3-Cyclopentene-1-acetaldehyde, 2,2,3-trimethyl-, reaction products with Me Et ketone, hydrogenated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March – 22 September 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted in compliance with OECD guideline 421 with minor deviations: temperature was dropped to 60.2 °F in an animal room; minor recording or procedural errors (end mix time of formulation and viability of rats before assigned to study were not recorded, body weight of one dam on a day was not recorded, no. of pups in a dam was incorrectly recorded on 2 days; unscheduled maternal observations were recorded for 2 dams on a day)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

temperature dropped to 60.2 °F in a room; minor recording or procedural errors (body weight of one dam on a day was not recorded, no. of pups in a dam was incorrectly recorded on 2 days; unscheduled maternal observations were recorded for 2 dams on a day)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentan-2-ol; 6-(2,2,3-trimethylcyclopent-3-en-1-yl)hexan-3-ol

EC Number:

939-525-3

Cas Number:

1471313-03-7

Molecular formula:

C14H26O

IUPAC Name:

3-methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentan-2-ol; 6-(2,2,3-trimethylcyclopent-3-en-1-yl)hexan-3-ol

Details on test material:

- Name of test material (as cited in study report): Sandalore
- CAS number: 65113-99-7
- Source: Givaudan Fragrances Corporation, East Hanover, USA
- Physical state: Pale yellow liquid
- Analytical purity: sum of C14H26O isomers 92.5 %
- Lot/batch No.: VE00061266
- Date received: 18 January 2010
- Expiration date of the lot/batch: 02 January 2012
- Storage condition of test material: Controlled room temperature, protected from light

Test animals

Species:

rat

Strain:

other: Crl:WI(Wistar Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, USA
- Approximate age at arrival: Male: 91 days; female: 88 days
- Weight at study assignment: Males: 329-362 g; females: 198-221 g
- Housing: Adult P (parental F0) generation rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of rats was housed in the male rat's cage. Beginning no later than DG 20 (day of gestation), female rats were individually housed in nesting boxes. Each dam and delivered litter was housed in a common nesting box during the postpartum period.
- Diet: Certified Rodent Diet® #5002 (PMI® Nutrition International, St. Louis, USA), ad libitum; chewable Nylabones® (Nylabone® Products, Neptune, NJ), enrichment devices for the maintenance of rodent oral health, were supplied to all rats during the course of the study.
- Water: Local water processed by passage through a reverse osmosis membrane (R.O. water), ad libitum
- Acclimation period: Approximately 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C (66-77 °F)
- Humidity: 30-70 %
- Air changes: 10/h
- Photoperiod: 12 h dark / 12 h light (fluorescent light)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Suspensions of the test substance were prepared once weekly at the testing facility and were stored refrigerated (2-8 °C).
- Vehicle was dispensed weekly as aliquots for daily use and stored at room temperature.
- Prepared formulations were stirred for at least 15 minutes prior to dosage administration and continuously during dosage administration.

VEHICLE
- Vehicle: Corn oil
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day
- Lot/batch no.: J-145
- Storage conditions: Room temperature

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum 13 days
- Proof of pregnancy: Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0.
- After successful mating each pregnant female was individually caged.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentrations (25, 75 and 250 mg/mL) were analysed on first and last day of preparation using GC/MS method.
- All analyses were performed after validation (Study No. 0020000519) of the analytical method.
- All dosage formulation samples met acceptance criteria for both concentration (the difference between the actual mean value and the targeted concentration was ≤ 15 % and homogeneity (the relative standard deviation [RSD] for the formulation, calculated as the RSD for the grand mean of the average values for top, middle and bottom locations, was ≤ 5 %.
- Stability of the dosage formulations for the low and high concentrations (25 and 250 mg/mL, respectively) was established under room temperature conditions after 48 h and after 6 days and under refrigerated conditions after 8 days. All results were within ± 10 % of the initial stability results.

Duration of treatment / exposure:

- Males: 14 days before cohabitation, through the cohabitation period (maximum 13 days) and continuing through the day before sacrifice (14 days after the completion of cohabitation).
- Females: 14 days before cohabitation, through the cohabitation period (maximum 13 days) and continuing through Day 4 of lactation. For female rats that did not deliver a litter, the last day of dosage administration was DG 24.

Frequency of treatment:

Once daily, 7 days/week

Details on study schedule:

None

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose-levels were selected based on the results of the previous studies conducted with the test substance (data not shown). The highest dosage level was expected to produce some toxicity (clinical signs or a decrease in body weight), but not death or severe suffering. The intermediate dosage level was expected to produce minimal observable toxicity, while the lowest dosage level was expected to produce no evidence of either systemic or reproductive toxicity. A descending sequence of dosages was selected with the purpose of demonstrating any dosage-related response and to establish a no-observed-adverse-effect level (NOAEL).
- Rationale for animal assignment (if not random): Animals were assigned to dosage groups using a computer-generated (weight-ordered) randomization procedure. At study initiation, the weight variation of the rats did not exceed ± 20 % of the mean weight of each sex.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations: Rats were observed for viability at least twice each day. Rats were also examined for clinical observations, abortions, premature deliveries and deaths before and after dosage administration. On the first two days of dosage administration (DS 1 and 2), postdosage observations were recorded at approximately hourly intervals for the first four hours and at the end of the normal working day. Starting on the third day of dosage administration (DS 3), postdosage clinical observations were recorded within two hours of dosage administration and at the end of the normal working day, and once on the day of sacrifice.

BODY WEIGHT: Yes
Time schedule for examinations:
- P Generation rats: Body weights were recorded weekly during the acclimation period, daily during the dosage period and on the day sacrifice occurred.

FOOD CONSUMPTION:
- Feed consumption values for male rats were recorded weekly during the dosage period, except during cohabitation.
- Feed consumption values for female rats were recorded weekly to cohabitation, on DGs 0, 7, 10, 14, 18, 21, 25 (when necessary) and on DLs 1 and 5.

PARTURITION:
- Females were allowed to litter normally and rear their progeny until Day 5 post-partum inclusive. Any sign of a difficult or prolonged parturition was recorded. The day of completed parturition was designated as Day 1 post-partum.

Oestrous cyclicity (parental animals):

Estrous cycling was evaluated daily by examination of vaginal cytology beginning 14 days before cohabitation and continuing until the female was positive for mating.

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: testes weight, epididymis weight and spermatogenesis in the testes

Litter observations:

PARAMETERS EXAMINED:
- Values for the duration of gestation (DG 0 to the day the first pup is observed), implantation sites per delivered litter, dams with stillborn pups, gestation index (number of rats with live offspring/number of pregnant rats), dams with all pups dying, pups delivered, liveborn and stillborn pups, pups found dead or presumed cannibalized, viability index, surviving pups per litter, percent male pups per number of pups sexed, live litter sizes at weighing and pup weights per litter were evaluated.
- Clinical signs and viability: Each litter was evaluated for viability at least twice daily. Clinical observations were recorded once daily during the postpartum period and the pups in each litter were counted once daily.
- Body weight: Weight of each pup was recorded on Days 1 and 5 post-partum (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam).

Postmortem examinations (parental animals):

SACRIFICE: All surviving animals were sacrificed by carbon dioxide asphyxiation.
- Male animals: Male rats were sacrificed after completion of the cohabitation period.
- Maternal animals: All surviving female rats were sacrificed after completion of the 5-day postpartum period. Rats that did not deliver a litter were sacrificed on DG 25.

UNSCHEDULED SACRIFICE
- A female rat in the 100 mg/kg bw/day dosage group was sacrificed on DL 2, before scheduled termination, due to no surviving pups. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed; the number and distribution of implantation sites were recorded.
- A female rat in the 1000 mg/kg bw/day dosage group was sacrificed on DG 23, before scheduled termination, due to adverse clinical condition resulting from difficulties in parturition. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed; the number and distribution of implantation sites were recorded. The lungs, trachea and esophagus were perfused and saved in neutral buffered 10 % formalin for possible future evaluation. Pregnancy status and uterine contents were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Gross lesions were retained in neutral buffered 10 % formalin for possible histological evaluation.

ORGAN WEIGHTS
- Epididymides, ovaries, prostate, seminal vesicles with coagulating gland, testes, uterus with cervix were weighed.

HISTOPATHOLOGY
- Epididymides, gross lesions, ovaries, prostate, seminal vesicles with coagulating gland, testes, uterus with cervix were evaluated microscopically.
- Tissues identified for microscopic evaluation from all treated P generation male and female rats were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Any remaining tissues were preserved in 10 % neutral buffered formalin for possible future evaluation.
- All gross lesions from rats in all dosage groups were evaluated microscopically.
- Special emphasis was placed on stages of spermatogenesis and histopathology of interstitial testicular cell structure.
- Histopathological examination was performed for all control and high dosage group rats.

Postmortem examinations (offspring):

SCHEDULED SACRIFICE: Surviving pups were sacrificed on Day 5 postpartum by an intraperitoneal injection of sodium pentobarbital.

GROSS NECROPSY
- Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly.
- Gross lesions were preserved in neutral buffered 10 % formalin for possible future evaluation.
- Pups that died before examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were identified as stillborn.
- Pups with gross lesions were preserved in Bouin's solution for possible future evaluation.
- Pups that died were examined for gross lesions and the cause of death or condition on the day the observation was made.
- Pups found dead on Days 2 to 5 postpartum were preserved in Bouin's solution for possible future evaluation.

Statistics:

- All data were tabulated, summarized and/or statistically analyzed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft® Excel (part of Microsoft® Office 97/2000/2003/XP/2007), Quattro Pro 8 and/or The SAS System (version 6.12).
- Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
- Continuous data (e.g., body weight changes and feed consumption values) were analyzed using Bartlett’s test of homogeneity of variances and the analysis of variance, when appropriate [i.e., Bartlett’s test was not significant (p> 0.001)]. If the analysis of variance was significant (p≤ 0.05), Dunnett’s test was used to identify the statistical significance of the individual groups.
- If the analysis of variance was not appropriate [i.e., Bartlett’s test was significant (p≤ 0.001)], the Kruskal-Wallis test was used, when less than or equal to 75 % ties were present. In cases where the Kruskal-Wallis test was statistically significant (p≤ 0.05), Dunn’s method of multiple comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75 % ties, Fisher’s Exact test was used to analyze the data. Count data were evaluated using the procedures described above for the Kruskal-Wallis test.

Reproductive indices:

Fertility Parameters:
- Fertility index (percentage of matings that resulted in pregnancies).
- Gestation index (percentage of pregnancies that resulted in birth of live litters).
- Number and sex of offspring per litter (live and dead pups).
- Number of implantation sites.
- General condition of dam and litter during the postpartum period.

Offspring viability indices:

Litter Data:
- Litter size and viability [at birth (DL 1) and on DL 5].
- Viability indices (percentages of pups born that survive to DLs 1 and 5).
- Percent survival and sex ratio [at birth (DL 1) and on DL 5].

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Test substance intake: not applicable

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- All male rats survived until scheduled sacrifice. One female rat in the 1000 mg/kg bw/day dosage group was sacrificed before scheduled termination due to adverse clinical condition resulting from difficulties during parturition (dystocia). All other female rats survived until scheduled sacrifice.
- Excess salivation occurred in both male and female rats at dosage levels of 300 and 1000 mg/kg bw/day.
- Urine-stained abdominal fur occurred in both male and female rats in the 1000 mg/kg bw/day dosage group.
- Piloerection, ptosis and red perivaginal substance occurred in female rats in the 1000 mg/kg bw/day dosage group during the gestation period.
All those clinical signs were not considered as adverse or related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Males:
- Reductions in mean body weight gains and feed consumption values occurred in the male rats in the 300 and 1000 mg/kg bw/day dosage groups.
- Mean body weight gains in the male rats during the entire dosage period (calculated as Days 1 through 42 of study [DSs 1 through 42]) were 93, 78 and 77 % of the vehicle control group value in the 100, 300 and 1000 mg/kg bw/day dosage groups, respectively. However, those changes were not statistically significant; at 1000 mg/kg bw/day, mean bodyweight gain was statistically lower than control during weeks 1 and 5 but was equivalent or higher during the other periods of treatment.
Females:
- Increases in mean body weights and body weight gains were observed in the female rats in the 1000 mg/kg bw/day dosage group during the premating period. These increases were not considered adverse effects of test substance because the increases were not consistent with changes in feed consumption and similar effects were not observed in the male rats.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- No effect on estrous cycle was observed at any of the dosage levels tested.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No treatment-related effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
- There were no significant differences in organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- No gross lesions in the female rats were caused by treatment with the test substance at dosage levels as high as 1000 mg/kg bw/day.
- Mucosal surface in the pyloric region of the stomach contained numerous brown ulcerations in one dam in the 1000 mg/kg bw/day dosage group; this dam was euthanized on DG 23 due to difficulty during parturition. Histopathological evaluation confirmed that the gastric lesions were ulcers. Because this rat was in adverse clinical condition due to dystocia, the ulcers were considered stress related and not attributed to treatment with the test substance.
- Mucosal surface in the cecum contained two red areas and one tan area in one dam in the 1000 mg/kg bw/day dosage group. Histopathological evaluation confirmed that the discolored areas were ulcers. These ulcers were considered incidental findings that were unrelated to treatment with the test substance because the finding was observed in a single rat on the entire study.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- There were no histopathologic lesions in the reproductive organs (ovaries and uterus with cervix) that could be attributed to treatment with the test substance.

OTHER FINDINGS (PARENTAL ANIMALS)
- In the male rats, spermatogenesis was progressing normally with all stages of the spermatogenic cycle presented.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

for reproductive toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: - no effect on the ability of male and female rats to mate and produce viable litters at any dosage level tested. - no treatment-related microscopic changes were observed in reproductive organs

2

Dose descriptor:

NOAEL

Remarks:

for general toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects, that can be considered as adverse and/or related to treatment, could have been detected in general toxicity (clinical signs; mortality; body weight; food consumption; gross pathology)

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

No clinical signs or gross lesions were observed in the F1 generation pups attributed to maternal treatment with the test substance.

Non-treatment related findings:
Clinical observations:
- One male pup in the litter from one dam in the vehicle control group had a scab on the left hindleg throughout the abbreviated postpartum period, as well as a laceration with concurrent swelling. This persistent clinical observation was presumed to be related to an incidental injury.
- Pup clinical observations in the 300 mg/kg bw/day dosage group included purple discoloration on the nose on DLs 1 and 2 in one male pup and cold to touch on DL 1 in another male pup. These clinical observations were transient and unrelated to maternal treatment with the test substance.
- Right hindpaw and tail were missing and were presumed cannibalized on DL 1 in one male pup in the 1000 mg/kg bw/day dosage group. On DL 2, one male pup was missing from this litter and was presumed cannibalized; it was presumed that the missing male pup was the partially cannibalized but live male pup from the previous day. The incidence of missing pups/presumed cannibalization in the 1000 mg/kg bw/day dosage group was within the range of historical control data and unrelated to maternal treatment with the test substance.
- All other dams and respective litters appeared normal during the lactation period.
Necropsy observations:
- The stomach contained no milk, and the intestines appeared large in one male pup that was found dead on DL 2 in the 100 mg/kg bw/day dosage group. The intestinal finding, which was first observed by in-life personnel during the standard abbreviated necropsy examination, could not be confirmed by necropsy personnel via the wet tissues.
- Necropsy examination of the F1 generation pups revealed no other gross lesions.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test conditions, it was considered that the No Observed Adverse Effect Level (NOAEL) for general toxicity was 1000 mg/kg bw/day (see document attached), and the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was 1000 mg/kg bw/day.

Executive summary:

In a reproduction / developmental toxicity screening test conducted according to the OECD Guideline 421 and in compliance with GLP, the substance was administered daily by oral gavage to groups of Crl:WI (Wistar Han) rats (10/sex/dose) at the dose levels of 0 (vehicle), 100, 300 and 1000 mg/kg bw/day. The substance and/or the vehicle, corn oil, was administered to male rats once daily beginning 14 days before cohabitation, through the cohabitation period (maximum 13 days) and continuing through the day before sacrifice (14 days after the completion of cohabitation). Female rats were given the substance and/or the vehicle once daily beginning 14 days before cohabitation, through the cohabitation period (maximum 13 days) and continuing through day 4 of lactation. The following parameters were evaluated: viabilities, clinical observations and body weights (P and F1 generations), feed consumption values, estrous cyclicity, mating and fertility parameters, natural delivery and litter observations and maternal behavior, necropsy observations (P and F1 generations), organ weights and microscopic evaluation. P (parental F0) generation male rats were sacrificed after the completion of cohabitation, and all surviving P generation female rats were sacrificed on Day 5 of lactation (DL 5). A complete necropsy was performed; testes, epididymides, prostate, seminal vesicles with coagulating gland, ovaries and uterus with cervix were weighed, retained and processed for microscopic evaluation. All surviving F1 generation pups were sacrificed on Day 5 postpartum and examined for gross lesions.

All male rats survived until scheduled sacrifice. One female rat in the 1000 mg/kg bw/day dosage group was sacrificed before scheduled termination due to adverse clinical condition resulting from difficulties during parturition (dystocia). All other female rats survived until scheduled sacrifice. Excess salivation occurred in both male and female rats at dosage levels of 300 and 1000 mg/kg bw/day. Urine-stained abdominal fur occurred in both male and female rats in the 1000 mg/kg bw/day dosage group. Piloerection, ptosis and red perivaginal substance occurred in female rats in the 1000 mg/kg bw/day dosage group during the gestation period. All those clinical signs were considered neither as treatment related nor as adverse effects. Reductions in mean body weight gains and feed consumption values occurred in the male rats in the 300 and 1000 mg/kg bw/day dosage groups. Mean body weight gains in the male rats during the entire dosage period (calculated as Days 1 through 42 of study) were 93, 78 and 77 % of the vehicle control group value in the 100, 300 and 1000 mg/kg bw/day dosage groups, respectively. This decrease in bodyweight gain was not considered as adverse as it is not statistically significant from control and that bodyweight and bodyweight gain evolutions are similar between all groups throughout the study. Increases in mean body weights and body weight gains were observed in the female rats in the 1000 mg/kg bw/day dosage group during the premating period. These increases were not considered as treatment related because, the increases were not consistent with changes in feed consumption and similar effects were not observed in the male rats. Mating and fertility parameters and natural delivery and litter observation parameters were unaffected by treatment with the substance at dosage levels as high as 1000 mg/kg bw/day. No gross lesions were caused by treatment with the substance at dosage levels as high as 1000 mg/kg bw/day. Terminal body weights, absolute organ weights and ratios of organ weights to terminal body weights were unaffected by treatment with the substance at dosage levels as high as 1000 mg/kg bw/day. There were no histopathological lesions in the reproductive organs that could be attributed to treatment with the substance. In the male rats, spermatogenesis was progressing normally with all stages of the spermatogenic cycle presented. There were no clinical signs or gross lesions observed in the F1 generation pups attributed to maternal treatment with the substance.

Under the test conditions, it was considered that the No Observed Adverse Effect Level (NOAEL) for general toxicity was 1000 mg/kg bw/day and the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was 1000 mg/kg bw/day.