Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl propionate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Jul 2010 to 23 Nov 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Isobornyl propionate.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Jul 2010 to 23 Nov 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Isobornyl propionate.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat was selected as the Test System because of known response to toxic effects on reproductive capacity and history of use as a rodent species in these evaluations

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: Approximately 6 weeks (Males) or 11 weeks (females) at the initiation of the treatment.
- Weight at study initiation: 133-159 (Males) and 212-253 g (Females) (value represent weights at study assignment)
- Housing (P generation): P generation rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female rats was housed in the male rat's cage. Beginning no later than DG 20, P generation female rats were individually housed in nesting boxes until they either naturally delivered litters or were euthanized on DG 25. Each dam and delivered litter was housed in a common nesting box during the postpartum period. Bed-o’cobs bedding (The Andersons Industrial Products Group, Maumee, OH) was used as the nesting material. Chewable Nylabones® were supplied to all rats during the course of the study.
- Housing (F1 generation): After weaning, F1 generation pups selected for continued evaluation were individually housed in stainless steel wire-bottomed cages. Pups selected for continued evaluation that had a body weights of less than 28 grams were pair housed for approximately one week in a nesting box to optimize the conditions under which pups are transitioned from group to individual housing
Bed-o’cobs bedding (The Andersons Industrial Products Group, Maumee, OH) was used as the nesting material. Chewable Nylabones® were supplied to all rats during the course of the study.
- Diet: Rats were given ad libitum access to Certified Rodent Diet #5002 meal (PMI Nutrition International, Inc., St. Louis, MO) in individual feeders.
- Water: Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: Appr. 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (suspensions) for Groups 1 through 3 were prepared at least once weekly at the Testing Facility. Formulations (suspensions) for Group 4 were prepared at least once daily at the Testing Facility until 10-day stability of this formulation was established, after which Group 4 formulations were prepared at least once weekly at the Testing Facility. All prepared formulations were stored at room temperature, protected from light. Prepared formulations were stirred continuously prior to and during dosage administration.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 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 DG 0

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Information to document or certify the identity, composition, strength, and activity/purity, of the test substance was provided by the Sponsor to the Testing Facility. Stability data for lower concentrations used in this study was established before the conduct of the study.
Samples of dose formulations were analysed by high-performance liquid chromatography with ultraviolet detection (HPLC-UV). The method was validated for the analysis of dose formulations at concentrations ranging from 1 mg/mL to 100 mg/mL of test substance in corn oil. Stability of the dose formulations for Group IV, 75 mg/mL were evaluated at 20°C to 25°C for 25 hours and 14 days. In addition, the actual concentrations and the relative standard deviation (RSD) of the mean of the average concentration values for the top, middle, and bottom of each dose formulation were calculated to assess homogeneity and the actual concentration was determined.

Duration of treatment / exposure:

- P generation male rats were given the test substance and/or the vehicle once daily beginning 84 days before cohabitation, through cohabitation and continuing until the day before scheduled euthanasia for a total of 113 to 116 dosages.
- P generation female rats were given the test substance and/or the vehicle once daily beginning two weeks before cohabitation, through cohabitation, and continuing through DG 25 (rats that did not deliver) or Day 22 postpartum (rats that delivered a litter).
- F1 generation pups were not directly administered the test substance and/or vehicle, but may have possibly been exposed to the test substance and/or the vehicle during maternal gestation (in utero exposure) or via maternal milk during the lactation period.

Frequency of treatment:

once daily

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected on the basis of a dosage-range reproduction study. Eight rats per sex per group were dosed via gavage with 0 (Vehicle), 5, 15, 45, or 135 mg/kg/day in a corn oil vehicle. Dosing occurred 14 days prior to cohabitation, through cohabitation, gestation, and postpartum day 5 or the day of euthanasia. There were no treatment-related effects on viability or clinical observations. In male rats, body weights and feed consumption were comparable among all groups. In female rats, treatment with isobornyl acetate reduced body weight gains during the precohabitation period in a non-dosage dependent manner. During gestation, maternal body weight gains were reduced in the 45 and 135 mg/kg/day dosage groups. Body weight gains during the 5-day lactation period were highly variable. Feed consumption was unaffected by treatment. All pregnant rats delivered a litter and there were no effects on natural delivery or litter observations. There were no effects on pup body weights. Based on these data in which borderline maternal toxicity was observed at 45 and 135 mg/kg/day (reduced gestational weight gain), a higher dose of 300 mg/kg/day was selected to ensure that sufficient toxicity will be observed. The middle and low dosages were selected in 3-fold increments.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
All rats were observed for viability twice each day of the study and for clinical observations and general appearance, at least once weekly during the acclimation period. Observations for clinical signs, abortions, premature deliveries and death were made once daily before dosage administration, between one and two hours following dosage administration and at the end of the normal working day during the dosage period, and on the day euthanasia occurred.
Female rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed). Litter size (all pups delivered), and pup viability at birth. Female rats were examined for maternal behavior on DLs 1, 5, 8, 15 18, and 22.

BODY WEIGHT:
Body weights were recorded at least weekly during the acclimation for all rats, and on the first day of dosage administration, weekly thereafter and on the day of euthanasia for male rats. Body weights were also recorded for female rats at least weekly during the precohabitation and cohabitation periods, and on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11, 15, 18 and 22 (terminal body weight).

FOOD CONSUMPTION
Feed consumption values were recorded weekly during the acclimation period for rats not selected for study, and weekly during the acclimation and dosage periods for all rats selected for study. Values were recorded for female rats on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11 and 15. Because pups begin to consume maternal feed on or about DL 15, feed consumption values were not tabulated after DL 15.

OTHER:
Female rats were examined for maternal behavior on DLs 1, 5, 8, 15 18, and 22

Oestrous cyclicity (parental animals):

Estrous cycling was evaluated daily by examination of vaginal cytology beginning 14 days before scheduled cohabitation and continuing through cohabitation until mating was confirmed.

Sperm parameters (parental animals):

Sperm concentration and motility were evaluated using computer-assisted sperm analysis (CASA). Motility was evaluated by the Hamilton Thorne IVOS by collection of a sample from the left and right vas deferens. A homogenate was prepared from the left
cauda epididymis for evaluation by the Hamilton Thorne IVOS to determine sperm concentration/density (sperm per gram of tissue weight).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED PRE-WEANING
- pup body weights were recorded after all pups in a litter were delivered and groomed by the dam
- Litters were examined after delivery to identify the number and sex of pups, stillbirths, live births and gross alterations.
- Each litter was evaluated for viability twice daily. The pups in each litter were counted
once daily.
- Clinical observations were recorded once daily during the preweaning period. Pup body weights were recorded on Days 1 (birth), 5, 8, 11, 15, 18 and 22 postpartum.
- Anogenital distance was measured for all live F1 generation pups on Days 1 and 22 postpartum.
- Nipple eruption was evaluated for all live F1 generation pups once on Day 12 postpartum.

PARAMETERS EXAMINED POST-WEANING
- Rats were observed for viability twice daily and for clinical observations once daily during the postweaning period.
- Body weights were recorded weekly during the postweaning period, on the day sexual maturation was achieved and on the day euthanasia occurred (terminal weight).
- Feed consumption values were recorded weekly during the postweaning period (only for individually housed rats).
- Female rats were evaluated for the age of vaginal patency, beginning on Day 28 postpartum and continuing until achievement of this developmental parameter or Day 40 postpartum.
- Male rats were evaluated for the age of preputial separation, beginning on Day 39 postpartum until achievement of this developmental parameter or Day 50 postpartum.

GROSS EXAMINATION OF DEAD PUPS:
- Day 1 Postpartum: 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 sunk were identified as stillborn; pups with lungs that floated were identified as liveborn and to have died shortly after birth. Pups with gross lesions were preserved in Bouin's solution for possible future evaluation.
- Day 2 to 22 Postpartum: Pups that died before scheduled termination were examined for gross lesions and the cause of death or condition on the day the observation was made. Pups found on Days 2 to 5 postpartum were preserved in Bouin's solution for possible future evaluation; pups found on Days 6 to 22 postpartum were preserved in neutral buffered 10% formalin.
- After day 22 Postpartum: F1 generation male rats that were found dead or did not survive until scheduled euthanasia and the F1 generation female rat that was found dead were examined for the cause of death or condition on the day the observation was made. The rats were examined for gross lesions, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: After completion of the cohabitation period, all surviving P generation male rats were euthanized by carbon dioxide asphyxiation
- Maternal animals: After completion of the 22-day postpartum period, all surviving P generation female rats were euthanized by carbon dioxide asphyxiation. Rats that did not deliver a litter were euthanized by carbon dioxide asphyxiation on Day 25 of presumed gestation.

GROSS NECROPSY
- A complete necropsy examination was performed in situ, which included an evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain and spinal column; the nasal cavity and neck with associated organs and tissues; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Rats were examined for gross lesions. Gross lesions were retained in neutral buffered 10% formalin (NBF) and shipped to the Principal Investigator for histological examination. The number and distribution of implantation sites was recorded.

EARLY DEATHS
- Male rats that died or were euthanized before scheduled termination and female rats that were euthanized before scheduled termination were examined for the cause of death or condition on the day the observation was made. The rats were examined for gross lesions.
- Tissues were weighed and retained, and microscopic evaluations were conducted . The lungs, trachea and esophagus were perfused and saved in 10% NBF for microscopic evaluation.
- Pregnancy status and uterine contents of female rats were recorded (when appropriate). Conceptuses in utero were examined to the extent possible. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites, and retained in 10% NBF for microscopic evaluation.

HISTOPATHOLOGY
-The following tissues from all P generation male and/or female rats in the vehicle control and high dosage groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin: Brain, Epididymides, Esophagus, Glands adrenals, Glands mammary, Glands pituitary, Gross lesions/masses, Heart, Kidneys, Liver, Lungs, Ovaries, Prostate, Seminal Vesicles with coagulating gland, Spleen, Stomach, Testes, Trachea, Uterus with cervix and Vagina.
- Gross lesions from all P generation male and female rats in all dosage groups were processed for microscopic evaluation. In addition, tissues collected from any rats that did not survive to scheduled euthanasia were processed and evaluated microscopically. A quantitative evaluation of primordial follicles was conducted for P generation female rats.

ORGAN WEIGHTS
The following organs were weighed at necropsy for all P generation rats that survived to scheduled euthanasia: Brain, Epididymides (individually weighted for scheduled euthanasia), Glands, adrenals, Glands, pituitary, Kidneys, Liver, Ovaries (individually weighted for scheduled euthanasia), Prostate, Seminal Vesicles with coagulating gland, Testes (individually weighted for scheduled euthanasia) and Uterus with cervix. Organ to body weight ratio (using the terminal body weight obtained at necropsy) were calculated.

Postmortem examinations (offspring):

SACRIFICE
- All pups not selected for continued evaluation were euthanized on Day 22 postpartum via carbon dioxide asphyxiation. All gross lesions were preserved in 10% NBF for possible future evaluation. Necropsy included a single crosssection of the head at the level of the frontal-parietal suture and examination of the crosssectioned brain for apparent hydrocephaly. Carcasses of pups not selected for continued observation were discarded without further evaluation.
- Schedueled euthanasia: Male and female F1 generation rats were euthanized on Days 57 to 63 postpartum by carbon dioxide.

GROSS NECROPSY
Sacrificed animals were subjected to a gross necropsy of the thoracic, abdominal and pelvic viscera. Gross lesions were preserved in 10% NBF.

ORGAN WEIGHTS
The following organs were weighed at necropsy for all scheduled euthanasia F1 generation rats: Brain, Epididymides (individually weighted), Glands, adrenal, Gland, pituitary, Kidneys, Ovaries (individually weighted), Prostate, Testes (individually weighted), Seminal Vesicles and Uterus with cervix. Organ to body weight ratio (using the terminal body weight obtained at necropsy) were calculated.

Statistics:

- Clinical observations and other proportion data were analysed using the Variance Test for Homogeneity of the Binomial Distribution.
- Variables with interval or ratio scales of measurement, such as body weights, feed consumption values and percent mortality per litter were analysed as described under the Parametric heading of the schematic. Bartlett’s Test of Homogeneity of Variances was used to estimate the probability that the dosage groups have different variances. A nonsignificant result (p> 0.001) indicated that an assumption of homogeneity of variance was
not inappropriate, and the data were compared using the Analysis of Variance. If that test was significant (p≤ 0.05), the groups given the test substance was compared with the control group using Dunnett’s Test. If Bartlett’s Test was significant (p≤ 0.001), the Analysis of Variance Test would not be appropriate, and the data were analysed as described under the Nonparametric heading of the schematic. When 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis Test was used to analyse the data, and in the event of a significant result (p≤ 0.05), Dunn’s Test was used to compare the groups given the test substance with the control group. When more than 75% of the scores in any dosage group were tied, Fisher’s Exact Test was used to analyse the data.
- Count data were analysed using the procedures described under the Nonparametric heading of the schematic.
- Group means and standard deviations of primordial follicles was calculated and compared across groups using the Kruskal-Wallis non-parametric ANOVA test. If a significant result occurred (p<0.05), the Wilcoxon (Mann-Whitney U) test was used for pair-wise group comparison to the control group. Statistical analyses were performed using an IBMTM compatible computer with SAS computer programs (Version 9.1.3, Service Pack 4; SAS/STAT User's Guide, 1989).

Reproductive indices:

- Fertility Index (percentage of matings that result in pregnancies): Reported for each sex.
- Gestation Index (percentage of pregnancies that result 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 size and viability [tabulated at birth (day 1) and on days 5, 8, 11, 15, 18 and 22 postpartum].
- Viability Indices (percentages of pups born that survive to days 1 and 5 postpartum).
- Lactation Index (percentage of pups that survive from day 5 to day 22 postpartum).
- Percent Survival an Sex Ratio [tabulated at birth (day 1) and on days 5, 8, 11, 15, 18 and 22 postpartum].

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males:
- Clinical signs in P generation male rats that were attributed to treatment included slight and moderate excess salivation at 300 mg/kg/day. Slight excess salivation was observed as early as DS 27 and occurred in 22 of 25 males in the 300 mg/kg/day dosage group. Moderate excess salivation occurred in 6 of 25 males in the same dosage group. Each of these clinical observations occurred in significantly more (p≤0.01) P generation male rats in comparison to the vehicle control group values.
- All other clinical observations were considered unrelated to treatment. These clinical observations included sparse hair coat (head, neck, limbs or underside), a scab at one or more sites on the body (head, neck, right side of the neck, left side of the neck, right and/or left forepaw, right forelimb, back and/or left hindpaw), chromodacryorrhea, missing/broken/misaligned incisors, chromorhinorrhea, hyperreactivity, soft or liquid feces, an ulceration at one or more sites on the body (neck, right side of the neck and/or left side of the neck), mild or moderate dehydration (based on skin turgor), swollen snout, localized alopecia (neck, limbs or underside), erythema on the left forepaw, swollen left hindpaw or both forepaws, scant feces, brown perioral substance, an abrasion on the neck, brown fur on the right forelimb and lower midline, red and/or clear discharge, piloerection, apprehensive when handled, over groomed, excessive grooming, excessive licking, aggressive when handled, red substance in the cage, ungroomed coat, dyspnea, bent snout, lacrimation, a laceration on the left hindpaw, an elongated snout, gasping and a red ulceration. No other clinical signs occurred in the F0 generation male rats.

Females:
- Similar to male rats, the number of P generation female rats with excess salivation (slight and/or moderate) was significantly increased (p≤0.01) in the 300 mg/kg/day dosage group, in comparison to the vehicle control group values. At 300 mg/kg/day, slight excess salivation was observed as early as DS 2 and persisted into the lactation period; the clinical sign was more pronounced during the precohabitation and gestation periods. At 100 mg/kg/day, the number of P generation female rats with slight excess salivation was significantly increased (p≤0.01) only during the gestation dosage period, in comparison to the vehicle control group value.
- In addition, a low incidence of urine-stained abdominal fur (p≤0.01; N=4 out of 25) occurred in the 300 mg/kg/day dosage group during the gestation period. This clinical sign was not considered an adverse finding of isobornyl acetate because the observation was transient and did not persist.
- All other clinical observations during the precohabitation, gestation and lactation periods
were considered unrelated to treatmentbecause: 1) the incidences were not dosage-dependent; and/or 2) the number of rats affected did not differ significantly from the vehicle control group values. These clinical observations included extreme excess salivation, a scab on the mouth, mild dehydration (based on skin turgor), red substance in the cage or cage pan, red and dried red substance on the fur, a broken toenail on the right hindpaw, dried red perioral substance, chromodacryorrhea, missing/broken/misaligned incisors, vocalization in the cage or to touch, chromorhinorrhea, soft or liquid feces, a mass in the left inguinal area or left axilla, purple and/or black appearance to the left inguinal area, a scab present in the left inguinal area and left hindlimb, pale ears, red perivaginal substance, hyperpnea, piloerection, sparse hair coat on the neck or limbs, a portion of the tail was missing, localized alopecia on the limbs, a mass along the left axilla, a swollen third eyelid, ptosis and lacrimation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- Males: No mortality related to the test substance occurred during this study. One male rat in the vehicle control group was found dead on Day 42 of study (DS 42) following an apparent gavage accident and one male rat in the 100 mg/kg/day dosage group was euthanized on DS 107 because of adverse clinical signs unrelated to administration of the test substance. All other F0 generation male rats survived to scheduled euthanasia.
- Females: No mortality related to the test substance occurred during this study. One female rat in each of the 100 and 300 mg/kg/day dosage groups was euthanized during the gestation period because of adverse clinical observations, including an apparent tail injury and signs of dystocia. All other F0 generation female rats survived to scheduled euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
- Body weights and body weight gains were unaffected by dosages of substance as high as 300 mg/kg/day.
- Transient, but statistically significant increases (p≤0.05 or p≤0.01) in body weight gains occurred in each treated group during the first week of the dosage period (DSs 1 to 8), and again on DSs 36 to 43 in the 100 mg/kg/day dosage group, in comparison to the vehicle control group values. These findings were not attributed to administration because: 1) the increases were transient; 2) the increases were not dosage-dependent; and/or 3) the increases were minimal and had no overall impact on the cumulative body weight gains (DSs 1 to 78 or DSs 1 to 112) or the average body weight at any dosage level.
- The average body weight on DS 112 was 103%, 104% and 103% of the vehicle control group value in the 30, 100 and 300 mg/kg/day dosage groups, respectively.
- Terminal body weights were comparable among the four dosage groups and did not significantly differ from the vehicle control group value.

Females:
- Body weights and body weight gains during the precohabitation, gestation and lactation periods were unaffected by dosages as high as 300 mg/kg/day. All values were comparable among the four dosage groups. The following statistically significant findings occurred:
• A statistically significant increase (p≤0.05) in the average body weight at 100 mg/kg/day on DS 8
• Statistically significant increases (p≤0.05 or p≤0.01) in body weight gains at 100 and 300 mg/kg/day on DGs 5 to 8
• A statistically significant increase (p≤0.01) in body weight gains at 300 mg/kg/day for the cumulative gestation period (DGs 1 to 22)
These increases in body weight or body weight gain were considered unrelated to substance because: 1) the increase was not dosage-dependent; and/or 2) the increase reflected an overall reduction in body weight gains or a net loss in body weight in the corresponding vehicle control group.
- Terminal body weights were comparable among the four dosage groups and did not significantly differ from the vehicle control group value.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males:
- Absolute (g/day) and relative (g/kg/day) feed consumption values were unaffected by dosages of substance as high as 300 mg/kg/day.
- Transient, but statistically significant increases (p≤0.05 or p≤0.01) in absolute and relative feed consumption occurred in the 100 and 300 mg/kg/day dosage groups during the first week of the dosage period (DSs 1 to 8), and again on DSs 15 to 22 in the 300 mg/kg/day dosage group, in comparison to the vehicle control group values. These findings were not attributed to administration of treatment because: 1) the increases were transient; and/or 2) the increases were minimal and had no overall impact on the cumulative feed consumption (DSs 1 to 78 or DSs 1 to 112).

Females:
- Absolute (g/day) and relative (g/kg/day) feed consumption values during the precohabitation, gestation and lactation periods were unaffected by dosages of substance as high as 300 mg/kg/day. All values were comparable among the four dosage groups.
- The statistically significant increase (p≤0.05) in absolute and relative feed consumption that occurred in the 100 and 300 mg/kg/day dosage groups on Days 8 to 11 of lactation (DLs 8 to 11) were considered unrelated to treatment because the increases were transient and not dosage-dependent.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated rats and were considered unrelated to administration of the substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Females
- There were no statistically significant differences (p<0.05) in the number of primordial ovarian follicles in either left, right, or both ovaries combined, in P generation female rats administered 300 mg/kg/day compared to the vehicle control rats. Corpora lutea were present in all rats evaluated.
- Natural delivery and litter observations were unaffected by dosages of substance as high as 300 mg/kg/day.
- Values for the numbers of dams delivering litters, the duration of gestation, averages for implantation sites per delivered litter, the gestation index (number of dams with one or more liveborn pups/number of pregnant rats), the numbers of dams with stillborn pups and of dams with all pups dying, litter sizes, surviving pups per litter, the viability index (number of live pups on day 5 postpartum/ number of liveborn pups on Day 1 postpartum), the lactation index (number of live pups on Day 22 postpartum/ number of live pups on day 5 postpartum), the percentage of male pups per number of pups sexed per litter, live litter size at weighing and pup weight per litter were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The number of estrous stages per 14 days was comparable among the four dosage groups during the precohabitation period.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

All sperm parameters evaluated were unaffected by dosages as high as 300 mg/kg/day. Values for number and percent motile sperm, number of nonmotile sperm and total sperm count from the vas deferens and cauda epididymal sperm count
and density were comparable among the four dosage groups and did not significantly differ from the vehicle control group values.

Reproductive performance:

no effects observed

Description (incidence and severity):

Males:
- All mating and fertility parameters [numbers of days in cohabitation, rats that mated, the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during the first or second weeks of cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages of substance as high as 300 mg/kg/day. All values were comparable among the four dosage groups and did not significantly differ.
- The apparent increase in the mean number of days to mating that occurred in the 100 and 300 mg/kg/day dosage groups was attributed to one or two rats that were in cohabitation for 10 or 14 days prior to mating. Otherwise, the remaining rats in the 100 or 300 mg/kg/day dosage groups mated with their respective cohort females within 1 to 4 days.

Females:
- All mating and fertility parameters [numbers of days in cohabitation, rats that mated, the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during the first or second week of cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages as high as 300 mg/kg/day. All values were comparable among the four dosage groups and did not significantly differ.
- The apparent increase in the mean number of days to mating that occurred in the 100 and 300 mg/kg/day dosage groups was attributed to one or two rats that were in cohabitation for 10 or 14 days prior to mating. Otherwise, the remaining rats in the 100 or 300 mg/kg/day dosage groups mated with their respective cohort males within 1 to 4 days.
- Pregnancy occurred in 23, 22, 24 and 22 of the 25 mated P generation female rats in the 0 (Vehicle), 30, 100 and 300 mg/kg/day dosage groups, respectively. All pregnant dams delivered litters.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related adverse effects up to the highest dose tested

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

other: No effects on fertility up to the highest dose tested

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Preweaning
- There were no treatment-related clinical signs observed in the F1 generation pups following treatment of the P generation rats with dosages as high as
300 mg/kg/day. All transient and persistent clinical observations were considered unrelated to treatment because: 1) the incidences were not dosage-d pendent; and/or 2) the number of litters affected did not differ significantly from the vehicle control group values.
- Transient clinical observations that occurred and were associated with poor maternal care included dehydration (mild to severe), not nesting, and cold to touch. Other transient clinical observations included swelling (right or left hindpaw, right hindlimb, Anogenital region or tail), scab formation (right hindpaw or lower midline), a fissure along the lower midline, a black appearance to the tail, a constricted tail, an umbilical hernia and limited use of the fore- and hindlimbs.
- Persistent clinical observations that occurred in the F1 generation pups included missing digits on the left or right hindpaw and a necrotic appearance to the right hindpaw. No other transient or persistent clinical observations occurred.

Postweaning
All clinical observations that occurred in male or female rats during the postweaning period were considered unrelated to treatment of P generation rats because: 1) the incidences were not dosage-dependent; 2) the observations occurred in one to three rats in any dosage group; and/or 3) the observations occurred only in the vehicle control group. These clinical observations included a slight excess salivation, scab formation at one or more sites on the body, mild dehydration (based on skin turgor), an abrasion on the right and/or left side of the neck or right forelimb, red perioral substance, chromodacryorrhea, misaligned/missing/broken incisors, a bent tail, lacrimation, swelling of the nictitating membrane in the right eye, a red appearance to the nictitating membrane in the right eye, a protruding palate, tachypnea, piloerection, urine-stained abdominal fur, sparse hair coat on the neck, vocalization to touch and ungroomed coat.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Preweaning
- There was a statistically significant reduction (p≤0.05 or p≤0.01) in the number of stillborn pups in the 30 and 300 mg/kg/day dosage groups that were evaluated at necropsy examination, in comparison to the vehicle control group value. These significant reductions (p≤0.05 or p≤0.01) were based on the overall number of pups that were found dead or stillborn in the vehicle control group, and were not attributed to administration test substance to the P generation rats.

Postweaning
- No deaths related to treatment occurred in the F1 generation rats.
- One F1 generation male rat (no. 5972) in the 100 mg/kg/day dosage group was euthanized on Day 63 postpartum because of a broken palate. Clinical signs that
occurred on the day of euthanasia included chromodacryorrhea, misaligned incisors, a protruding palate, tachypnea and piloerection. Body weight gains and feed consumption values were unremarkable. Necropsy confirmed the broken palate; all other tissues appeared normal.
- All other F1 generation rats selected for continued evaluation postweaning survived until scheduled euthanasia.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Body weights and body weight gains of the F1 generation male and female rats during the postweaning period were unaffected by treatment of the P generation rats at dosages as high as 300 mg/kg/day. Body weight gains in F1 male rats in the 30, 100 and 300 mg/kg/day dosage groups were 103%, 105% and 103% of the vehicle control group value, respectively, on Day postpartum to termination.
- Body weight gains in F1 female rats in the 30, 100 and 300 mg/kg/day dosage groups were 101%, 104% and 100% of the vehicle control group value, respectively, on day 23 postpartum to termination.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Absolute and relative feed consumption values for F1 generation male and female rats during the postweaning period were unaffected by treatment of the P generation rats with dosages as high as 300 mg/kg/day. Absolute feed consumption values for F1 male rats in the 30, 100 and 300 mg/kg/day dosage groups were 99%, 104% and 100% of the vehicle control group value, respectively, on days 23 to 51 postpartum.
- Absolute feed consumption values for F1 female rats in the 30, 100 and 300 mg/kg/day dosage groups were 98%, 101% and 96% of the vehicle control group value, respectively, on Days 23 to 51 postpartum.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

- Anogenital distance on days 1 or 22 postpartum in F1 male and female pups was not affected by treatment of the P generation rats at any dosage level tested.
- Nipple eruption did not occur in any male pup at any dosage level tested. All female pups had nipples present on day 12 postpartum.
- Sexual maturation in F1 rats was unaffected by treatment of P generation rats with dosages as high as 300 mg/kg/day. The average day on which preputial separation was observed in F1 male rats and vaginal opening was observed in F1 female rats was comparable among the dosage groups. In addition, the average body weight on the day sexual maturation was achieved in F1 male and female rats was comparable among the dosage groups and did not differ significantly from the vehicle control group values.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no apparent effects of treatment of P generation rats with substance on the reproductive and nonreproductive tissue weights (brain, paired kidneys, paired adrenals) of the F1 male or female rats at any dosage level.
- A statistically significant reduction (p≤0.05) in the relative (% terminal body weight) weight of the paired adrenal glands occurred in F1 male rats at 100 mg/kg/day, in comparison to the vehicle control group value. This reduction was considered unrelated to treatment of P generation rats with isobornyl acetate because: 1) there was no statistically significant change in the absolute weight of this organ; 2) the reduction did not occur in the F1 female rats; and 3) the increase was not dosage-dependent.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Preweaning
There were no test substance-related gross lesions observed in the F1 generation pups that survived to scheduled necropsy on day 22 postpartum. One pup in the 300 mg/kg/day dosage group had complete situs inversus; all other pups appeared normal. In the pups that were stillborn or found dead, no milk was present in the stomach of 2 F1 generation pups in the 300 mg/kg/day dosage group.

Postweaning
There were no test substance-related necropsy observations. All necropsy observations were considered unrelated to treatment of P generation rats because: 1) the incidences were not dosage-dependent; 2) the observations occurred in only one rat in any dosage group; and/or 3) the observations were limited to the vehicle control group. In F1 male rats, these necropsy observations included slight or moderate dilation of the pelvis in the right kidney and small testes. Gross lesions observed in F1 female rats included slight dilation of the pelvis in the right kidney. No other gross lesions occurred in the F1 generation rats.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested

Critical effects observed:

no

Reproductive effects observed:

no

Verification of dosing solutions

- Concentration: Mean measured concentrations for all dose formulations were within the acceptable limits (≤15% difference from nominal concentration).

- Homogeneity: Homogeneity was acceptable (≤5% RSD) for all dose formulations.

- Stability: Stability of dose formulations for Group IV, 75 mg/mL were evaluated at 20°C to 25°C for 25 hours and 14 days. Mean measured concentrations for stored samples were within the acceptable limits (±10% of the initial mean measured concentration).

Conclusions:

Based on the results of this study, the parental NOAEL for systemic toxicity is 300 mg/kg/day, because no treatment related adverse effects have been observed up to the highest concentration tested. The NOAEL for fertility and F1 development is exceeding 300 mg/kg bw/day since no effects on the relevant parameters were observed up to the highest dose tested.

Executive summary:

A one-generation study was performed according to OECD guideline 415 and GLP principles. One hundred male and 100 female rats were assigned to four dosage groups, 25 rats per sex per group, for treatment with the test substance. Suspensions of the test substance, or the vehicle, corn oil, were administered via gavage to the male rats once daily beginning 84 days before the cohabitation period, through the cohabitation period (maximum of 14 days) and continuing until the day before euthanasia; and to the female rats once daily beginning 14 days before the cohabitation period, through the cohabitation period (maximum of 14 days) and continuing through the day of euthanasia (through Day 25 of presumed gestation [rats that do not deliver] or Day 22 of lactation [rats that deliver a litter]). Dosage levels were 0 (Vehicle), 30, 100 and 300 mg/kg/day, which was based on a dose-range finder study. The dosage volume was 4 mL/kg, which was adjusted weekly on the basis of the individual body weights. The following parameters were evaluated: P generation males: viability, clinical observations, body weights, feed consumption; mating and fertility, organ weights, gross and microscopic observations, sperm assessments (motility and concentration); P generation females: viability, clinical observations, body weights, feed consumption, estrous cycling, mating and fertility, natural delivery and litter observations, organ weights, gross and microscopic observations, and ovarian follicle counts; F1 generation pups/rats: viability, body weights, anogenital distance (days 1 and 22 postpartum), sexual maturation, nipple eruption (day 12 postpartum), and gross observations. Surviving male rats were euthanized after completion of the cohabitation period (Days 114 through 117 of study). Female rats that delivered a litter and pups not selected for continued evaluation were euthanized on Day 22 of lactation (DL 22). Female rats that did not deliver a litter were euthanized on Day 25 of presumed gestation (DG 25). F1 generation rats selected for continued evaluation were euthanized on Days 57 through 63 postpartum.

Results: Analytical verification of dosing solutions

Analytical verification of the dosing solution revealed that all prepared dose formulations were acceptable for use on this study.

Results: P Generation Male Rats

No mortality related to the test substance occurred during this study. Clinical signs in P generation male rats that were attributed to treatment included slight and moderate excess salivation at 300 mg/kg/day. Slight excess salivation was observed as early as DS 27 and occurred in 22 of 25 males in the 300 mg/kg/day dosage group. Moderate excess salivation occurred in 6 of 25 males in the same dosage group. The substance did not affect body weights, body weight gains or feed consumption values (g/day or g/kg/day) at any dosage level, and did not increase the incidence of gross or microscopic findings at dosages as high as 300 mg/kg/day. There were no apparent effects on mating and fertility, reproductive (epididymides, caudal epididymis, testes, seminal vesicles [with and without fluid] and prostate) or non-reproductive (pituitary, brain, paired kidneys and paired adrenals) organ weights or sperm motility and concentration at any dosage level tested.

Results: P Generation Female Rats

No mortality related to the test substance occurred during this study.Similar to male rats, the number of P generation female rats with excess salivation (slight and/or moderate) was significantly increased at 300 mg/kg/day during the premating, gestation and lactation periods, in comparison to the vehicle control group values. At 100 mg/kg/day, the number of P generation female rats with slight excess salivation was significantly increased only during the gestation dosage period, in comparison to the vehicle control group value. In addition, low incidences of urine-stained abdominal fur occurred at 300 mg/kg/day during the gestation period. Body weights, body weight gains and absolute (g/day) and relative (g/kg/day) feed consumption in P generation female rats were unaffected by dosages as high as 300 mg/kg/day prior to mating and during the gestation and lactation periods. In addition, there were no apparent effects on the estrous cycle, mating and fertility parameters or natural delivery and there were no treatment-related gross lesions or microscopic changes at any dosage level tested. Terminal body weights were comparable among the four dosage groups and did not significantly differ from the vehicle control group value. Reproductive (left and right ovaries and the uterus with cervix) and non-reproductive (i.e., pituitary, brain, liver, paired kidneys and paired adrenals) were unaffected by oral administration at dosages as high as 300 mg/kg/day. In addition, there were no microscopic findings, including ovarian follicle evaluation, that were attributed to the test substance at any dosage level.

Results: F1 Generation Rats

No deaths related to the test substance occurred in the F1 generation rats. One F1 generation male rat in the 100 mg/kg/day dosage group was euthanized on day 63 postpartum because of a broken palate. All other F1 generation rats selected for continued evaluation postweaning survived until scheduled euthanasia. There were no treatment-related clinical signs, gross lesions or changes in body weight, body weight gains, feed consumption (g/day or g/kg/day) or organ weights (reproductive or nonreproductive) in the male and female F1 generation rats at any dosage level tested. Anogenital distance on day 1 or 22 postpartum, nipple eruption on day 12 postpartum and sexual maturation was unaffected in either sex following treatment of the P generation male and female rats with dosages of up to and including 300 mg/kg/day.

Conclusion

Based on the results of this study, the NOAEL for parental systemic toxicity is 300 mg/kg/day. Increased incidences of excess salivation occurred in P generation male and female rats at 100 and/or 300 mg/kg/day throughout the dosage period, and low incidences of urine-stained abdominal fur occurred in females at 300 mg/kg/day during the gestation period. However, these clinical signs were not considered an adverse effect test item. The reproductive NOAEL in the P generation rats and the NOAEL for viability and growth of the F1 generation offspring is greater than or equal to 300 mg/kg/day. There were no apparent effects on estrous cycling, mating and fertility, reproductive organ weights or natural delivery parameters in the P generation, and growth and development (including anogenital distance, nipple eruption or sexual maturation) in the F1 generation rats at the highest dosage level tested (300 mg/kg/day).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate

EC Number:

204-727-6

EC Name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate

Cas Number:

125-12-2

Molecular formula:

C12H20O2

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: Approximately 6 weeks (Males) or 11 weeks (females) at the initiation of the treatment.
- Weight at study initiation: 133-159 (Males) and 212-253 g (Females) (value represent weights at study assignment)
- Housing (P generation): P generation rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female rats was housed in the male rat's cage. Beginning no later than DG 20, P generation female rats were individually housed in nesting boxes until they either naturally delivered litters or were euthanized on DG 25. Each dam and delivered litter was housed in a common nesting box during the postpartum period. Bed-o’cobs bedding (The Andersons Industrial Products Group, Maumee, OH) was used as the nesting material. Chewable Nylabones® were supplied to all rats during the course of the study.
- Housing (F1 generation): After weaning, F1 generation pups selected for continued evaluation were individually housed in stainless steel wire-bottomed cages. Pups selected for continued evaluation that had a body weights of less than 28 grams were pair housed for approximately one week in a nesting box to optimize the conditions under which pups are transitioned from group to individual housing
Bed-o’cobs bedding (The Andersons Industrial Products Group, Maumee, OH) was used as the nesting material. Chewable Nylabones® were supplied to all rats during the course of the study.
- Diet: Rats were given ad libitum access to Certified Rodent Diet #5002 meal (PMI Nutrition International, Inc., St. Louis, MO) in individual feeders.
- Water: Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: Appr. 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (suspensions) for Groups 1 through 3 were prepared at least once weekly at the Testing Facility. Formulations (suspensions) for Group 4 were prepared at least once daily at the Testing Facility until 10-day stability of this formulation was established, after which Group 4 formulations were prepared at least once weekly at the Testing Facility. All prepared formulations were stored at room temperature, protected from light. Prepared formulations were stirred continuously prior to and during dosage administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Information to document or certify the identity, composition, strength, and activity/purity, of the test substance was provided by the Sponsor to the Testing Facility. Stability data for lower concentrations used in this study was established before the conduct of the study.
Samples of dose formulations were analysed by high-performance liquid chromatography with ultraviolet detection (HPLC-UV). The method was validated for the analysis of dose formulations at concentrations ranging from 1 mg/mL to 100 mg/mL of test substance in corn oil. Stability of the dose formulations for Group IV, 75 mg/mL were evaluated at 20°C to 25°C for 25 hours and 14 days. In addition, the actual concentrations and the relative standard deviation (RSD) of the mean of the average concentration values for the top, middle, and bottom of each dose formulation were calculated to assess homogeneity and the actual concentration was determined.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 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 DG 0

Duration of treatment / exposure:

- P generation male rats were given the test substance and/or the vehicle once daily beginning 84 days before cohabitation, through cohabitation and continuing until the day before scheduled euthanasia for a total of 113 to 116 dosages.
- P generation female rats were given the test substance and/or the vehicle once daily beginning two weeks before cohabitation, through cohabitation, and continuing through DG 25 (rats that did not deliver) or Day 22 postpartum (rats that delivered a litter).
- F1 generation pups were not directly administered the test substance and/or vehicle, but may have possibly been exposed to the test substance and/or the vehicle during maternal gestation (in utero exposure) or via maternal milk during the lactation period.

Frequency of treatment:

once daily

Duration of test:

Males: Day 1 to Day 116
Females: Day 1 to DG 22 or DG 25
F1: PPD1 to PPD 57- 63

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected on the basis of a dosage-range reproduction study. Eight rats per sex per group were dosed via gavage with 0 (Vehicle), 5, 15, 45, or 135 mg/kg/day in a corn oil vehicle. Dosing occurred 14 days prior to cohabitation, through cohabitation, gestation, and postpartum day 5 or the day of euthanasia. There were no treatment-related effects on viability or clinical observations. In male rats, body weights and feed consumption were comparable among all groups. In female rats, treatment with isobornyl acetate reduced body weight gains during the precohabitation period in a non-dosage dependent manner. During gestation, maternal body weight gains were reduced in the 45 and 135 mg/kg/day dosage groups. Body weight gains during the 5-day lactation period were highly variable. Feed consumption was unaffected by treatment. All pregnant rats delivered alitter and there were no effects on natural delivery or litter observations. There were no effects on pup body weights. Based on these data in which borderline maternal toxicity was observed at 45 and 135 mg/kg/day (reduced gestational weight gain), a higher dose of 300 mg/kg/day was selected to ensure that sufficient toxicity will be observed. The middle and low dosages were selected in 3-fold increments.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
All rats were observed for viability twice each day of the study and for clinical observations and general appearance, at least once weekly during the acclimation period. Observations for clinical signs, abortions, premature deliveries and death were made once daily before dosage administration, between one and two hours following dosage administration and at the end of the normal working day during the dosage period, and on the day euthanasia occurred.
Female rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed). Litter size (all pups delivered), and pup viability at birth. Female rats were examined for maternal behavior on DLs 1, 5, 8, 15 18, and 22.

BODY WEIGHT:
Body weights were recorded at least weekly during the acclimation for all rats, and on the first day of dosage administration and weekly thereafter. Body weights were also recorded for female rats at least weekly during the precohabitation and cohabitation periods, and on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11, 15, 18 and 22 (terminal body weight).

FOOD CONSUMPTION
Feed consumption values were recorded weekly during the acclimation period for rats not selected for study, and weekly during the acclimation and dosage periods for all rats selected for study. Values were recorded for female rats on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11 and 15. Because pups begin to consume maternal feed on or about DL 15, feed consumption values were not tabulated after DL 15.

OTHER:
Female rats were examined for maternal behavior on DLs 1, 5, 8, 15 18, and 22

SACRIFICE
- After completion of the 22-day postpartum period, all surviving P generation female rats were euthanized by carbon dioxide asphyxiation. Rats that did not deliver a litter were euthanized by carbon dioxide asphyxiation on DG 25.

GROSS NECROPSY
- A complete necropsy examination was performed in situ, which included an evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain and spinal column; the nasal cavity and neck with associated organs and tissues; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Rats were examined for gross lesions. Gross lesions were retained in neutral buffered 10% formalin (NBF) and shipped to the Principal Investigator for histological examination.

EARLY DEATHS
- Female rats that were euthanized before scheduled termination were examined for the cause of death or condition on the day the observation was made. The rats were examined for gross lesions.
- Tissues were weighed and retained, and microscopic evaluations were conducted . The lungs, trachea and esophagus were perfused and saved in 10% NBF for microscopic evaluation.

HISTOPATHOLOGY
The following tissues from all P generation female rats in the vehicle control and high dosage groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin: Brain, Esophagus, Glands, adrenals, Glands mammary Glands pituitary, Gross lesions/masses, Heart, Kidneys, Liver, Lungs, Ovaries, Spleen, Stomach, Trachea, Uterus with cervix and Vagina. Gross lesions from all P generation female rats in all dosage groups were processed for microscopic evaluation. In addition, tissues collected from any rats that did not survive to scheduled euthanasia were processed and evaluated microscopically. A quantitative evaluation of primordial follicles was conducted for P generation female rats.

ORGAN WEIGHTS
The following organs were weighed at necropsy for all P generation rats that survived to scheduled euthanasia: Brain, Epididymides (individually weighted for scheduled euthanasia), Glands, adrenals, Glands, pituitary, Kidneys, Liver, Ovaries (individually weighted for scheduled euthanasia), and Uterus with cervix. Organ to body weight ratio (using the terminal body weight obtained at necropsy) were calculated.

Ovaries and uterine content:

- Early deaths: Pregnancy status and uterine contents of female rats were recorded (when appropriate). Conceptuses in utero were examined to the extent possible. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites, and retained in 10% NBF for microscopic evaluation.
- Scheduled euthanasia: The number and distribution of implantation sites was recorded.

Statistics:

- Clinical observations and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
- Variables with interval or ratio scales of measurement, such as body weights, feed consumption values and percent mortality per litter were analyzed as described under the Parametric heading of the schematic. Bartlett’s Test of Homogeneity of Variances was used to estimate the probability that the dosage groups have different variances. A nonsignificant result (p> 0.001) indicated that an assumption of homogeneity of variance was
not inappropriate, and the data were compared using the Analysis of Variance. If that test was significant (p≤ 0.05), the groups given the test substance was compared with the control group using Dunnett’s Test. If Bartlett’s Test was significant (p≤ 0.001), the Analysis of Variance Test would not be appropriate, and the data were analyzed as described under the Nonparametric heading of the schematic. When 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis Test was used to analyze the data, and in the event of a significant result (p≤ 0.05), Dunn’s Test was used to compare the groups given the test substance with the control group. When more than 75% of the scores in any dosage group were tied, Fisher’s Exact Test was used to analyze the data.
- Count data were analyzed using the procedures described under the Nonparametric heading of the schematic.
- Group means and standard deviations of primordial follicles was calculated and compared across groups using the Kruskal-Wallis non-parametric ANOVA test. If a significant result occurred (p<0.05), the Wilcoxon (Mann-Whitney U) test was used for pair-wise group comparison to the control group. Statistical analyses were performed using an IBMTM compatible computer with SAS computer programs (Version 9.1.3, Service Pack 4; SAS/STAT User's Guide, 1989).

Indices:

- Litter size and viability [tabulated at birth (day 1) and on days 5, 8, 11, 15, 18 and 22 postpartum].
- Viability Indices (percentages of pups born that survive to days 1 and 5 postpartum).
- Lactation Index (percentage of pups that survive from day 5 to day 22 postpartum).
- Percent Survival an Sex Ratio [tabulated at birth (day 1) and on days 5, 8, 11, 15, 18 and 22 postpartum].

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- The number of P generation female rats with excess salivation (slight and/or moderate) was significantly increased (p≤0.01) in the 300 mg/kg/day dosage group, in comparison to the vehicle control group values. At 300 mg/kg/day, slight excess salivation was observed as early as DS 2 and persisted into the lactation period; the clinical sign was more pronounced during the precohabitation and gestation periods. At 100 mg/kg/day, the number of P generation female rats with slight excess salivation was significantly increased (p≤0.01) only during the gestation dosage period, in comparison to the vehicle control group value.
- In addition, a low incidence of urine-stained abdominal fur (p≤0.01; N=4 out of 25) occurred in the 300 mg/kg/day dosage group during the gestation period. This clinical signs was not considered an adverse finding of isobornyl acetate because the observation was transient and did not persist.
- All other clinical observations during the precohabitation, gestation and lactation periods
were considered unrelated to treatment because: 1) the incidences were not dosage-dependent; and/or 2) the number of rats affected did not differ significantly from the vehicle control group values. These clinical observations included extreme excess salivation, a scab on the mouth, mild dehydration (based on skin turgor), red substance in the cage or cage pan, red and dried red substance on the fur, a broken toenail on the right hindpaw, dried red perioral substance, chromodacryorrhea, missing/broken/misaligned incisors, vocalization in the cage or to touch, chromorhinorrhea, soft or liquid feces, a mass in the left inguinal area or left axilla, purple and/or black appearance to the left inguinal area, a scab present in the left inguinal area and left hindlimb, pale ears, red perivaginal substance, hyperpnea, piloerection, sparse hair coat on the neck or limbs, a portion of the tail was missing, localized alopecia on the limbs, a mass along the left axilla, a swollen third eyelid, ptosis and lacrimation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality related to the test substance occurred during this study. One female rat in each of the 100 and 300 mg/kg/day dosage groups was euthanized during the gestation period because of adverse clinical observations, including an apparent tail injury and signs of dystocia. All other F0 generation female rats survived to scheduled euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Body weights and body weight gains during the precohabitation, gestation and lactation periods were unaffected by dosages as high as 300 mg/kg/day. All values were comparable among the four dosage groups. The following statistically significant findings occurred:
• A statistically significant increase (p≤0.05) in the average body weight at 100 mg/kg/day on DS 8
• Statistically significant increases (p≤0.05 or p≤0.01) in body weight gains at 100 and 300 mg/kg/day on DGs 5 to 8
• A statistically significant increase (p≤0.01) in body weight gains at 300 mg/kg/day for the cumulative gestation period (DGs 1 to 22)
These increases in body weight or body weight gain were considered unrelated to substance because: 1) the increase was not dosage-dependent; and/or 2) the increase reflected an overall reduction in body weight gains or a net loss in body weight in the corresponding vehicle control group.
- Terminal body weights were comparable among the four dosage groups and did not significantly differ from the vehicle control group value.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Absolute (g/day) and relative (g/kg/day) feed consumption values during the precohabitation, gestation and lactation periods were unaffected by dosages of substance as high as 300 mg/kg/day. All values were comparable among the four dosage groups.
- The statistically significant increase (p≤0.05) in absolute and relative feed consumption that occurred in the 100 and 300 mg/kg/day dosage groups on Days 8 to 11 of lactation (DLs 8 to 11) were considered unrelated to isobornyl acetate because the increases were transient and not dosage-dependent.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- The weights of the left and right ovaries and the uterus with cervix and the ratios of these organ weights to terminal body were unaffected by dosages as high as 300 mg/kg/day. In addition, there were no apparent effects on the weights of the nonreproductive organs that were evaluated (i.e., pituitary, brain, liver, paired kidneys and paired adrenals) at scheduled euthanasia.
- The following statistically significant changes occurred: decrease (p≤0.05) in the absolute weight of the brain at 30 mg/kg/day and increase (p≤0.05) in the absolute weight of the left ovary at 100 mg/kg/day. These differences in organ weights were not attributed to administration of the substance: 1) the increase was not dosage-dependent; 2) there was no change in the relative weight of the affected organ; and/or 3) there was no change in the contralateral organ.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related necropsy observations. All gross lesions were considered unrelated to treatment because: 1) the incidences were not dosage-dependent; or 2) the observations occurred in only one rat in any dosage group. These necropsy observations included moderate dilation of the pelvis in the left kidney, a small left kidneys and the presence of tissue in the stomach (presumed cannibalized conceptuses). No other gross lesions occurred in the P generation female rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated rats and were considered unrelated to administration of substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- There were no statistically significant differences (p<0.05) in the number of primordial ovarian follicles in either left, right, or both ovaries combined, in P generation female rats administered 300 mg/kg/day compared to the vehicle control rats. Corpora lutea were present in all rats evaluated.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The averages for implantation sites per delivered litter were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Values for the duration of gestation were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Values for the numbers of dams delivering litters were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Other effects:

no effects observed

Description (incidence and severity):

- Natural delivery and litter observations were unaffected by dosages of substance as high as 300 mg/kg/day.
- The gestation index (number of dams with one or more liveborn pups/number of pregnant rats), the numbers of dams with stillborn pups and of dams with all pups dying, litter sizes, surviving pups per litter, the viability index (number of live pups on day 5 postpartum/ number of liveborn pups on Day 1 postpartum) and the lactation index (number of live pups on Day 22 postpartum/ number of live pups on day 5 postpartum) were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No treatment related adverse effects up to the highest dose tested

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The pup weight per litter were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The gestation index (number of dams with one or more liveborn pups/number of pregnant rats), the numbers of dams with stillborn pups and of dams with all pups dying, litter sizes and surviving pups per litter were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The percentage of male pups per number of pups sexed per litter were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The live litter size at weighing was comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The viability index (number of live pups on day 5 postpartum/ number of liveborn pups on Day 1 postpartum) and the lactation index (number of live pups on Day 22 postpartum/ number of live pups on day 5 postpartum) were comparable among the four dosage groups and did not differ significantly from the vehicle control group values.

External malformations:

no effects observed

Description (incidence and severity):

There were no test substance-related gross lesions observed in the F1 generation pups that survived to scheduled necropsy on day 22 postpartum.

Skeletal malformations:

not examined

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no test substance-related gross lesions observed in the F1 generation pups that survived to scheduled necropsy on day 22 postpartum. One pup in the 300 mg/kg/day dosage group had complete situs inversus

Other effects:

not examined

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Verification of dosing solutions

- Concentration: Mean measured concentrations for all dose formulations were within the acceptable limits (≤15% difference from nominal concentration).

- Homogeneity: Homogeneity was acceptable (≤5% RSD) for all dose formulations.

- Stability: Stability of dose formulations for Group IV, 75 mg/mL were evaluated at 20°C to 25°C for 25 hours and 14 days. Mean measured concentrations for stored samples were within the acceptable limits (±10% of the initial mean measured concentration).

Applicant's summary and conclusion

Conclusions:

Based on the results of this GLP-compliant study according to OECD TG 415, the maternal NOAEL for systemic toxicity is 300 mg/kg/day, because no significant treatment related adverse effects have been observed up to the highest concentration tested. The NOAEL for developmental toxicity is exceeding 300 mg/kg bw/day since no effects on the relevant parameters were observed up to the highest dose tested.

Executive summary:

A one-generation study was performed according to OECD guideline 415 and GLP principles.

Methods

One hundred male and 100 female rats were assigned to four dosage groups, 25 rats per sex per group, for treatment with the test substance. Suspensions of the test substance, or the vehicle, corn oil, were administered via gavage to the male rats once daily beginning 84 days before the cohabitation period, through the cohabitation period (maximum of 14 days) and continuing until the day before euthanasia; and to the female rats once daily beginning 14 days before the cohabitation period, through the cohabitation period (maximum of 14 days) and continuing through the day of euthanasia (through Day 25 of presumed gestation [rats that do not deliver] or Day 22 of lactation [rats that deliver a litter]). Dosage levels were 0 (Vehicle), 30, 100 and 300 mg/kg/day, which was based on a dose-range finder study. The dosage volume was 4 mL/kg, which was adjusted weekly on the basis of the individual body weights. The following parameters were evaluated: P1 generation females: viability, clinical observations, body weights, feed consumption, estrous cycling, mating and fertility, natural delivery and litter observations, organ weights, gross and microscopic observations, and ovarian follicle counts; F1 generation pups/rats: viability, body weights, anogenital distance (days 1 and 22 postpartum), sexual maturation, nipple eruption (day 12 postpartum), and gross observations. Surviving male rats were euthanized after completion of the cohabitation period (Days 114 through 117 of study). Female rats that delivered a litter and pups not selected for continued evaluation were euthanized on Day 22 of lactation (DL 22). Female rats that did not deliver a litter were euthanized on Day 25 of presumed gestation (DG 25). F1 generation rats selected for continued evaluation were euthanized on Days 57 through 63 postpartum.

Results: Analytical verification of dosing solutions

Analytical verification of the dosing solution revealed that all prepared dose formulations were acceptable for use on this study.

Results: P1 Generation Female Rats

No mortality related to the test substance occurred during this study. The number of P1 generation female rats with excess salivation (slight and/or moderate) was significantly increased at 300 mg/kg/day during the premating, gestation and lactation periods, in comparison to the vehicle control group values. At 100 mg/kg/day, the number of P generation female rats with slight excess salivation was significantly increased only during the gestation dosage period, in comparison to the vehicle control group value. In addition, low incidences of urine-stained abdominal fur occurred at 300 mg/kg/day during the gestation period. Body weights, body weight gains and absolute (g/day) and relative (g/kg/day) feed consumption in P generation female rats were unaffected by dosages as high as 300 mg/kg/day prior to mating and during the gestation and lactation periods. In addition, there were no apparent effects on the estrous cycle, mating and fertility parameters or natural delivery and there were no treatment-related gross lesions or microscopic changes at any dosage level tested. Terminal body weights were comparable among the four dosage groups and did not significantly differ from the vehicle control group value. Reproductive (left and right ovaries and the uterus with cervix) and non-reproductive (i.e., pituitary, brain, liver, paired kidneys and paired adrenals) were unaffected by oral administration at dosages as high as 300 mg/kg/day. In addition, there were no microscopic findings, including ovarian follicle evaluation, that were attributed to the test substance at any dosage level.

Results: F1 Generation Rats

No deaths related to the test substance occurred in the F1 generation rats. One F1 generation male rat in the 100 mg/kg/day dosage group was euthanized on day 63 postpartum because of a broken palate. All other F1 generation rats selected for continued evaluation postweaning survived until scheduled euthanasia. There were no treatment-related clinical signs, gross lesions or changes in body weight, body weight gains, feed consumption (g/day or g/kg/day) or organ weights (reproductive or nonreproductive) in the male and female F1 generation rats at any dosage level tested. Anogenital distance on day 1 or 22 postpartum, nipple eruption on day 12 postpartum and sexual maturation was unaffected in either sex following treatment of the P generation male and female rats with dosages of up to and including 300 mg/kg/day.

Conclusion

Based on the results of this study, the NOAEL for maternal systemic toxicity is 300 mg/kg/day. Increased incidences of excess salivation occurred in P1 generation female rats at 100 and/or 300 mg/kg/day throughout the dosage period, and low incidences of urine-stained abdominal fur occurred in females at 300 mg/kg/day during the gestation period. However, these clinical signs were not considered an adverse effect test item. The reproductive NOAEL in the P generation rats and the NOAEL for viability and growth of the F1 generation offspring is greater than or equal to 300 mg/kg/day. There were no apparent effects on estrous cycling, mating and fertility, reproductive organ weights or natural delivery parameters in the P generation, and growth and development (including anogenital distance, nipple eruption or sexual maturation) in the F1 generation rats at the highest dosage level tested (300 mg/kg/day).