2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Test item: 2-Ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate
Test item identity (including alternative names): MCS-1562
Appearance: Liquid
Storage conditions: In a refrigerator (2 to 8C)
Supplier: Sponsor
Batch number: AH03201
Expiry date: February 2019
Purity: 100%

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

SD rats are proven test subjects for this sort of testing

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd.
Number of animals ordered 44 males and 48 females.

Duration of acclimatisation At least 5 days before commencement of estrous cycle evaluation for females or start of treatment for males.
Age of the animals at the start of the study Males 10 to 11 weeks old. Females 12 to 13 weeks old.
Weight range of the animals at the start of the study Males 338 to 400 g. Females 229 to 290 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages. On Day 1 of study all animals were weighed and body weights and estrous cycles were reviewed by Study Management before dosing commenced. Body weight of animals did not exceed 20% of the mean for each sex.
Identification of animals Each adult animal was assigned a number and identified uniquely within the study by microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Environmental Control

Rodent facility Limited access - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%. Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation

Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage Pre-pairing: up to five animals of one sex, Pairing: one male and one female , Males after mating: up to five animals, Gestation: one female, Lactation: one female + litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation), replaced when necessary and returned following removal of offspring.
Plastic shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Administration
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.

Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth. If female was in parturition, dosing was not performed on one day. Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage
as a check of correct administration.

Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Details on mating procedure:

Pairing commenced After a minimum of two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males were treated for 2 weeks pre-mating, up to 2 weeks of mating and through week 5 of gestation. Females were treated for 2 weeks pre-mating, for up to 2 weeks of mating and then through gestation and to necropsy at lactation day 14.

Frequency of treatment:

daily

Details on study schedule:

Males were treated for 2 weeks pre-mating, up to 2 weeks of mating and through week 5 of gestation. Females were treated for 2 weeks pre-mating, for up to 2 weeks of mating and then through gestation and to necropsy at lactation day 14.

Dose / conc.:

350 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

no

Parental animals: Observations and examinations:

Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs
Wet smears Using pipette lavage during the following phases:
For 14 days before treatment; animals that failed to exhibit 4-5 cycles were not allocated to the study.
After pairing until mating.
For four days before scheduled termination (Days 11 to 14 of lactation).

Sperm parameters (parental animals):

none

Litter observations:

Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Recorded on Day 1 for all offspring.
Nipple/areolae count On Day 13 of age for male offspring.

Postmortem examinations (parental animals):

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

F0 males A fter Week 5 investigations completed.
F0 females failing to produce a viable litter Day 25 after mating.
F0 females Day 14 of lactation.
F1 offspring Selected offspring for thyroid hormone analysis – Day 4 of age
Scheduled kill - Day 13 of age.

Where the F0 female was found dead or sacrificed during lactation, the offspring were killed on the same day.

Postmortem examinations (offspring):

Premature deaths Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination (excluding offspring for thyroid retention) Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.

Statistics:

Statistics are not presented as the system will not allow enough characters to be entered. There is not reason why the space for statistics is limited, and this should be changed.

Reproductive indices:

Values such as mating performance, litter size, weight, sex ratio and survival were collected.

Offspring viability indices:

Viability was documented.

Clinical signs:

no effects observed

Description (incidence and severity):

The distribution of minor signs noted at the detailed physical examination and arena observations showed no test item related effects.
There were no clinical signs associated with dose administration

Mortality:

mortality observed, treatment-related

Description (incidence):

There were six decedent females; 4 females given 100 mg/kg/day and 2 females given 350 mg/kg/day, all occurring between Days 9 and 13 of lactation.

Female 135, given 350 mg/kg/day, was found dead on Day 13 of lactation with no adverse signs noted previously apart from a firm area on upper ventral surface from Day 20 of gestation, and had macroscopic findings of bedding material present in the oral cavity and a distended stomach. On microscopic examination, most tissues showed changes associated with autolysis. There was slight, multifocal, sub-capsular necrosis seen in the liver and a slight decrease in white pulp cellularity of the spleen.

Female 148, given 350 mg/kg/day was sacrificed due to poor condition including decreased activity, piloerection and cold to touch on Day 9 of lactation and had findings of pale color and pale material in the middle section of the jejunum. Microscopic findings consisted of minimal atrophy of the thymus.

Female 105, given 100 mg/kg/day, was sacrificed due to poor condition including prostate posture and unresponsiveness on Day 9 of lactation. There were no macroscopic findings. Microscopic findings included slight, generalised, decreased cellularity within the mesenteric and axillary lymph nodes, a diffuse cortical hypertrophy within the adrenal glands, and a slight bilateral dilatation of the lateral ventricles of the brain. There was also a minimal, decreased red pulp cellularity within the spleen.

Female 106, given 100 mg/kg/day, was sacrificed due to poor condition including circling behaviour, uncoordinated gait and piloerection on Day 9 of lactation. Macroscopic findings comprised depression of the glandular portion of the stomach which correlated histologically with slight, multifocal erosion in the same area.

Female 107, given 100 mg/kg/day was sacrificed due to poor condition including unresponsiveness, hunched posture, prominent eyes and irregular breathing on Day 11 of lactation. There were no macroscopic findings. Microscopically, there was a slight decrease in cellularity within the mesenteric lymph node and of the red pulp within the spleen. There was also minimal, cortico-medullary mineralisation seen in the kidneys of this female.

Female 141, given 100 mg/kg/day, was found dead on Day 10 of lactation. No prior adverse clinical signs were noted. The only macroscopic finding of pelvic dilatation within both kidneys which was confirmed microscopically. Other microscopic changes included a slight decrease in cellularity within the bone marrow, axillary lymph node and red pulp of the spleen.

No macroscopic or microscopic changes were observed which were considered to have contributed to the poor clinical condition or death of these animals. Factors contributing to poor clinical condition and the cause of death in could not be determined. For all of these decedents, no previous clinical signs had been seen, there were no adverse effects during parturition and no effects on their litters from birth.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weight gain over Weeks 0-5 for males given 350 mg/kg/day was statistically significantly lower than the concurrent control group (0.76x Control).
There were no test item related effects on body weight over Weeks 0-5 in the males given 30 or 100 mg/kg/day.
There were no test item related effects on group mean body weight gain of females over the first 2 weeks of treatment, or during gestation or lactation. Absolute group mean body weight on Day 1 of lactation in females given 100 mg/kg/day and on Days 1, 4 and 13 of lactation in females given 350 mg/kg/day was statistically significantly lower than the concurrent control but was similar to the controls on Gestation Day 20. However, as body weight gain during lactation was similar to the controls this is considered to be of no toxicological significance.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The incidence and distribution of microscopic findings were considered to be unrelated to treatment.
Detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. No stage specific testicular abnormalities were found.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females assigned to the study had regular estrous cycles prior to the start of treatment.
There were no treatment related findings on estrous cycles in the first 2 weeks of treatment prior to pairing.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. No stage specific testicular abnormalities were found.

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects - systemic

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects - reproduction

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Critical effects observed:

no

Reproductive effects observed:

no

Executive summary:

The purpose of this study was to assess the general systemic toxicity potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate by oral gavage administration for at least 5 weeks.

Four groups of ten male and ten female rats received 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate at doses of 30, 100 or 350 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, nipple counts (male only) and macropathology for all offspring were also assessed. 

Results

F0 responses

Four females given 100 mg/kg/day and two females given 350 mg/kg/day died or were sacrificed due to poor clinical condition including decreased activity, unresponsiveness, prostate posture, uncoordinated gait, circling behaviour or hunched posture between lactation Days 9 and 13. There were no pathological findings that elucidated the cause of demise.

There were no clinical signs seen during the weekly detailed physical examinations that were considered to be test item related and no clinical signs were recorded in association with dose administration. Sensory reactivity, grip strength and motor activity were unaffected by treatment.

Body weight gain of males given 350 mg/kg/day was statistically significantly lower than controls (0.76x) and food consumption of females given 100 or 350 mg/kg/day was lower (up to 0.88x) than controls, occasionally attaining statistical significance.

Estrous cyclicity, pre-coital interval, gestation length, mating performance and fertility were unaffected by treatment.

 

 

Haematology and blood chemistry investigations revealed no effect of treatment and there was no effect upon circulating levels of thyroxine (T4) in adult males.

 

After five weeks of treatment for males and on Day 14 of lactation for females, liver weights were higher in males and females given 100 and 350 mg/kg/day and kidney weights were higher in males and females given 350 mg/kg/day. There were no test item related macropathological or micropathological findings in the full list of tissues examined.

 

F1 responses

Body weight gain of male and female offspring from the 100 and 350 mg/kg/day groups were statistically significantly lower (0.75X to 0.82x) than the Controls.

The clinical condition, litter size, sex ratio and survival indices of offspring were unaffected by parental treatment.

 

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

 

The ano-genital distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

 

No macroscopic findings in the offspring were considered to be related to paternal treatment.

Conclusion

It was concluded that the oral administration of 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate to parental Sprague Dawley male rats at dose levels of 30, 100 or 350 mg/kg/day for two weeks before pairing, during pairing and then up to termination after five weeks of treatment was associated with statistically significantly lower bodyweight gain (0.76x). Oral administration of the same dose levels to female rats for two weeks before pairing, during pairing and during gestation and lactation up to termination on Day 14 of lactation was associated with 4 decedent females during lactation at 100 mg/kg/day and 2 decedent females during lactation at 350 mg/kg/day. There were no pathological changes noted and the cause of the demise was not established. Food consumption was slightly and occasionally statistically significantly reduced in females given 100 (up to 0.88x, gestation and lactation only) or 350 mg/kg/day (up to 0.89x) and considered non adverse.

Growth of offspring from parental doses of 100 or 350 mg/kg/day was slightly and statistically significantly lower (up to0.75x), but considered non adverse. There were no other effects on reproductive performance, fertility, litter size or offspring survival.

In the context of this study,2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylateshowed no evidence of being an endocrine disruptor, as assessed by measurement of thyroxine (T4) in adult males and offspring, organ weights, ano-genital distance and external examination of offspring, and nipple counts in male offspring,

The no-observed-adverse-effect level (NOAEL) of2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylatefor systemic toxicity of males was considered to be 350 mg/kg/day.

The no-observed-adverse-effect level (NOAEL) of2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylatefor systemic toxicity of females was considered to be 30 mg/kg/day, based on mortality

The no-observed-adverse-effect level (NOAEL) of2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylatefor reproductive/developmental toxicity was considered to be 350 mg/kg/day.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Justification for classification or non-classification

No adverse effects were found in an OECD 422 combined repeat dose/reproductive developmental screen, so no classification was required.