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Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD TG 421
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one
EC Number:
251-649-3
EC Name:
1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one
Cas Number:
33704-61-9
Molecular formula:
C14H22O
IUPAC Name:
1,1,2,3,3-pentamethyl-2,3,4,5,6,7-hexahydro-1H-inden-4-one

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Labs, Wilmington, Massachusetts
- Weight at study initiation: 150 - 280 grams
- Fasting period before study: 18 hours
- Housing: Individually in stainless steel wire mesh cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 C +/- 3 C
- Humidity (%): 30 - 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12h light, 12h dark

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- Rate of preparation of diet (frequency): Experimental diets were prepared (mixing in a mechanical blender) one week before the start of the study and at 3 weeks during the study. After preparation, the experimental diets were divided into daily amounts and stored in plastic bags in a freezer (≤-18°C). Each day, one bag per day was removed for use
- Mixing appropriate amounts with (Type of food): cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3, supplied by SDS Special Diet Service, Witham, Englang)
- Storage temperature of food: in a freezer: (≤-18°C)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet samples were extracted with acetonitrile. The resulting samples were analyzed with HPLC with UV detection at 210nm, using a Phenomenex Luna 5 μm C18 150 mm/ 4.6 mm column with a MilliQ water/acetonitrile gradient. Cashmeran concentration was quantified by comparing the peak area of in the sample extracts with those in calibration solutions containing known amounts of Cashmeran. Validation of the method was performed by analyzing three spiked samples per low dose level and three samples per high dose level, to conform linearity, selectivity, recovery and repeatability.
Details on mating procedure:
Details on mating procedure
- M/F ratio per cage: 1/1
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually for the birth and rearing of their pups
Duration of treatment / exposure:
Males: 30 days
Females: up to 44 days
Frequency of treatment:
Continuously in the diet
Duration of test:
- Treatment schedule males: 14 days premating, maximally 4 days mating, up to sacrifice
- Treatment schedule females: 14 days premating, maximally 4 days mating, 21/22 days gestation, 4 days lactation
- Age at mating of the mated animals in the study: approximately 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg diet
Remarks:
Nominal expected dose level 0 mg/kg bw/day
Dose / conc.:
150 mg/kg diet
Remarks:
Nominal expected dose level 10 mg/kg bw/day
Dose / conc.:
600 mg/kg diet
Remarks:
Nominal expected dose level 40 mg/kg bw/day
Dose / conc.:
1 875 mg/kg diet
Remarks:
Nominal expected dose level 125 mg/kg bw/day
No. of animals per sex per dose:
12 animals/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on the results of a 2-week dose-range finding study with Cashmeran in rats
- Rationale for animal assignment (if not random): computer randomization proportionally to body weight

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: signs of toxicity, dead or moribund animals, abnormalities, signs of ill health and reaction to treatment

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: One day before the start of the experiment (at randomization), at the start of the study (Day 0) and weekly thereafter for males and females. Females were also weighed on days 1 and 4 of lactation.
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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

SACRIFICE
- Male animals: All surviving animals after the mating period
- Maternal animals: All surviving animals at or shortly after day 4 of lactation
GROSS NECROPSY
- Gross necropsy consisted of gross examination for pathological changes.
HISTOPATHOLOGY / ORGAN WEIGHTS
-The following tissues were weighed and prepared for microscopic examination: Testes, Epididymides, Liver, Kidneys, Seminal vesicles, Prostate, All gross lesions
-The following tissued were prepared for microscopic examination: Ovaries (after counting of the corpora lutea), Uterus (after counting of the implantation sites)
Fetal examinations:
SACRIFICE: At or shortly after day 4 of lactation, all surviving pups were killed by hypothermia in a freezer (-18°C) whilst under CO2/O2 anaesthesia
GROSS NECROPSY: All surviving pups were examined externally for gross abnormalities and stored in a freezer for possible further skeletal and visceral analyses
HISTOPATHOLOGY / ORGAN WEIGTHS: Not applicable.
Statistics:
The following methods were used to analyse the data. P < 0.05 was considered as the level of significance.
- Clinical findings: Fisher's exact probability test.
- Body weight, body weight gain, food consumption and organ weights data: one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Number of mated and pregnant females, the number of pregnant females with implants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely: Fisher's exact probability test
- Pre-coital time (mean number of days), the duration of gestation, the number of corpora lutea and implantation sites, the total number of pups delivered (mean), the mean number of live pups per litter 99 and pre- and post-implantation loss (%): Kruskal-Wallis nonparametric analysis of variance and the Mann-Whitney U test.
- Mortality data and data of the pathology of parent animals: Fisher’s exact probability test.
Indices:
Reproductive indices
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sire/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100

Offspring viability indices
- viability index day n-m = (number of pup surviving m days/number of liveborn on day n) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of
implantation sites] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Not applicable
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Mean body weight of male
animals of the high dose group were slightly but statistically significantly decreased during the dosing period, as well as the mean body weight gain. Furthermore, a slightly but statistically significantly decrease in food consumption was observed in both male and female animals of the high dose group, during the first week of the study.
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): Related to food consumption (see above)
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): No effects observed
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No effects observed
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No effects observed
ORGAN WEIGHTS (PARENTAL ANIMALS): A dose-related increase in absolute or relative weight of the kidneys and live was observed in the male animals of all dosing groups. The increase was minimal at low dose.
GROSS PATHOLOGY (PARENTAL ANIMALS): No effects observed
HISTOPATHOLOGY (PARENTAL ANIMALS): No treatment-related changes observed. Additional
histopathological investigation of the kidneys for specific assessment of alpha-2u-microglobulins did not demonstrate treatment-related differences, Although an increased incidence of basophilic tubul es was observed in males at the highest dose group, this could not be related to an underlying histopathological change. Therefore it was concluded that no histopathological changes were observed that could explain the increased kidney weights in the treated animals.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
females
Effect level:
1 875 mg/kg diet
Based on:
test mat.
Basis for effect level:
other: No effects
Remarks on result:
other: Corresponds to 121.83 mg/kg bw/day (actual intake).
Key result
Dose descriptor:
NOAEL
Remarks:
males
Effect level:
150 mg/kg diet
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: Corresponds to 9.76 mg/kg bw/day (actual intake).

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
no effects observed

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 875 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No toxicological effects of the test substance on fertility and development.
Remarks on result:
other: Corresponds to 121.83 mg/kg bw/day (actual dose received).
Key result
Dose descriptor:
NOAEL
Effect level:
1 875 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No toxicological effects of the test substance on fertility and development.
Remarks on result:
other: Corresponds to 115.24 mg/kg bw/day (actual dose received).

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

VIABILITY (OFFSPRING): A statistically significantly increase in pre-implantation loss in the females of the high-dose group was observed, however this couls be related mainly to 2 females and was considered to be not related to treatment
CLINICAL SIGNS (OFFSPRING): On postnatal day 1, a high incidence of pups that were cold (all groups) or had no milk in the stomach (control group) were observed. These findings were considered to be not related to treatment
BODY WEIGHT (OFFSPRING): No effects observed
GROSS PATHOLOGY (OFFSPRING): Stillborn pups that died during the lactation period showed no milk in the stomach, undistended lungs or were partly cannibalized. These findings were considered to be not related to treatment

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test (OECD TG 421, GLP), the systemic NOAEL is determined to be 150 mg/kg food, corresponding to 9.76 mg/kg bw based dose related effects on organ weights (liver and kidneys) in males. In females no adverse systemic effects were seen.
The NOAEL for fertility and developmental toxicity was determined to be 1875 mg/kg food, corresponding to 115.24 mg/kg bw/d for males and 121.83 mg/kg bw/d for females, respectively. The lower value will be used for the risk assessment.
Executive summary:

This summary is the same as for toxicity to reproduction. 


In a reproscreening study (OECD TG 421, GLP) test substance was given orally in the diet to Wistar rats at 0, 150, 600 or 1875 mg/kg food during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. Doses were selected based in a 2-week dose-range finding study with the test substance in rats. The control group received the vehicle only (diet).


Results:


Clinical signs: Daily clinical observations during the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Mean body weight of male animals of the high dose group were slightly (<10%) but statistically significantly decreased during the dosing period and the mean body weight gain of these animals was statistically significantly decreased between days 7-14, 21-28 and 0-28.


A slightly, but statistiscally significantly decrease in food consumption was observed in the male and female animals of the high dose group during the first week of the study, most probably related to the palatability of the diets containing a high concentration of the test substance. Since the effect on body weight gain in the male animals could only be partly explained by the lower food consumption during the first week of the dosing period, the effects on body weight gain of the high dose males were considered treatment related.


Organ effects: In males increased relative liver weight was seen: 9, 20 and 31% in the low, mid and high dose respectively of which the high dose may be considered adverse but not histopathology was performed and makes it difficult to assess its adversity. In females the increase was only 9% at the high dose and not considered adverse.


In males increased relative kidney weight were seen: 7, 14 and 22%, in the low, mid and high dose, respectively,In females this increase was 6%. In males, histopathology showed an increase in basophilic tubili: 3, 6, 6 and 12/12 animals, reaching statistical significance at the high dose. This high dose effects are considered adverse. Specific assessment of alpha-2u-microglobulins did not demonstrate treatment-related differences.


In females of all dose groups no effects on liver or kidney weigh were observed.


Fertility: No treatment related effects were observed in reproduction indices, except for a statistically significant increase in pre-implantation loss in the highest dose group. However, as this effect was mainly due to 2 females in this group and the mean pre-implantation loss was within the historical control range, this effect was considered to be not related to treatment.


Developmental: No treatment-related effects on pups were observed.


Conclusion: Based on the results, the NOAELparental is considered to be 150 mg/kg food, corresponding to an overall intake of 9.76 mg/kg bw/d for males. No effects were observed on fertility and the development. Therefore, the NOAELfertility/developmental toxicity is 1875 mg/kg food, corresponding to 115.24 mg/kg bw/d for males and 121.83 mg/kg bw/d for females.