week), F0 females during pregnancy, F0 females from the second week of lactation on.
Food consumption determined twice weekly: F0 females during first week of lactation
Food consumption was not determined between days 14 and 21 after parturition as required in the test guidelines, since during this time pups will begin to consume considerable amounts of solid food offered, and therefore there was no point for such a measurement.

Body weight data
Body weights were determined once weekly and before the start of the study., with the following exceptions:
a) During the mating period the F0 generation parental females were weighed on the day of positive evidence of sperm and weekly thereafter
b) Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
C) Females with litter were weighed on the day of parturition, twice within the first 7 days thereafter and once weekly in the remaining time
d) Females without litter were not weighed during the lactation phase.


Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated daily for all F0 female parental rats for a minimum of 3 weeks prior to mating and these evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at necropsy a vaginal smear was examined to determine the stage of the estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
Sperm parameters
lmmediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from the F0 males of all dose groups. The following parameters were determined in the F0 males of all dose groups:
Sperm motility, sperm morphology, sperm head count (cauda epididymis and testis)
Litter observations:
Pup number and status at delivery
All pups derived from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter.

Pup viability/mortality
In general, a check was made for any dead or moribund pups once or twice daily. Viability and lactation indices were calculated according to the following formulas:

Viability lndex (%) = ((number of live pups on day 4 after birth)/(number of live pups on the day of birth)) x 100
Lactation lndex (%) = ((number of live pups on day 21 after birth)/(number of live pups on day 4 after birth)) x 100

Sex ratio
On the day of birth (day 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. The sex of the pups was assessed by the external appearance of the anogenital region and/or the mammary line of the animals and was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 21 after birth according to the following formula:

Sex ratio =((number of live male or female pups on day 0/21)/ (number of live male and female pups on day 0/21)) x 100

Pup body weight data
The pups were weighed twice within the first week and weekly thereafter.

Pup clinical observations
All live pups were examined each day for clinical symptoms (including gross-morphological findings).

Selected F1 animals (reared F1 weanlings)
Mortality
At least once daily a check was made for dead or moribund animals.

Clinical observations
All selected F1 animals were checked daily for clinical signs of toxicity.

Food consumption
Food consumption was determined once a week (each time for a period of 7 days).

Body weight data
Body weight was determined once a week at the same time of the day (in the morning); if possible, the weighing were carried out until nearly the end of the respective study period. The body weight change of the animals was calculated from these results. Additionally, at the day of vaginal opening/preputial separation the body weights of the respective animals were also determined.

Sexual maturation data
Vaginal opening
All female F1 pups selected to become the F1 animals (25/group) were evaluated daily for vaginal opening with examinations initiating on day 27 p.p.
Preputial separation
All male F1 pups selected to become the F1 animals (25/group) were evaluated daily for preputial separation with examinations initiating on day 40 p.p.
Postmortem examinations (parental animals):
Necropsy
The F0 parental animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animals that died prematurely were necropsied and assessed as soon as possible after death to prevent autolysis.

Organ weights
The following weight parameters F0 group were deterrnined:
anesthetized animals, liver, kidneys, epididymides (total, tail), testes, uterus with oviducts and cervix uten, ovaries, seminal vesicle (including coagulating gland and its liquid), prostate gland, brain, pituitary gland, adrenal glands, spleen, thyroid glands including parathyroid glands

Histopathology
The following organs or tissues were fixed in 4% formaline solution:
vagina, cervix uteri, uterus, ovaries (fixed in BOUIN's solution), oviducts, left testis (fixed in BOUIN's solution), left epididymidis (fixed in BOUIN's solution), seminal vesicles, coagulating glands, prostate gland, pituitary gland, liver, kidneys, spleen, brain, adrenal glands, all gross lesions, thyroid glands, including parathyroid glands
After fixation, the organs were hematoxylin-eosin stained, examined light-microscopically and assessed. A correlation between gross lesions and histopathological findings was performed. An attempt was made to evaluate testes and epididymides by staging the spermatogenesis according to the methods described by HESS.
Postmortem examinations (offspring):
Pup necropsy observations
All pups with scheduled sacrifice were killed by means of CO2. These pups were examined externally and eviscerated, their organs were assessed macroscopically. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated, and their organs assessed macroscopically.

Pup organ weights
After scheduled sacrifice brain, spleen and thymus of 1 pup/sex and litter were weighed.

Statistics:
CLINICALEXAMINATIONS
Food consumption, parental animals' body weight and body weight change (parental animals and pups),estrous cycle duration, number of mating days, duration of gestation, number of pups delivered per litter, sexual maturation: dose group comparison with the control using two-sided Dunnett's test
Male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy, sexual maturation: comparison of dose groups with the control group using the Fisher's exact test
Proportions of affected pups per litter with necropsy observations: Pairwise comparison of the different dose groups using the one-sided Wilcoxon test
Pup organ weights: Non parametric analysis using two-sided Kruskal-Wallis test, comparison of the dose groups with the control group using the two-sided Wilcoxon test
Males with >4% abnormal sperm: Fisher's exact test
Total spermatids/g testis, Total sperm/g cauda epididymides, % motility: comparison of the dose groups with the control group using the Wilcoxon test with Bonferi-Holm adjustment.

PATHOLOGY
Weight parameters: two-sided Kruskal-Wallis test for non-parametric analysis; Wilcoxon test for comparison of dose groups with the control group
Reproductive indices:
Male reproduction data
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating index (%) = ((number of males with confirmed mating)/ (number of males placed with females)) x 100
Male fertility index (%) = ((number of males proving their fertility)/ (number of males placed with females)) x 100

Female reproduction and delivery data
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

Female mating index (%) = ((number of females mated) / (number of females placed with males)) x 100
Female fertility index (%) = ((number of females pregnant)/(number of females mated)) x 100´
Gestation index (%) = ((number of females with live pups on the day of birth)/ (number of females pregnant)) x100

Offspring viability indices:
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:
Live birth index (%) = ((number of liveborn pups at birth) / (total number of pups born)) x 100

The implantations were counted and the postimplantation loss (in %) was calculated according to the following formula:
Postimplantation loss (%) = ((number of implantations - number of pups delivered)/ (number of implantations)) x 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
36 mg/kg bw: salivation after treatment in 22 out of 25 male and 6 out of 24 female animals; no mortalities
6 mg/kg bw: salivation after treatment in 5 out of 25 male and 2 out of 24 female animals

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
36 mg/kg bw: significantly decreased mean body weights and body weight gain in males (from 2. week onwards)
6 mg/kg bw: significant decrease of the terminal body weights of male rats

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no test substance-related adverse effects observed.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
36 mg/kg bw: decrease of epididymal sperm head count by nearly 50%, increase of morphologically abnormal sperms (approx. 79%), almost no motile sperms (9%)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
36 mg/kg bw: no influence of the compound on mating behavior (mating index 100%), but none of the female mating partners became pregnant (fertility index 0%); no pups in this dose group

GROSS PATHOLOGY (PARENTAL ANIMALS)
36 mg/kg bw: significant decrease of absolute testes , epididymides and cauda epididymides weight

HISTOPATHOLOGY (PARENTAL ANIMALS)
36 mg/kg bw: reduced sperm density in the epididymides

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
6 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: fertility index; epididymides and spermatogenesis
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
1 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: decreased terminal body weight

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
36 mg/kg bw: no pups born; none of the females became pregnant

CLINICAL SIGNS (OFFSPRING)
no test substance-related adverse effects observed.

BODY WEIGHT (OFFSPRING)
no test substance-related adverse effects observed.

SEXUAL MATURATION (OFFSPRING)
no test substance-related adverse effects observed.

GROSS PATHOLOGY (OFFSPRING)
no test substance-related adverse effects observed.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
6 mg/kg bw/day
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Under the test conditions chosen, 625 -Triketone displays toxicity to reproduction and development. Target organs were epididymides, in particular spermatogenesis.

Applicant's summary and conclusion