A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 2011 - 14 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: The study is also summarized in Sections 7.5 and 7.8.2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]

EC Number:

422-040-1

EC Name:

A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]

Cas Number:

426218-78-2

Molecular formula:

C16H26O

IUPAC Name:

1-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-2-oxabicyclo[2.2.2]octane; 3,6',6',6'a-tetramethyl-2',3'a,4',5',6',6'a-hexahydrospiro[cyclohexane-1,3'-cyclopenta[b]fura]-3-ene; 4,6',6',6'a-tetramethyl-2',3'a,4',5',6',6'a-hexahydrospiro[cyclohexane-1,3'-cyclopenta[b]fura]-3-ene; 5-methyl-1-(2,2,3-trimethylcyclopent-3-en-1-yl)-6-oxabicyclo[3.2.1]octane

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:WI(Han).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 288 gr (males) or 203 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

On Day 16 of study, the lights were turned off for 20 minutes in the afternoon due to power outage.
Evaluation: Laboratory historical data do not indicate an effect of this deviation.

IN-LIFE DATES: From: 15 December 2011 to 14 March 2012

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

- Method of formulation: The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.
- Storage conditions of diets: In the freezer until day of use. Twice weekly the pellets were defrosted and offered to the animals for a maximum of four days.
- Stability of pelleted diet: At least 14 days stable in the freezer and 4 days at room temperature (concentration range of 1.000 to 10.000 ppm; determined during NOTOX Project 498280).

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
- Detection of mating was not confirmed for five Group 1 females which did deliver live offspring. The mating dates of these animals were based on re-evaluation of the vaginal smears.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (07 February 2012), according to a validated method (NOTOX Project 498280). Dietary samples were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

In addition, random samples were taken and stored at ≤-15°C for possible future analysis. Any remaining samples will be discarded after approval by the sponsor, or at finalization of the study report.

The accuracy of diet preparations was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the diets of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).
In the chromatograms of the Group1 diet, a small response was observed at the retention time of the test substance but this did not derive from the test substance.
The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

Males were exposed for 30 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 5 days of lactation.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Ad libitum.

Details on study schedule:

- Age at mating : Approximately 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

actual ingested

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Remarks:

ca. 70 mg/kg bw, actual ingested

Doses / concentrations 3

Dose / conc.:

2 500 ppm

Remarks:

ca. 170 mg/kg bw, actual ingested

Doses / concentrations 4

Dose / conc.:

8 000 ppm

Remarks:

ca. 550 mg/kg bw, actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-Day dose range finding study.
- Based on the results of this dose range finding study, dose levels for the main study were selected to be 1.000, 2.500 and 8.000 ppm (being 550 mg/kg bw based on analysed concentrations)
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT:
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Twice weekly, except for males and females which were housed together for mating and for females without evidence of mating.
Food consumption of mated females was measured on Days 0, 3, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Five female animals of Group 4 were provided with insufficient diet in the night of Day 7 to 8 of study. This was caused by an unexpected compensatory increase in food consumption for these animals.
Evaluation: Overall (Days 4-8 of study) a normal mean food consumption was noted and all animals showed a body weight increase.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

From the males of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis.

Litter observations:

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

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
- The animals were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- The number of former implantation sites and corpora lutea were recorded for all paired females.
- According to test guidelines

ORGAN WEIGHTS
- Adrenal glands, Epididymides, Kidneys, Liver and Testes.

HISTOPATHOLOGY
According to test guidelines

Organ weights were determined for the Group 1 Female with total litter loss (should have been done for planned necropsies only).
Evaluation: Additional information.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection and pale appearance for femalal at 550 mg/kg bw

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Transient effect, not regarded toxicologically relevant

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Transient effect, not regarded toxicologically relevant

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

MORTALITY
No mortality occurred during the study period.
On Group 1 female was terminated on Day 4 of lactation due to total litter loss.

CLINICAL SIGNS
Treatment related clinical signs were noted for females at 550 mg/kg bw.
Hunched posture was noted for six animals for 2 to 5 days during the first week of treatment. Piloerection was noted for three females during the first week of treatment (1-3 days) and for all females from the fourth week of treatment onwards. In addition, one female showed a pale appearance for two days at the end of pregnancy.

Piloerection was also noted for one female treated at 1.000 ppm (circa 125 mg/kg bw) for one day. At this low incidence and as it was not noted at 2.500 ppm (circa 300 mg/kg bw, it was not considered toxicologically significant.

BODY WEIGHTS
At 550 mg/kg bw, body weight loss was noted for the males (slight) and females (marked to severe) during the first four days of treatment. Normal body weight gains were noted during the remainder of the treatment period, however body weights remained slightly reduced (especially for the females).

At circa 125 and 300 mg/kg bw body weights and body weight gain remained in the same range as controls over the treatment period.

FOOD CONSUMPTION
At 550 mg/kg bw, food consumption was decreased during the first four days of treatment: moderately for the males and severely for the females. Normal food consumption values were noted during the remainder of the study.

Slightly decreased food consumption (statistical significant) was noted for females at 550 mg/kg bw on Days 7-11 post-coitum. As these values were still within normal limits, this change was not considered toxicologically significant.

Food consumption before or after allowance for body weight was similar between animals treated at circa 125 and 300 mg/kg bw and control animals.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings consisted of pelvic dilation of the kidneys, nodule at the epididymides, alopecia, enlarged liver and foci at the thymus. These findings were within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend, and were therefore not considered to be of any toxicological relevance.

ORGAN WEIGHTS
A treatment related effect was noted for the weights of the liver and adrenals.

Dose related increased liver weights were noted at circa 300 and 550 mg/kg bw for the males, and at all dose levels for the females. Decreased adrenals weights were noted for both sexes at 550 mg/kg bw.

MICROSCOPIC EXAMINATION
No toxicologically significant microscopic findings were noted up to 550 mg/kg bw ppm.

There were treatment related findings in the female liver. Hepatocellular hypertrophy was noted at minimal degree in 1/10 0 ppm (Group 1), in 3/10 circa 125 mg/kg bw (Group 2), in 3/10 circa 300 mg/kg bw (Group 3) and in 6/10 550 mg/kg bw (Group 4) treated females. Minimal hepatocellular hypertrophy was considered to be an adaptive non adverse finding.

The remainder of the microscopic findings recorded, including the requested adrenals, were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

No abnormalities were seen in the reproductive organs of the rats who failed to sire or deliver healthy pups, which could account for their infertility.

Further, the spermatogenic staging profiles were normal for all males evaluated.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted.

Of the control group, one female did not mate and one female mated within 13 days. All other females mated within 4 days of pairing.

Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

GESTATION
Gestation index and duration of gestation were within normal limits for all groups.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(parental)

Effect level:

> 550 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Non-adverse effects at the highest dose for the females were observed. In the absence of functional or morphological disturbances supporting the slight changes a NOAEL of at least 550 mg/kg bw was established.

2

Key result

Dose descriptor:

NOAEL

Remarks:

(for reproduction)

Effect level:

> 550 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No reproductive toxicity was observed up to 550 mg/kg bw

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Details on results (F1)

EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

The statistical significant increased value of number of living pups at first litter check at 1.000 ppm was considered due to the slightly low control value, and was not considered toxicologically relevant.

MORTALITY PUPS
Two pups of the control group, two pups at 1.000 ppm and one pup at 2.500 ppm were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Total litter loss was noted for one Group 1 litter consisting of only one pup. This pup was missing on Day 4 of lactation. As this was a control animal, it was not treatment related.

CLINICAL SIGNS PUPS
Scabs on the back was noted for one pup. As this was a control animal, it was not treatment related.

BODY WEIGHT PUPS
Body weights of pups were considered to have been unaffected by treatment.

The observed statistical significant changes were caused by the relatively high control values (mainly due to two litters with only 2-5 pups) and were therefore not considered toxicologically relevant.

MACROSCOPY PUPS
Absence of milk in the stomach was noted for three pups that were found dead. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 550 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: See remarks

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

See details above

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Tables and further information can be found in the attached study report.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD 421, GLP), the parental NOAEL was determined to be >=550 mg/kg bw based on the absence of toxic adverse effects in the parental animals. The fertility NOAEL is >= 550 mg/kg bw based on the absence of reproductive toxicity.

Executive summary:

In a Reproscreen study (OECD TG 421, GLP), the test substance was administered via the diet to Sprague-Dawley rats at dosages of 70, 170 or 550 mg/kg bw. Control animals received a plain diet. All parameters from the OECD TG 421 have been recorded. The following results were found:

Clinical signs: No effects were seen on body weight (gain) and food consumption. Some piloerection was seen in the females. (Relative) liver weights were increased in males and females in the high dose and minimal liver hepatocellular hypertrophy was noted at minimal degree and was considered to be an adaptive non adverse finding. Adrenal weight were decreased but no macroscopic or microscopic findings related to treatment were noted.

Fertility: Mating index, fertility index, conception index, gestation index and duration of gestation index were not affected by the substance.

Based on the absence of toxicologically relevant effects, the parental NOEAL and the NOAEL for reproduction were determined to be >= 550 mg/kg bw.