Cyclopentane, methoxy-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A reproductive toxicity screening study was conducted by the oral route (Huntingdon Life Sciences, 2012, Study VHJ0076) in accordance with OECD and EC test guidelines, and in compliance with GLP. The results suggest the substance does not have an adverse effect on reproductive performance and development in rats at dose level up to 450 mg/kg/day.

A Two-generation reproduction toxicity study in rats was conducted on the test substance according to OECD Guideline 416, non-GLP study. Under this study condition, the no observed adverse effect concentration for two-generation reproduction toxicity study was estimated to be 1250 mg/kg 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 May 2012 - 07 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD test guidelines, and in compliance with GLP; on this basis the data is considered reliable without restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 99.97%
- Lot/batch No.: 1820146

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) approximately 10 weeks
- Weight at study initiation: (P) Males: 317 - 346 g; Females: 228 - 254 g
- Housing: Polycarbonate cage with solid polycarbonate flooring; note that during pairing polycarbonate cages with stainless steel grid floors were used.
- Diet (e.g. ad libitum): Unrestricted access
- Water (e.g. ad libitum): Unrestricted access
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark.

IN-LIFE DATES: From: 09 May 2012 (animal arrival) To: 07 July 2012 (necropsy completed)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: A series of solutions at the required concentrations were prepared by dilution of individual weighings of the test substance. Fresh formulations were prepared weekly and were stored refirigerated. A previous study (Huntingdon Life Sciences, 2002, ZCE095; refer to section 7.5.1 for details) had demonstrated that formulations were stable for up to 8 days' refirigerated storage.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used for consistency with a previous study conducted on CPME (Refer to section7.5.1, ZCE095).
- Concentration in vehicle: 0 (Vehicle control group), 10, 30, or 90 mg/mL.

Details on mating procedure:

- M/F ratio per cage: 1 to 1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As noted above, a previous study performed at Huntingdon Life Sciences, ZCE095, determined that corn oil formulations of CPME were stable for up to 2 days when stored at room temperature, and for up to 8 days when refrigerated.

During Weeks 1, 2, 3 and 4 of treatment and last week of dosing, samples from each concentration were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from each group. During Week 1 following a low result for the Group 2 achieved concentration, the dose was reformulated and an additional four samples taken for analysis. Two samples were assayed from each test group and one sample assayed from the Control group. The remaining samples were frozen and retained as contingency for analysis if any result required confirmation

Duration of treatment / exposure:

A minimum of 4 weeks for males, and for 15 days before pairing until day 6 after birth of F1 generation for females.

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: at least 12 weeks

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

450 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The toxicity of CPME was previously assessed at 700, 150, and 15 mg/kg/day in a 4-week repeat dose toxicity study (refer to section 7.5.1, ZCE095). 700 mg/kg/day was not well tolerated by males, with the majority of the animals having to be terminated before day 16 of treatment; females dosed at this level showed les reaction to treatment. 150 mg/kg/day was identified as the No Observed Adverse Effect Level in both sexes. Dose levels in this study were chosen to be 50, 150, and 450 mg/kg/day on this basis.
- Rationale for animal assignment (if not random): Random

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during week 1; from week 2 onwards once each week

BODY WEIGHT: Yes
- Time schedule for examinations:Bodyweigts recorded during acclimatisation, before dosing on the day on which treatment commenced, and weekly thereafter. In addition, F0 females were weighed on days 0,3,7,10,14,17 and 20 after mating and on days 1,4 and 7 of lactation.


Food consumption was recorded according to the following schedule: F0 males and females: Weekly before pairing (from first day of treatment until pairing). F0 females: Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6 of lactation

Oestrous cyclicity (parental animals):

Dry smears taken for 15 days before pairing, using cotton swabs. Wet smears taken after pairing until mating, using pipette lavage.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
Organ weights (Adjusted for terminal bodyweight): epididymides (L and R), Pituitary, testes (L and R).

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Clinical observations, litter size, sex ratio, and individual offspring bodyweights.

GROSS EXAMINATION OF DEAD PUPS:
Where possiblefresh macroscopic examination (external and internal) with an assessment of the stomach for milk content was made.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals during week 5 of treatment.
- Maternal animals: All surviving animals : Day 25 after the last day of pairing for females failing to mate; on day 25 after mating for females failing to produce a viable litter; on or after the day that the last offspring dies for females whose litter dies before day 7, or on day 7 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- All offspring were sacrificed on Day 7 of age

For offspring surviving to scheduled termination, a careful external examination was performed for gross abnormalities and externally normal offspring were discarded without internal examination. Externally abnormal offspring were internally examined and any abnormal tissues were retained in an appropriate fixative.

Additionally, the following procedures were applicable:
Premature deaths: Missing offspring, and those grossly autolysed or grossly cannibalised, could not be examined. All other offspring dying before Day 7 were examined as detailed. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.

Reproductive indices:

Mating performance and fertility
Percentage mating = (Number animals mating/ animals paired) x 100
Conception rate (%) = (Number animals achieving pregnancy/ animals mated) x 100
Fertility index (%) = Number animals achieving pregnancy/ animals pairing) x 100

Gestation length
Gestation index (%) =( Number of live litters born/Number pregnant) x 100

Offspring viability indices:

Survival indices, the following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day1 after littering/total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 7/Number live offspring on Day 1 after littering) x 100
Group mean values were calculated from individual litter values.

Sex ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1 and 7 of age.
Percentage males = (Number of males in litter/Total number of offspring in Litter)x 100
Group mean values were calculated from individual litter values.

Clinical signs:

effects observed, treatment-related

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male receiving 450 mg/kg/day was killed for welfare reasons on Day 18 of treatment. Terminal signs included underactive behaviour, hunched posture, fast breathing, poor reflexes and partially closed eyelids. Macroscopic examination revealed dark liver and adrenals, reduced stomach and caecal content, abnormal orange viscous fluid in the jejunum with colon and rectum devoid of content. Microscopic examination of abnormalities revealed no treatment related lesions. The factor contributing to the death of this animal was poor clinical condition.

On the first day of treatment, approximately 30 minutes after dosing, three males receiving 450 mg/kg/day had an unsteady gait and one of these animals was also observed as underactive. On Day 2 at 30 minutes, only two males at 450 mg/kg/day had unsteady gait and on Day 3 only one animal was observed as underactive. From Day 4 of treatment these signs were no longer observed.

Males and females receiving 150 or 450 mg/kg/day showed an increased incidence of chin rubbing and/or increased salivation after dose administration. These signs are often associated with oral gavage administration and are considered to relate to the palatability of the dose formulations rather than a direct effect of the test substance. There were no other signs observed in association with dose administration or at routine physical examination that could be related to treatment with the substance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Overall bodyweight gain for males receiving 450 mg/kg/day was significantly low when compared with the Controls (p<0.01). Bodyweight gain of males receiving 50 or 150 mg/kg/day was essentially similar to the Controls and was considered to be unaffected by treatment.
Bodyweight gain of females for the two week period before pairing, during gestation and up to Day 7 of lactation showed no adverse effects of treatment.
During the two week period before pairing both males and females receiving 450 mg/kg/day had low food consumption when compared with the Controls; approximately 93% and 88 % of Controls during Week 1 and 85% and 88% of Controls during Week 2, for males and females respectively. Food consumption for animals receiving 50 or 150 mg/kg/day was similar to Controls and unaffected by treatment.
During gestation and up to Day 3 of lactation food consumption for treated females was similar to the Controls. However during Days 4 to 6 of lactation food consumption of treated females was slightly but significantly high when compared with Controls (p<0.05); a dose response was not apparent

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Oestrous cycles, mating performance, fertility and gestation index were unaffected by treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
After 4 weeks of treatment assessment of the organ weights for males, namely epididymides, testes and pituitary, showed no adverse effects of treatment with the substance.
Microscopic examination of the testes, epididymis and abnormalities after 4 weeks of treatment revealed no test substance related lesions.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The gestation length of treated females was within the normal range of 22 to 23.5 days. However, females receiving 450 mg/kg/day showed a slight but significant shift in the duration of gestation (p<0.05) with a higher proportion showing longer gestation lengths than the Controls. The length of
gestation was unaffected at 50 or 150 mg/kg/day.

ORGAN WEIGHTS (PARENTAL ANIMALS)
After 4 weeks of treatment assessment of the organ weights for males, namely epididymides, testes and pituitary, showed no adverse effects of treatment with the substance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The macroscopic examination of males after 4 weeks of treatment and of females on Day 7 of lactation revealed no test substance related lesions.
The incidence and distribution of all the other findings were consistent with the common background.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic examination of the testes, epididymis and abnormalities after 4 weeks of treatment revealed no test substance related lesions.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted.
Microscopic examination of the ovaries and abnormalities performed on Day 7 of lactation in females revealed no test substance related lesions.

OTHER FINDINGS (PARENTAL ANIMALS)

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

Critical effects observed:

not specified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

marginally low bodyweight gain at 450mg/kg/day

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
The viability index on Day 7 of lactation at 450 mg/kg/day was slightly but not significantly low when compared with the Controls. This was attributed solely to one litter, (no. 79) that died on Day 4 of age. In the absence of any other effect on litter survival at this dose level, it is considered not to relate to treatment with CPME.

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs that could be attributed to maternal treatment.

BODY WEIGHT (OFFSPRING)
Offspring bodyweight on Day 1 of age was essentially similar amongst the groups and did not show any adverse effects of maternal treatment.
Subsequent bodyweight gain during Days 1 to 7 of age for offspring in the 450 mg/kg/day group was marginally but not significantly low when compared with the Controls; approximately 92% and 90% of Controls for male and female offspring, respectively.
Offspring weight gain at 50 or 150 mg/kg/day however was marginally superior to Controls showing no adverse effects of maternal treatment.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring that either died before scheduled termination or were examined as scheduled on Day 7 of age did not show any findings that could be related to parental treatment.

OTHER FINDINGS (OFFSPRING)
Litter size, and sex ratio were unaffected by treatment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

450 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Dosing of parental generation had no discernable effect on the development of offspring.

Critical effects observed:

not specified

Reproductive effects observed:

not specified

Conclusions:

It is concluded that oral administration of the substance to Sprague-Dawley rats at doses of 50, 150 and 450 mg/kg/day caused mild toxicity at the highest dose in adult animals, evident as reduced weight gain for males, low food consumption for both males and females and a shift in the distribution of gestation length towards a longer gestation. There was no effect of treatment on reproductive performance, including mating performance, fertility and offspring survival and development up to Day 7 of age. Effects on the offspring were restricted to marginally low bodyweight gain at the high dose.

The no-observed-adverse-effect level (NOAEL) for reproductive function was therefore considered to be 450 mg/kg/day and, in the absence of any effects at the intermediate dose level, the no-observed-effect-level (NOEL) was considered to be 150 mg/kg/day.

Reference 2

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.

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

Specific details on test material used for the study:

Batch No.: 2820246
Purity: 99.9%

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:

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.

Dose / conc.:

0 mg/L drinking water

Dose / conc.:

313 mg/L drinking water

Dose / conc.:

1 250 mg/L drinking water

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:

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%

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 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.

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

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

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

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

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

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

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.

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

169.18 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study, non GLP, but in compliance with China National Metrology Accreditation.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A subacute oral and a subchronic inhalation toxicity studies were conducted (Mitsubishi Chemical Safety Institute Ltd., 2004, Study B031366 and Huntingdon Life Sciences, 2003, Study ZCE095) to assess the systemic toxicity of the substance in the rat, in accordance with OECD and EC test guidelines, and in compliance with GLP. In the subacute study, the substance was administered by oral gavage in corn oil, once daily, to three groups of five male and five female rats for up to twenty-eight consecutive days at dosage levels of 0, 15, 150 or 700 mg/kg/day. In addition, a further two groups of five male and five female rats (Recovery groups) received the test substance at dosage levels of 0 (Control) or 700 mg/kg/day for up to 28 days and surviving animals were then maintained for a further 14 days untreated. In the subchronic study, the substance was administered by whole-body inhalation exposure, 6 hr/day and 5days/week, to five groups of 10 male and 10 female rats for up to thirteen weeks at concentrations of 0 (Control) , 0.2, 0.4, 0.8 and 4.0 mg/L. In addition, a further two groups of 6 male and 6 female rats received the test substance at concentration of 0 or 4.0 mg/L for up to 90 days and were subjected to a further 28 days untreated as a recovery period. In both studies, testes, ovaries and uterus of animals subjected to necropsy were weighed and prepared for histopathological examination. No dose related differences in organ weight or histopathological findings were observed in the reproductive organs of the rats in these studies.

In order to provide an initial screening assessment of the influence of the substance on reproductive performance and development, a reproductive toxicity screening was conducted by the oral route (Huntingdon Life Sciences, 2012, Study VHJ0076) in accordance with OECD and EC test guidelines, and in compliance with GLP. Three groups of ten male and ten female rats received the substance by oral gavage administration at doses of 50, 150 or 450 mg/kg/day. The F0 adults were treated daily for a minimum of 15 days before pairing; males then continued treatment until necropsy after 4 weeks of treatment and females continued until Day 6 after the birth of the F1 generation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose for the same duration.The F1 generation received no direct administration of the test substance; any exposure was in utero or via the mothers’ breast milk. Oral administration of the substance caused mild toxicity at the highest dose in adult animals, evident as reduced weight gain for males, low food consumption for both males and females. Oestrous cycles, mating performance, fertility and gestation index were unaffected by treatment. The gestation length of treated females was within the normal range of 22 to 23.5 days. However, females receiving 450 mg/kg/day showed a slight but significant shift in the duration of gestation (p<0.05) with a higher proportion showing longer gestation lengths than the Controls. The length of gestation was unaffected at 50 or 150 mg/kg/day.After 4 weeks of treatment assessment of the organ weights for males, namely epididymides, testes and pituitary, showed no adverse effects of treatment with the substance. The macroscopic examination of males after 4 weeks of treatment and of females on Day 7 of lactation revealed no test substance related lesions. Microscopic examination of the testes, epididymis and abnormalities of males after 4 weeks of treatment and examination of the ovaries of females on Day 7 of lactation revealed no test substance related lesions. There were no clinical signs that could be attributed to maternal treatment. Litter size, offspring survival and sex ratio were unaffected by parental treatment. Offspring bodyweight on Day 1 of age was essentially similar amongst the groups and did not show any adverse effects of maternal treatment. Subsequent bodyweight gain during Days 1 to 7 of age for offspring in the 450 mg/kg/day group was marginally but not significantly low when compared with the Controls; approximately 92% and 90% of Controls for male and female offspring, respectively. Macroscopic examination of offspring that either died before scheduled termination or were examined as scheduled on Day 7 of age did not show any findings that could be related to parental treatment. Therefore these results suggest that the substance does not have an adverse effect on reproductive performance and development in rats at dose level up to 450 mg/kg/day.

A Two-generation reproduction toxicity study in rats was conducted on the test substance according to OECD Guideline 416, non-GLP study. 192 SD rats were assigned to four groups, 48 animals in each group, half females and half males. Administration via drinking water was used, the concentrations of sample in drinking water were 0, 313, 1250 and 5000 mg/kg respectively.

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.

The results of repeated oral exposure of test substance showed that 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/kg 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.

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-11-06 to 2013-10-31

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

no

Remarks:

non GLP, but in compliance with China National Metrology Accreditation

Limit test:

no

Specific details on test material used for the study:

Batch No.: 2820246
Purity: 99.9%

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zhejiang center of laboratory animals
- Age at study initiation: 3 months old
- Weight at study initiation: 200-240 g for female and 350-450 g for male
- Fasting period before study:
- Housing: Suspended, wire bottom, stainless steel cage, no more than 5 rats per cage
- Diet (e.g. ad libitum): Conventional laboratory diet
- Water (e.g. ad libitum): Tap water by aseptic filtration, ad libitum
- Acclimation period: more than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70%
- Air changes (per hr): 12 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The weighed sample is added to tween-80 and distilled water to form emulsion, add distilled water to the nominal dose.
Exposure pathway is gavage exposure and gavage volume is 5 mL/kg.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Female and male rats should be mated in a cage on the ration of 1:1 at 4:00 pm daily, check the vaginal plug or vaginal smear the next morning.
Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found.

Duration of treatment / exposure:

6-15 days after conception

Frequency of treatment:

daily

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

800 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

140 SD rats with 40 male and 100 female were applied. Four groups were assigned.
The number of pregnant rats in control group, low dose level group, intermediate dose level group and high dose level group was 22, 24, 25 and 21 respectively.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on acute LD50 value 3160 mg/kg in female rats
- Rationale for animal assignment (if not random): randomly
- Other:

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weigh female rats once every 3 days from conception

POST-MORTEM EXAMINATIONS: Yes
Females showing signs of abortion or premature delivery prior to scheduled termination should be killed and subjected to a thorough macroscopic examination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

- External examinations: Yes, each fetus is examined for external anomalies during caesarean section.
- Soft tissue examinations: Yes, one third of each litter was immersed in Bouin's solution and examined for viscera anomalies.
- Skeletal examinations: Yes, the remainder was immersed in 90% ethanol and examined for skeletal alterations.

Statistics:

Method
Each group of pregnant rats’ body weight, body length, tail length and body weight was compared with the control group applying Dennett’s t test. The rate of fetal resorptions, mortality and teratogenic rate deal with the chi-square test. Significance level α=0.05, chi-square test corrected significance level a=0.0170 (low, intermediate and high dose group compared with the control group, respectively).
Analysis of indicators
The number of experimental animal, average weight, number of pregnant animals, the number of corpus luteum, implantation, absorption fetus, number of live births, the number and percentage of stillbirth, fetal situation (sex, weight, placental weight, body length, tail length), teratogenic types (including appearance, bones and internal organs), the number and percentage.
Teratogenic rate:
Total teratogenic rate of each treatment group (%) = (total live fetuses of teratogenic / total live fetuses) *100%
(It was treated as a terata when live fetuses had more than one malformation)
It should be calculated for the single teratogenic rate when terata based on one or a few defects.
Single teratogenic rate of each treatment group (%) = (total terata / total live fetuses) *100%
Teratogenic index:
If total teratogenic rate or single teratogenic rate of group was significantly higher than the control group. It indicates that the dose had teratogenic effect.
Teratogenic index = LD50 of female animal/minimum teratogenic dose.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight of high dose group from day 9 were lower than the control group, body weight gain during pregnancy and absolute gain of high dose group were lower than the control group, the differences were statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

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

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Formation of rat embryos and fetal growth and development status:
In high dose group the weight of uterus (with fetal), the average fetal body weight, body length and tail length were lower than the control group, the differences were statistically significant.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

maternal abnormalities

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

In high dose group the weight of uterus (with fetal), the average fetal body weight, body length and tail length were lower than the control group, the differences were statistically significant.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In high-dose group a fetal was found gastroschisis with no tail and left hind foot valgus. But litters or fetuses as statistical unit compared with the control group, the difference was not statistically significant.
In high dose group two fetal malformations were found (different nest), a fetal with gastroschisis, no tail and left hind foot valgus, other one with rib deletion. But litters or fetuses as statistical unit compared with the control group, the difference was not statistically significant.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Some fetuses appears with hypoplastic supraoccipitals, hypoplastic interparietals and agenesis of other skull bone in high, intermediate and low dose level group. But litters or fetuses as statistical unit compared with the control group, the differences were not statistically significant.
A fetal with the right rib 13 deletion was found in high-dose group. But litters or fetuses as statistical unit compared with the control group, the differences were not statistically significant.

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Total teratogenic rate of high dose group was 0.8%, the difference was not statistically significant compared with the control group.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

no

Lowest effective dose / conc.:

800 mg/kg bw/day (actual dose received)

Treatment related:

no

Conclusions:

The results of teratogenicity study for this substance to pregnant rats repeated exposed by oral in 6 to 15 days of pregnancy showed that no obvious symptoms of poisoning was observed in all dose group (including period of exposure and the observation period). Compared with the control group, in the high dose level group, body weight gain of pregnant rats in later pregnancy was slower, the weight of uterus (with fetal), the average fetal body weight, body length and tail length were lower, the differences were significant. In high dose group two fetal malformations were found (different nest), a fetal with gastroschisis, no tail and left hind foot valgus, other one with rib deletion. But litters or fetuses as statistical unit compared with the control group, the difference was not statistically significant. The low-dose group and intermediate dose level group showed no abnormal compared with the control group. The results show that under the experimental conditions, the tested sample to the SD rats had no significant teratogenic effects. The NOAEL for maternal toxicity and the NOAEL for offspring developmental toxicity were both 200mg/(kg.d).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study, non GLP, but in compliance with China National Metrology Accreditation.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A teratogenicity study was conducted (Zhejiang Academy of Medical Sciences, 2013) on rats for this substance, in accordance with OECD Guideline 414. Three months old SD rats of sexual maturity were chosen for test substance in teratogenicity study. 140 SD rats with 40 male and 100 female were applied. Four groups were assigned: control group, low dose level group, intermediate dose level group and high dose level group, with the dosage of 0.0, 50.0, 200.0, 800.0 mg/kg respectively. Young adult nulliparous female animals are mated with males (1:1). Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found. Female rats of copulation successful were randomly assigned into each experimental group daily. Finally,the number of pregnant rats in control group, low dose level group, intermediate dose level group and high dose level group was 22, 24, 25 and 21 respectively.Dosing begins from day 6 post mating for 10 days, the control group is treated by blank emulsion.

The main observation markers for pregnant rats were the general condition, the weight change, vaginal bleeding after exposure and toxic symptom. Females are killed on the 20th day after gestation. The uteri are removed and weighted. The uterine contents are examined for number of implantations, corpora lutea, resorptions, dead and live fetuses. The sex, bodyweight, body length and tail length of each fetus are determined. Each fetus is examined for external anomalies. Approximately one third of each litter was immersed in Bouin's solution and examined for viscera anomalies. The remainder was immersed in 90% ethanol and examined for skeletal alterations.

The results of teratogenicity study for this substance to pregnant rats repeated exposed by oral in 6 to 15 days of pregnancy showed that no obvious symptoms of poisoning was observed in all dose group (including period of exposure and the observation period). Compared with the control group, in the high dose level group, body weight gain of pregnant rats in later pregnancy was slower, the weight of uterus (with fetal), the average fetal body weight, body length and tail length were lower, the differences were significant. In high dose group two fetal malformations were found (different nest), a fetal with gastroschisis, no tail and left hind foot valgus, other one with rib deletion. But litters or fetuses as statistical unit compared with the control group, the difference was not statistically significant. The low-dose group and intermediate dose level group showed no abnormal compared with the control group. The results show that under the experimental conditions, the tested sample to the SD rats had no significant teratogenic effects. The NOAEL for maternal toxicity and the NOAEL for offspring developmental toxicity were both 200mg/(kg.d).

Justification for classification or non-classification

According to CLP Regulation (Commission Regulation 1272/2008), table 3.7.1, Substances should be classified  for reproductive toxicity when there is clear evidence from humans or experimental animals, of an adverse effect on sexual function and fertility, or on development.

For this substance, the two-generation reproduction study showed decrease of weight of pups of F1 generation and F2 generation and higher organ coefficient of testicle of paternal rats of P generation and F1 generation in high dose level group, no specific adverse reproductive effects was observed. The prenatal developmental study showed the tested sample to the SD rats had no significant teratogenic effects, no evidence of adverse effect on sexual function and fertility, or on development.

On this basis it is considered that the substance does not meet the criteria for classification as toxic to reproduction or development according to the CLP Regulation.