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]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

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:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

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)

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).

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%).

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).
- Age at mating of the mated animals in the study: Approximately 13 weeks
- 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 the appearance of an intravaginal copulatory plug and/or by determination of the estrous stage. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- Detection of mating was not confirmed for two Group 1 females which did deliver live offspring. The mating dates of these animals were based on re-evaluation of the vaginal smears.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

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

Duration of test:

Males: 30 days
Females: 41-53 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm

Remarks:

ca. 70 mg/kg bw, actual dose ingested

Doses / concentrations 2

Dose / conc.:

2 500 ppm

Remarks:

ca. 170 mg/kg bw, actual dose ingested

Doses / concentrations 3

Dose / conc.:

8 000 ppm

Remarks:

ca. 550 mg/kg bw, actual dose 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 circa 70, 170 and 550 mg/kg bw.

Examinations

Maternal 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.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

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.

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.

Indices:

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 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: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs were noted for females at 8.000 ppm.
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 for one day. At this low incidence and as it was not noted at 2.500 ppm, it was not considered toxicologically significant.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period, except for one control female who was terminated on Day 4 of lactation due to total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 8.000 ppm, 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 1.000 and 2.500 ppm, body weights and body weight gain remained in the same range as controls over the treatment period.

Description (incidence and severity):

At 8.000 ppm, 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 1.000 and 2.500 ppm, body weights and body weight gain remained in the same range as controls over the treatment period.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Description (incidence and severity):

No toxicologically significant microscopic findings were noted up to 8.000 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 1.000 ppm (Group 2), in 3/10 2.500 ppm (Group 3) and in 6/10 8.000 ppm (Group 4) treated females. Minimal hepatocellular hypertrophy was considered to most likely 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 deliver healthy pups, which could account for their infertility.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: See remarks

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Treatment with Cassiffix by dietary administration in male and female Wistar Han rats at dose levels of 70, 170 and 550 mg/kg bw revealed slight parental toxicity at 550 mg/kg bw for dams. In the absence of functional or morphological disturbances supporting the slight changes noted for the dams at 550 mg/kg bw, a maternal NOAEL of at least 550 mg/kg bw was established.

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: See remarks

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No developmental toxicity was observed up to the highest concentration tested: 550 mg/kg bw

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

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.
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 control group 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.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

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.

Other effects:

no effects observed

Description (incidence and severity):

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

Details on embryotoxic / teratogenic effects:

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.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 550 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See remarks

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

See details above

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Tables and further information can be found in the attached study report under IUCLID Chapter 7.8.1.

 

REPRODUCTIVE DATA

No toxicologically relevant effects onreproductive 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.

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 developmental NOAEL is >= 550 mg/kg bw based on the absence of developmental toxicity in the first generation animals.

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.

Developmental: Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment. 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. 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.

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