3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)pent-3-en-2-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Assigned reliability score of 2 on the basis that the test substance is being used for read-across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: approximately eight to nine weeks old
- Weight at study initiation: males weighed 219g to 253g, the females weighed 193g to 227g
- Fasting period before study: n/a
- Housing: Depending upon the phase of the study, the animals were housed in P2000 cages, RB3 cages or 2154 cages
- Diet (e.g. ad libitum): SDS VRF1 Certified diet
- Water (e.g. ad libitum): public supply
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23'C
- Humidity (%):40 to 70%
- Air changes (per hr): At least 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark; lights on at 06:00 hours GMT.

IN-LIFE DATES: From: 18 March 2010 To: 11 May 2010

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle for oral dosing
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5mL/kg

Details on mating procedure:

- M/F ratio per cage: Pre-pairing up to 4 per cage. During pairing, 1:1.
- Length of cohabitation: Were paired on a one-to-one basis until mating occurred or for a period of up 2 weeks.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes (for up to 2 weeks).
- After successful mating each pregnant female was caged (how): Females singularly.
- Any other deviations from standard protocol: None

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulations for this study were prepared by Huntingdon Life Sciences Pharmacy personnel. The analytical work was undertaken by Product
Chemistry personnel between 08 March 2010 and 18 May 2010. Report date: 26 July 2010.
The homogeneity and stability was confirmed for Ebanol in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during
distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 2 days and refrigerated storage for up to
15 days. The storage times represented the maximum time from preparation to completion of administration. The mean concentrations of Ebanol in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Three groups of ten male and ten female rats received Ebanol by oral gavage administration at doses of 30, 300 or 1000 mg/kg/day.
Males and females were treated daily for 15 days before pairing, throughout pairing until Day 6 after the birth of the F1 generation for females
and Day 48/49 of treatment for males. A similarly constituted Control group received the vehicle, corn oil, throughout the same periods.

Frequency of treatment:

All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day.
The volume administered to each animal was calculated from the most recently recorded bodyweight.

Details on study schedule:

- Age at mating of the mated animals in the study: 12-13 weeks

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

Three groups of ten male and ten female rats received Ebanol by oral gavage administration at doses of 30, 300 or 1000 mg/kg/day.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A Repeat dose administration for seven days at a dose up to 1000 mg/kg/day (Huntingdon Life Sciences study number: FVR0002) data suggested systemic exposure and that the test substance was well tolerated.

- Rationale for animal assignment (if not random):
Non-selective allocation to cages to equalise variation in bodyweight.
The cages were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was
practicable.

Positive control:

None.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily
- Cage side observations: Cages were inspected daily for evidence of animal ill-health.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. In addition, a more detailedphysical examination was performed weekly for F0 adults and Days 0, 6, 13 and 20 after mating and Days 1, 4 and 7 of lactation for F0 females to monitor general health. Detailed observations were also recorded in relation to dose administration. Males were observed daily during the first week of treatment, twice weekly during Week 2 and once a week from Week 3 onwards. Female reactions were recorded daily during the first week of
treatment, twice weekly during Week 2 of treatment, on Days 0, 6, 13 and 20 after mating and Days 1, 4 and 7 of lactation. Observations were recorded at the following times in relation to dose administration:

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males were weighed weekly throughout the study. F0 females were weighed weekly until mating was detected, on Days 0, 6, 13 and 20 after mating and Days 1, 4 and 7 of lactation.

Oestrous cyclicity (parental animals):

For 15 days before pairing, daily vaginal smears were taken from all females, using cotton swabs moistened with saline.
The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was
continued, using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
Testis weight, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] 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):

SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

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 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

F1 Offspring: Gross inspection. Abnormal tissues retained.
Sporadic deaths in early neonates: Gross external examination. Preserved in industrial methylated spirit pending examination. Assessment of stomach for presence of milk, where possible.

Statistics:

Quasar (version 1.1) in-house statistical analysis package used for bodyweight, food consumption and organ weights.
StarTox (version 3.2) in-house statistical analysis package used for litter size, survival indices data.
SAS (version 9.1.3) statistical analysis package used for sex ratio data.
StatXact 3 statistical analysis package used for mating performance and fertility and gestation length and gestation index data.

Reproductive indices:

Mating performance and fertility - group values (F0)
Group : 1 2 3 4
Dose (mg/kg/day) : 0 30 300 1000

Group No.: 1M, No. Paired: 10, No. Mated: 10, No. Achieving Pregnancy: 10, Percentage Mating: 100, Conception Rate (%): 100, Fertility Index (%): 100
Group No.: 2M, No. Paired: 10, No. Mated: 10, No. Achieving Pregnancy: 8, Percentage Mating: 100, Conception Rate (%): 80, Fertility Index (%): 80
Group No.: 3M, No. Paired: 10, No. Mated: 10, No. Achieving Pregnancy: 8, Percentage Mating: 100, Conception Rate (%): 80, Fertility Index (%): 80
Group No.: 4M, No. Paired: 10, No. Mated: 10, No. Achieving Pregnancy: 9, Percentage Mating: 100, Conception Rate (%): 90, Fertility Index (%): 90

Group No.: 1F, No. Paired: 10, No. Mated: 10, No. Achieving Pregnancy: 10, Percentage Mating: 100, Conception Rate (%): 100, Fertility Index (%): 100
Group No.: 2F, No. Paired: 10, No. Mated: 10, No. Achieving Pregnancy: 8, Percentage Mating: 100, Conception Rate (%): 80, Fertility Index (%): 80
Group No.: 3F, No. Paired: 10, No. Mated: 10, No. Achieving Pregnancy: 8, Percentage Mating: 100, Conception Rate (%): 80, Fertility Index (%): 80
Group No.: 4F, No. Paired: 10, No. Mated: 10, No. Achieving Pregnancy: 9, Percentage Mating: 100, Conception Rate (%): 90, Fertility Index (%): 90

Offspring viability indices:

Offspring survival indices - group mean values (F1)
Group : 1 2 3 4
Dose (mg/kg/day) : 0, 30, 300, 1000

Group No.: 1
Post implantation survival index (%): Live birth index (%): Viability index (%):
Mean: 95.1, 99.1, 100
No.: 10, 10, 10

Group No.: 2
Post implantation survival index (%): Live birth index (%): Viability index (%):
Mean: 88.6, 87.5, 100
No.: 8, 8, 7

Group No.: 3
Post implantation survival index (%): Live birth index (%): Viability index (%):
Mean: 92.3, 95.3, 98.1
No.: 8, 8, 8

Group No.: 4
Post implantation survival index (%): Live birth index (%): Viability index (%):
Mean: 97.1, 98.3, 100
No.: 9, 9, 9

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight, males at 1000 mg/kg/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slight, males at 1000 mg/kg/day

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths on study, or clinical signs at any dose level that were considered related to treatment with Ebanol.
Chin rubbing and post dose salivation were observed sporadically throughout the treatment period (from Day 3 and 5, respectively) for the majority of animals receiving 1000 mg/kg/day and to a lesser extent for animals receiving 300 mg/kg/day. Chin rubbing and salivation was observed during gestation for females receiving 300 or 1000 mg/kg/day, and chin rubbing and salivation were observed on Day 1 and 4 of lactation for females receiving 300 or 1000 mg/kg/day. These signs are all likely to relate to the dosing process and taste of the formulation rather than to systemic toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The mean bodyweight gain from Day 1 to Day 43 of treatment was slightly lower for males receiving 1000 mg/kg/day when compared with Controls, with statistical significance being attained during Days 22-29 of treatment. Bodyweight and bodyweight change for males receiving 30 or 300 mg/kg/day were similar to, or greater than, Controls and considered to be unaffected by treatment. Overall bodyweight gain for females receiving 1000 mg/kg/day was statistically significantly higher than Control before pairing (Days 1-15 of treatment) but the overall impression was that female weight gain before pairing, during gestation and lactation was not adversely affected by treatment.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
5 mL/kg/day - Oral gavage

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
All females had regular 4 or 5 day oestrous cycles that were considered to be unaffected by treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were considered unaffected by treatment. Mating performance was unaffected by treatment with all paired animals showing positive evidence of mating.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The mean adjusted and unadjusted organ weights for the F0 animals that received Ebanol showed no adverse effects after seven weeks of treatment for males and at least six weeks of treatment for females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no macroscopic abnormalities seen in males after seven weeks of treatment, or in females on Day 7 of lactation, that could be related to treatment with Ebanol.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The microscopic examination did not highlight any treatment related changes affecting the reproductive system in either male or female rats.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity for the various cell types present within different stages. No cell or stage abnormalities were noted. Most of the females killed on Day 7 of lactation showed microscopic changes in the vaginal
epithelium described as epithelial mucification/atrophy. The incidence of this finding was similar among the Controls (9/10) and in females receiving 1000 mg/kg/day (7/9). The described morphology was considered to be a physiological anestrus and is consistent with the stage of lactation.
In the animals sacrificed because of impaired reproductive performance there were no histopathological findings which were considered likely to cause reproductive failure in any animal.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects Body weight: Slight (non-significant) reduction in males at 1000 mg/kg/day

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly low for male and female offspring at 1000 mg/kg/day

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Litter size and offspring survival up to Day 7 of age was considered not to be affected by treatment with Ebanol.
There was no effect of treatment on pregnancy outcome. All pregnant females gave birth to a live litter.
One female receiving 30 mg/kg/day had total litter loss during the post-natal period, this female gave birth to one female pup but was subsequently found dead later that day. The dam was sent to necropsy as a total litter loss.
The following assessment of litter responses was made based on the 10, 7, 8 and 9 litters successfully reared to termination on Day 7 post partum in the 0 (Control), 30, 300 and 1000 mg/kg/day groups, respectively. Litter data, as assessed by mean numbers of implantations, total litter size, and live litter size on Day 1, 4 and Day 7 of age, were generally similar to the Control and are considered not to be affected by treatment.
Offspring survival during the pre- and post-natal period as assessed by the post implantation survival index, live birth index, viability index was unaffected by treatment.

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment with Ebanol.

BODY WEIGHT (OFFSPRING)
The mean bodyweight of male and female offspring were similar to respective Controls on Day 1 of age but bodyweight gain to Day 7 and absolute weight at Day 7 of age appeared slightly low for male and female offspring at 1000 mg/kg/day. F1 offspring mean bodyweight gain at 30 or 300 mg/kg/day was unaffected by treatment.

ORGAN WEIGHTS (OFFSPRING)
Not performed.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring that died prior to scheduled termination and those killed on Day 7 of age did not reveal any observations that could be related to parental treatment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

Conclusions:

It was concluded that 1000 mg/kg/day was the no observed adverse effect level (NOAEL) for parental growth, reproductive performance and offspring growth in this screening study.

Executive summary:

NOAEL = 1000 mg/kg/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

It was concluded that 1000 mg/kg/day was the no observed adverse effect level (NOAEL) for parental growth, reproductive performance and offspring growth in this screening study.

On the basis that the test substance (Ebanol) is being used to support MTCP Crude on the basis of read-across, the NOAEL for parental growth, reproductive performance and offspring growth for MTCP Crude was also considered to be 1000 mg/kg/day.

Short description of key information:
An OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) GLP study was conducted, a synopsis is given below.

Effects on developmental toxicity

Description of key information

No evidence was found for developmental effects in the OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) study.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Additional information

1000 mg.kg/day had no adverse effects on the progress or outcome of the pregnancies.

On the basis that the test substance (Ebanol) is being used to support 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)pent-3-en-2-one on the basis of read-across, the NOAEL for parental growth, reproductive performance and offspring grwoth for 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)pent-3-en-2-one was also considered to be 1000 mg/kg/day.

Justification for classification or non-classification

According to Directive 67/548/EEC and Regulation (EC) 1272/2008, no classification is warranted.

On the basis that the test substance (Ebanol) is being used to support MTCP Crude on the basis of read-across, the reproductive and developmental toxicity of MTCP Crude is also considered to be unclassified.