Cyclopentane, methoxy-

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-11-16 to 2014-08-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, non GLP, but in compliance with China National Metrology Accreditation, in which the test parameters documented are based on testing guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

no

Remarks:

non GLP, but in compliance with China National Metrology Accreditation

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-090-6

EC Name:

-

Cas Number:

5614-37-9

Molecular formula:

C6 H12 O

IUPAC Name:

Cyclopentyl methyl ether

Test material form:

liquid

Specific details on test material used for the study:

Batch No.: 2820246
Purity: 99.9%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zhejiang Center of Laboratory Animals, Hangzhou, Zhejiang
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males 4-5 weeks, females 8-9 weeks
- Weight at study initiation: Males: 177-211g; Females: 99-132g
- Fasting period before study:
- Housing: Polycarbonate cages, stainless steel racks, two rats per cage
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water by aseptic filtration, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 12 air changes/hours
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
According to set doses, emulsified test substance in the plant oil, tween-80 and water, then dissolved well in drinking water, makes different concentrations of test substance to achieve the designed doses.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 3 weeks
- Proof of pregnancy: presence of sperm or vaginal plugs, set as day 0 of gestation

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure for P generation:
Male rats were exposed for a period of 10 weeks pre-mating and continued to be exposed until successful mating, female rats were exposed for a period of 2 weeks pre-mating and continued to be exposed during the period of mating, pregnancy and lactation.
For F1 generation:
Male and female rats for mating were exposed after weaning, the male rats were exposed for a period of 10 weeks and continued to be exposed until mating was finished, the female rats were exposed for a period for 10 weeks and continued to be exposed during the period of mating, pregnancy and lactation.

Details on study schedule:

One male and one female of the F1 generation were selected randomly for mating after weaning from different litters in the same group to produce the F2 generation.
F1 generation offspring not utilized for mating were humanely sacrificed after weaning.
There should be about 20 pregnant rats in each group.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L drinking water

Doses / concentrations 2

Dose / conc.:

313 mg/L drinking water

Doses / concentrations 3

Dose / conc.:

1 250 mg/L drinking water

Doses / concentrations 4

Dose / conc.:

5 000 mg/L drinking water

No. of animals per sex per dose:

48 animals with 24 per sex

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based upon available information that acute oral LD50 values of the test substance were 3160 mg/kg in male and 2330 mg/kg in female rats.
- Rationale for animal assignment (if not random):
- Other:

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each day
A general clinical observation was made each day to each animal such as behavior change, abnormality of excretion, death and other toxic clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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, weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

Gross necropsy:
At the time of termination or death during the study, all parental animals with external abnormalities or clinical signs, were examined macroscopically for any structural abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. Dead pups or pups in a moribund condition were examined for possible defects of appearance and organs.

Histopathological examination:
At the time of termination, body weight and the weight of ovaries, testicle, and epididymis of P and F1 parental animals were determined. Uterus, ovaries, cervix, testicle, epididymis, seminal vesicle, prostate, penis, pituitary, brain and target organs were preserved for histopathological examination.

Postmortem examinations (offspring):

Gross necropsy:
At the time of termination or death during the study, all parental animals with external abnormalities or clinical signs, were examined macroscopically for any structural abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. Dead pups or pups in a moribund condition were examined for possible defects of appearance and organs.

Histopathological examination:
At the time of termination, body weight and the weight of ovaries, testicle, and epididymis of P and F1 parental animals were determined. Uterus, ovaries, cervix, testicle, epididymis, seminal vesicle, prostate, penis, pituitary, brain and target organs were preserved for histopathological examination.

Statistics:

A parameter or non-parameter test was selected based on the results of normality test and homogeneity of variance test. One-way analysis of variance and Dunnett's t test were used in parameter test, Kruskal-Wallis rank sum test and Wilcoxon-Wilcox rank sum test were used in non-parameter test, the test level of a was 0.05. Chi-square test and Fisher exact probability test were used in enumeration data, the test level of a was 0.05, corrected a' was 0.0170.

Reproductive indices:

rate of mating success (%) = (number of successful mating animals / number of female animal be mated) * 100%
pregnancy rate (%) = (number of pregnant animals / number of female animals be mated) * 100%
live birth rate (%) = (number of female animals producing live offspring / number of pregnant animals) * 100%
rate of birth livability (%) = (number of offspring survived on day 4 / number of offspring survived on birth day) * 100%
survival rate after weaning (%) = (number of offspring survived on 21 d after weaning / number of offspring survived on day 4) * 100%

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

During the total test period, eating and behavior of animals were normal and no clinical signs of toxicity were observed.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Before mating, for P generation of intermediate and high dose level groups, the body weight gain of male rats were lower than the control group, the body weight gain of female rats of high dose level group was lower than control group, there were statistically significant differences.
The body weights of pregnant rats at high dose level on gestation day 0, 7, 14, 20 were lower than control group, there were statistically significant differences.
The body weight of maternal rats at high dose level on day 0, 4, 7, 14, 21 and total body weight gain during lactation period were lower than control group, there were statistically significant differences.

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

The average drinking water consumption of parental rats of intermediate and high dose groups were lower than control group, there were statistically significant differences.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in rate of mating success, pregnancy rat, live birth rate, rate of birth livability and survival rate after weaning in each dose level group compared with control group.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

169.18 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

193.45 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

5 000 mg/L drinking water

System:

male reproductive system

Organ:

other: testicle

Treatment related:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

In the total test period, there was one pregnant rat that died in childbirth in high dose level group of F1 generation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Before mating, for F1 generation, in high dose level groups, the total body weight gain of male and female rats were lower than the control group, there were statistically significant differences.
The body weights of pregnant rats at high dose level on gestation day 0, 7, 14, 20 were lower than control group, there were statistically significant differences.
The body weight of maternal rats at high dose level on day 0, 4, 7, 14, 21 were lower than control group, there were statistically significant differences.
The average litter weight on day 14, 21 and the average body weights of pups on day 7, 14, 21 of high dose level group were lower than control group, there were statistically significant differences.

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

The average drinking water consumption of parental rats of intermediate and high dose groups were lower than control group, there were statistically significant differences.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The organ coefficient of testicle in high dose group of male rats was higher than control group, there were statistically significant differences.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 250 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

organ weights and organ / body weight ratios

Target system / organ toxicity (F1)

Critical effects observed:

yes

Lowest effective dose / conc.:

1 250 mg/L drinking water

System:

male reproductive system

Organ:

other: testicle

Treatment related:

yes

Relevant for humans:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average litter weight on day 14, 21 and the average body weights of pups on day 14, 21 of high dose level group were lower than control group, there were statistically significant differences.

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In summary, the main reproductive toxicity of test sample was shown below:
in high dose level group, weight of pups of F1 generation and F2 generation decreased; organ coefficient of testicle in high dose level group of paternal rats of P generation and F1 generation were higher than control group; in intermediate dose level group and low dose level group, no reproduction toxicity was observed.
Under this study condition, the no observed adverse effect concentration for two-generation reproduction toxicity study was estimated to be 1250 mg/L in drinking water for male and female rats, converted into average intake of sample, the NOAELs were 193.45 mg/kg/day for female rats and 169.18 mg/kg/day for male rats.