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:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DIMETHYLBENZYLIDENECYCLOPENTANONE 95%
- Physical state: Solid powder/ white
- Analytical purity: 96.6 %
- Lot/batch No.: COD-000462
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services, Germany GmbH
- Age at study initiation: 36 +- 1 days for males and females in the P/F0 generation
- Weight at study initiation: P/F0 Males: 111.5 – 141.0 g; Females: 96.6 – 118.6 g
- Housing: generally: individually in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany; exeptions: matings and GD 18 - LD 21
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP” (meal) supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): Drinking water was supplied from water bottles
- Acclimation period: ca. 8 days

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

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
The test substance was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentration. Mixing was carried out for about 10 minutes in a laboratory mixer.
Details on mating procedure:
In general, male and female animals were mated overnight at a ratio of 1:1 for a maximum of 2 weeks.Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
Mating occurred by placing the male in the cage of the female mating partner from about 4.00 p.m. until 7.00 - 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 of sperm detection was denoted "day 0" and the following day "day 1 p.c.” (post coitum).
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
in total ca. 18 weeks for the P/F0 generation; ca. 4 weeks for the F1 generation
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: ca. 16 weeks (ca. 36 days from birth to the start of application plus 74 days of application before mating procedure)
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 80, 360 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25 in both the P/F0 and the F1 generation
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The following dose levels were selected:
20 mg/kg body weight/day as the expected "no observed adverse effect level"
80 mg/kg body weight/day as intermediate dose level
360 mg/kg body weight/day as the high dose level
The oral route was selected since this has proven to be suitable for the detection of a toxicological risk.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All parental animals were checked daily for clinically evident signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the male and female parental animals was determined on the first day of the premating period and then once a week at the same time of the day (in the morning); if possible, weighing was carried out until the end of the respective study section.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, once a week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
After the 10th (F0 generation parental animals) test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for days 0-7, 7-14 and 14-20 p.c. During the lactation period (animals with litter) food consumption was determined for days
1-4, 4-7 and 7-14 p.p. 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 in such measurement.
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 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):
Immediately 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:
• sperm motility
• sperm morphology
• sperm head count (cauda epididymis)
• sperm head count (testis)
Sperm motility examinations were carried out in a randomized sequence.
Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group only.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Postmortem examinations (parental animals):
SACRIFICE
The following examinations were carried out in 12 animals per test group and sex

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
• leukocytes
• erythrocytes
• hemoglobin
• hematocrit
• mean corpuscular volume
• mean corpuscular hemoglobin
• mean corpuscular hemoglobin concentration
• platelets
• differential blood count
• reticulocytes
Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.
The clotting analyses were carried out using a ball coagulometer (KC 10 A model; Amelung, Lemgo, Germany). The following parameter was determined: prothrombin time (Hepato Quick's test).

Clinical chemistry
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. The following parameters were determined:
• alanine aminotransferase
• aspartate aminotransferase
• alkaline phosphatase
• serum-γ-glutamyltransferase
• sodium
• potassium
• chloride
• inorganic phosphate
• calcium
• urea
• creatinine
• glucose
• total bilirubin
• total protein
• albumin
• globulins
• triglycerides
• cholesterol
• magnesium

Necropsy
All F0 generation parental animals were sacrificed by decapitation under CO2-anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs.

Weight parameters
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
1. Anesthetized animals
2. Liver
3. Kidneys
4. Adrenal glands
5. Testes
6. Epididymides
7. Cauda epididymis
8. Prostate
9. Seminal vesicle with coagulation glands
10. Ovaries
11. Uterus
12. Spleen
13. Brain
14. Pituitary gland
15. Thyroid glands (with parathyroid gland)

Organ / Tissue preservation list
The following organs of all F0 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in Bouin’s solution, respectively:
1. Vagina
2. Cervix uteri
3. Uterus
4. Ovaries (fixed in Bouin’s solution) (incl. Differential Ovarian Follicle Count (DOFC))
5. Oviducts
6. Left testis (fixed in Bouin’s solution)
7. Left epididymis (fixed in Bouin’s solution)
8. Seminal vesicle
9. Coagulation gland
10. Prostate
11. Pituitary gland
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Report; Project No.: 75R0263/05040
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12. Adrenal glands
13. All gross lesions
14. Liver
15. Kidneys
16. Spleen
17. Brain
18. Thyroid glands (with parathyroid glands)
All organs fixed in Bouin’s solution were embedded in paraplast.
Postmortem examinations (offspring):
Pup organ weight
After scheduled sacrifice brain, spleen and thymus of 1 pup/sex and litter from the F1 pups were weighed. Normally, the first male and first female pup/litter were taken for these determinations. For the calculation of the respective relative organ weights, the pup body weights, determined routinely during the in-life phase on day 21 p.p., were taken.
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic) as follows:
pups were examined externally and eviscerated; their organs were assessed macroscopically

GROSS NECROPSY
- Gross necropsy consisted of external examinations
If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g. skeletal staining according to a modified method of KIMMEL and TRAMMELL [Kimmel, C.A., and Trammell, C., 1981]) and/or further processing of head according to WILSON’s method (Wilson, J.G., and Warkany, J.,
1965).
The stained skeletons were evaluated under a stereomicroscope or a magnifying glass.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

HISTOPATHOLOGY / ORGAN WEIGTHS
After scheduled sacrifice brain, spleen and thymus of 1 pup/sex and litter from the F1 pups were weighed.
Statistics:
- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided): Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), estrous cycle duration, number of mating days, duration of gestation, number of pups delivered per litter, numbers of implantation sites, postimplantation loss, duration of sexual maturation (days to preputial separation, days to vaginal opening)
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT 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 data (preputial separation, vaginal opening), males with >4% abnormal sperm
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided): Proportions of affected pups per litter with necropsy observations; total spermatids/g testis, total sperm/g cauda epididymides, Follicles: primordial, growing, and primordial + growing
- Pairwise comparison of the dose group with the control group using the WILCOXON-test (onesided) with Bonferoni-Holm-Adjustment: % motility
- Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided).If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON-test (twosided) for the equal medians: Pup organ weights, Clinical path. parameters, weight parameters
Reproductive indices:
Male mating index (%)
Female mating index (%)
Female fertility index (%)
Gestation index (%)
Live birth index (%)
Postimplantation loss (%)
Offspring viability indices:
Viability index (%)
Lactation index (%)
Sex ratio (on d0/d21)

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY
360 mg/kg body weight/day
• Statistically significantly decreased mean body weights in week 7 and during weeks 9 to 14 (final weight 7% below controls) and impaired body weight gain (10% below control) in the male rats
• Decreased mean body weights during gestation (7% below control on day 20 p.c.) and lactation (3% below control on day 21 p.p.)
• Decreased body weight gain during premating (7% below control) and gestation (12% below control) in female rats
• Increased cholesterol and decreased glucose levels in the rats of both sexes
• Increased activity of the γ-glutamyltransferase as well as decreased albumin levels in the males
• Increased total protein as well as globulin levels in the females
• Significantly decreased terminal body weight in males and females
• Significantly increased absolute and relative liver weight in males and female
• Liver: hypertrophy, central in 17 males and 7 females
• Reduced number of implantation sites per dam (10.6 versus 12.8 in the control)
• Reduced number of pups delivered per dam (10.0 versus 11.8 in the control)

80 mg/kg body weight/day
• Decreased body weights during gestation (6% below control on day 20 p.c.) and lactation (4% below controls on day 21 p.p.) in females
• Decreased body weight gain during premating period (5% below control) and gestation
(9% below control) in females
• Decreased glucose levels in the male rats
• Significantly decreased terminal body weight in females
• Significantly increased relative weights of the liver in males
• Liver: hypertrophy, central in 2 males and 2 females, respectively

20 mg/kg body weight/day
• No test substance-related adverse findings

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: significant impairment of body weight gain and pathological changes the liver in higher doses; significant disturbance of maternal homeostasis during pregnancy in females
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The slightly lower litter size at a dose of 360 mg/kg body weight/day is considered to be treatment-related, but secondary to maternal toxicity.

Results: F1 generation

Details on results (F1)

360 mg/kg body weight/day
CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
• Statistically significantly decreased mean body weights (6% below control at weaning) and body weight gain (8% below control, postnatal days 4-21) of F1 male and female pups
F1 reared animals
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS
• No test substance-related adverse findings

80 mg/kg body weight/day
CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
• No test substance-related adverse findings
F1 reared animals
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS
• No test substance-related adverse findings

20 mg/kg body weight/day
CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
• No test substance-related adverse findings
F1 reared animals
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS
• No test substance-related adverse findings

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: slightly decreased body weight gain at 360 mg/kg body weight/day lactation

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Under the conditions of this extended-One-Generation Reproduction Toxicity Study the NOAEL (no observed adverse effect level) for general, systemic toxicity of the test substance is 20 mg/kg body weight/day for the F0 parental rats of both sexes. At dose levels of 80 mg/kg body weight/day and above the test compound caused a significant impairment of body weight gain, which was most continuous in the F0 parental females during gestation, as well as pathological changes the liver, which were considered correlated to several clinical pathological changes. All clinical, clinical pathological and pathological findings together were indicative of a significant disturbance of maternal homeostasis during pregnancy.

The NOAEL for reproductive performance and fertility is 80 mg/kg body weight/day for the F0 parental rats. The slightly lower litter size at a dose of 360 mg/kg body weight/day is considered to be treatment-related, but secondary to maternal toxicity.

The NOAEL for developmental toxicity in the F1 progeny of the test substance is 80 mg/kg body weight/day. The slightly decreased body weight gain of F1 offspring at 360 mg/kg body weight/day lactation is considered to be treatment-related. However, the test compound did not adversely affect post-weaning development and sexual maturation.

---

The various analyses:

- demonstrated the stability of the test substance preparations over a period of up to 42 days at room temperature

- confirmed the homogeneous distribution of the test substance in the diet

- showed the correctness of the prepared concentrations.

 

Applicant's summary and conclusion