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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 September 2011-21 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD 421 and under GLP-conditions.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
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): Cashmeran
- Molecular formula: C14H22O
- Molecular weight: 206.167
- Physical state: Liquid
- Storage condition of test material: ambient temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: the initial body weight variation did not exceed 20% of the mean weight for each sex
- Housing: macrolon cages with a bedding of wood shavings, 4 per sex pre-mating, onr male and one female during mating, individual housing after mating
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 45-65%
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 14 September 2011 To: 21 October 2011 (males)/ 31 October - 4 November 2011

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- 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)
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
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.
Duration of treatment / exposure:
Males: 30 days
Females: up to 44 days
Frequency of treatment:
Continuously in the diet
Details on study schedule:
- 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
Remarks:
Doses / Concentrations:
0, 150, 600, 1875 mg/kg food
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
<0.5, 140, 571, 1817 mg/kg food
Basis:
analytical conc.
Concentration of Cashmeran was "close to intended" (relative difference <10%) for all dose levels
Remarks:
Doses / Concentrations:
0, 10, 40, 125 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
12 animals/sex/dose
Control animals:
yes, concurrent vehicle
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

Parental animals: Observations and 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
Estrous cyclicity (parental animals):
Number of females placed with males
Number of successful copulations (=number of females mated)
Number of pregnant females as demonstrated by the presence of implantation sites observed at necropsy
Number of females surviving delivery
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight, nr of males mated with females, number of males that became sire
Litter observations:
STANDARDISATION OF LITTERS: not applicable

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups delivered (live- and stillborn), number of live pups on day 1 and 4, number of pups lost, number of litters lost entirely, number of male pups on day 1 and 4, litter size

GROSS EXAMINATION OF DEAD PUPS:
Necropsy was performed on stillborn pups and on pups that died during the study.
Postmortem examinations (parental animals):
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)
Postmortem examinations (offspring):
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 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.
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: P0 (first parental animals)

General toxicity (P0)

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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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 signidicantly 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 tubules 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 (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Dose-related effects on organ weights (liver and kidney) in the mid and high dose groups
Dose descriptor:
NOAEL
Effect level:
ca. 120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related effects observed at all doses

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

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

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicological effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a reproscreening study in which Cashmeran was given orally in the diet to Wistar rats at 0, 150, 600 or 1875 mg Cashmeran/kg food. Increased organ weights (kidney and liver) were observed in male rats at the mid- and high-dose. No treatment-related effects were observed in females. The resulting NOAELparental is 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. Based on these results, Cashmeran is considered to be not reprotoxic/toxic for the development.
Executive summary:

A reproscreening study in accordance with OECD 421 was performed to determine the reprotoxic/developmental toxic potential of Cashmeran. Cashmeran was given orally in the diet to Wistar rats at 0, 150, 600 or 1875 mg Cashmeran/kg food during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. The homogeneity, stability and content of the test substance in the diets were confirmed by analysis.

No treatment-related changes in appearance, general condition or behaviour of the animals were observed. Mean body weight and body weight change were slightly decreased in the high dose group of the male animals. Also, food consumption was slightly decreased in this dose group, in both male and female animals. The effects on body weight could only partly be explained by the decrease in food consumption, therefore, this effect was considered to be treatment-related.

Increased organ weights (kidney and liver) were observed in male rats at the mid- and high-dose. At the low dose level also an increase was observed, however, this increase was considered to be minimal. No treatment-related effects on organ weight were observed in females. Histopathologically, an increased incidence of basophilic tubules was observed at the low-, mid- and high-dose. In the low- and mid-dose this increase was considered not toxicologically relevant due to the high incidence in the control and absence of increase in severity. Specific assessment of alpha-2u-microglobulins did not demonstrate treatment-related differences. Therefore it was concluded that no histopathological changes were observed that could explain the increased kidney weights in the treated animals.

No treatment related effects were observed in reproduction indices, except for a statistically significant increase in pre-implantion 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. No treatment-related effects on pups were observed.

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. Based on these results, Cashmeran is considered to be not reprotoxic/toxic for the development.