Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-07-11 to 2010-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2009-07-11 to 2010-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 415 (One-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Deviations did not affect the outcome or interpretation of this study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: International Flavors and Fragnances, Inc. (Union Beach, NJ); Lot# 2814692
- Expiration date of the lot/batch: August 2010
- Purity test date: August - October 2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, Protected from light
- Stability under test conditions: Stable at room temperature, protected from light

FORM AS APPLIED IN THE TEST (if different from that of starting material): Yellow liquid
Species:
rat
Strain:
Crj: 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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, MI)
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: (P): Males: 72 days, Females: 66 days
- Weight at study initiation: (P) Males: 316-342 g; Females: 201-230 g;
- Fasting period before study: Not specified
- Housing: Upon arrival, P generation rats were assigned to individual housing on the basis of computer-generated random units. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals.
- Diet (e.g. ad libitum): Rats were given ad libitum access to Certified Rodent Diet® #5002 meal (PMI® Nutrition International, Inc., St. Louis, MO) in individual feeders.
- Water (e.g. ad libitum): Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to 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: 7 days
- Sanitization: Cage pan liners were changed at least three times weekly. Cages were changed approximately every other week. Bedding was changed as often as necessary to keep the rats dry and clean.
- Bedding Material: Bed-o'cobs® bedding (The Andersons Industrial Products Group, Maumee, OH) was used as the nesting material.
- Enrichment: Chewable Nylabones® (Nylabone®Products, Neptune, NJ, USA) were supplied to all rats during the course of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64°F to 79°F (18°C to 26°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): minimum of ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters
- Photoperiod (hrs dark / hrs light): An automatically controlled 12-hours light:12-hours dark fluorescent light cycle was maintained. Each dark period began at 1900 (± 30 minutes).

IN-LIFE DATES: From: 2009-08-11 To: 2009-10-02
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
25% active in Corn Oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Suspensions of the test substance were prepared daily at the Testing Facility. Prepared formulations were stored at room temperature, protected from light. Each aliquot was stirred continuously during dosage administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn Oil (Charkit Chemical Corporation (South Norwalk, CT)).
- Concentration in vehicle: 0, 3.125, 6.25, 12.5, and 25 mg/mL
- Amount of vehicle (if gavage): 4 mL/Kg
- Lot/batch no. (if required): J-145
- Purity: Not specified
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 7 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing female rats not mated were considered to be at DG 0 on the last day of cohabitation and assigned to individual housing.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): assigned to individual housing
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration and Homogeneity:
Concentration and homogeneity of the prepared formulations were verified during the course of this study; concentration and homogeneity were assessed for all test article groups (Groups II through V), while only concentration was verified for the vehicle group (Group I). Quadruplicate samples (1 mL each) were taken from the top, middle and bottom of each concentration on the first day prepared. Two samples from each quadruplicate set were shipped for analysis; the remaining samples were retained at the Testing Facility as backup samples. Quadruplicate samples (1 mL each) were taken from each concentration at the mid-point of the study period and on the last day of preparation. Two samples from each quadruplicate set were shipped for analysis; the remaining samples were retained as backup samples. Backup samples were stored and discarded at the Testing Facility following issue of the final report.

Stability:
Stability of the prepared formulations was documented during this study in conjunction with a concentration analysis. Stability was determined for the lowest and highest concentration after storage for at least 10 days.

For the stability condition and timepoint listed in the paragraph above, a quadruplicate set of samples (1 mL each) were taken from the lowest and highest concentrations. All stability samples were shipped for analysis and stored. At the scheduled timepoints after the initial concentration analysis, a duplicate set of samples was analyzed. The remaining stability samples served as backups to be analyzed if the analytical results were not accepted from one of the stability analyses. The backup samples were discarded after issue of the final report.
Duration of treatment / exposure:
P Generation Male Rats
Male rats were given the test substance and/or the vehicle once daily beginning 14 days before cohabitation, through cohabitation, and continuing through the day before sacrifice. The dosage volume was adjusted daily on the basis of individual body weights recorded before administration. Dosages were given at approximately the same time each day.

P Generation Female Rats
Female rats were given the test substance and/or the vehicle once daily beginning 14 days before cohabitation, through cohabitation, and continuing through PPD 4 (rats that delivered a litter) or DS 45. Any dam in the process of parturition was not given the test substance and/or vehicle until the following work day; no dams missed more than one dosagea. The dosage volume was adjusted daily on the basis of individual body weights recorded before administration. Dosages were given at approximately the same time each day.

F1 Generation Litters
Pups were not administered the test substance directly, but may have been exposed in utero during gestation or via maternal milk and maternal feed during the postpartum period.
Frequency of treatment:
Once daily
Details on study schedule:
P Generation Male and Female Rats
Within each dosage group, consecutive order was used to assign P generation rats to cohabitation, one male rat per female rat. The cohabitation period consisted of a maximum of seven days. 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 and assigned to individual housing. Female rats not mated after completion of the 7-day cohabitation period were considered to be at DG 0 on the last day of cohabitation and assigned to individual housing.

Rats were observed for viability at least twice each day of the study and for clinical observations and general appearance at least weekly during the acclimation period.

On the first five days of dosage, clinical observations were recorded prior to dosage administration and at approximately hourly intervals for the first four hours after dosage administration, and at the end of the normal working day. Beginning on the sixth day of dosage administration, clinical observations were recorded prior to dosage administration, between one and two hours after dosage administration and at the end of the normal working day as well as once on the day sacrifice occurred.a In addition to clinical observations, female rats were observed for abortions and premature deliveries. During the postdosage periods, clinical observations were recorded once daily.

Body weights for male and female rats were recorded at least weekly during the acclimation period, daily during the dosage period and on the day sacrifice occurred. Feed consumption values for male rats were recorded at least weekly during the acclimation and dosage periods, except during cohabitation, and on the day sacrifice occurred.a Feed consumption values for female rats were recorded at least weekly during the acclimation and precohabitation periods, on DGs 0, 7, 8, 9, 10, 14, 18, 21 and 25, and on PPDs 1 and 5.

Estrous cycling was evaluated by examination of vaginal cytology for 14 days before cohabitation, and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. Rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter sizes (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on PPDs 1 and 5.

F1 Generation Litters
Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam).

Each litter was evaluated for viability at least twice daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily. Pup body weights were recorded on PPDs 1 and 5.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control (vehicle)
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
8/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: There were no available data on developmental or reproductive effects of hexyl cinnamic aldehyde in rats. In a 14-day repeat-dose study, hexyl cinnamic aldehyde was administered orally by gavage to 2 rats/sex/dosage at 150, 375, 750, 1000, and 1500 mg/kg/day. One male died after three days of treatment at 1500 mg/kg/day. Profuse salivation occurred in the majority of animals following daily gavage. Body weights did not appear to be affected by the administration of hexyl cinnamic aldehyde at the dose levels investigated. Gross pathological observation revealed mild to moderate irritation of the gastric mucosa in all groups with the exception of three animals treated with 150 mg/kg/day.

In a 90-day subchronic dermal study in 5 rats/sex at 25 mg/kg/day hexyl cinnamic aldehyde, no effects were observed in the limited tissues examined. In a second 90-day subchronic dermal study, 15 rats/sex/dosage were administered 125, 250, 500, or 1000 mg/kg/day hexyl cinnamic aldehyde. Dose-dependent dermal irritation was observed. Mortality was observed at the 1000 mg/kg/day dosage. Body weights were significantly decreased at 500 and 1000 mg/kg/day. Relative food consumption was increased at all dosage levels. Gross examination at necropsy revealed dose-related irritation of the gastrointestinal tract mucosa. The liver and kidney weights of treated females were significantly increased at 250, 500, and 1000 mg/kg/day. Histopathological examination revealed morphological alterations at 1000 mg/kg/day.

Due to the gastric irritation observed, the dosages of 12.5, 25, 50, and 100 mg/kg/day were selected for this dosage-range finding study.

- Rationale for animal assignment (if not random):
P Generation Rats:
Upon arrival, P generation rats were assigned to individual housing on the basis of computer-generated random units. After acclimation, rats were selected for study on the basis of physical appearance and body weight. The rats were assigned to five dosage groups (Groups I through V), eight rats per sex per group, using a computer-generated (weight-ordered) randomization procedure.

F1 Generation Litters:
Litters were not culled during the lactation period, because random selection of pups for culling could have resulted in potential biases in pup viabilities and body weight gains during this period.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least weekly during the acclimation period. On the first five days of dosage, clinical observations were recorded prior to dosage administration and at approximately hourly intervals for the first four hours after dosage administration, and at the end of the normal working day. Beginning on the sixth day of dosage administration, clinical observations were recorded prior to dosage administration, between one and two hours after dosage administration and at the end of the normal working day as well as once on the day sacrifice occurred. In addition to clinical observations, female rats were observed for abortions and premature deliveries. During the postdosage periods, clinical observations were recorded once daily.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
Feed consumption values for male rats were recorded at least weekly during the acclimation and dosage periods, except during cohabitation, and on the day sacrifice occurred.a Feed consumption values for female rats were recorded at least weekly during the acclimation and precohabitation periods, on DGs 0, 7, 8, 9, 10, 14, 18, 21 and 25, and on PPDs 1 and 5.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Estrous cyclicity (parental animals):
Estrous cycling was evaluated by examination of vaginal cytology for 14 days before cohabitation, and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period Rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter sizes (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on PPDs 1 and 5.
Sperm parameters (parental animals):
Testes and epididymides were excised and individual organ weights were recorded. The epididymides were retained in NBF. The testes were fixed in modified Davidson’s fixative, rinsed per Testing Facility SOP, and then retained in ethyl alcohol.
Litter observations:
Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam). Each litter was evaluated for viability at least twice daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily. Pup body weights were recorded on PPDs 1 and 5.
Postmortem examinations (parental animals):
P generation rats were sacrificed by carbon dioxide asphyxiation.

After completion of the cohabitation period, all male rats were sacrificed and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Testes and epididymides were excised and individual organ weights were recorded. The epididymides were retained in NBF. The testes were fixed in modified Davidson’s fixative, rinsed per Testing Facility SOP, and then retained in ethyl alcohol.

Female rats that delivered a litter were sacrificed after completion of the 5-day postpartum period. The number and distribution of implantation sites were recorded. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed.

Female rats that did not deliver a litter were sacrificed on an estimated DG 25. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed, and the rats were examined for pregnancy status. Uteri of apparently nonpregnant rats were examined, while being pressed between glass plates, to confirm the absence of implantation sites; uteri and ovaries were retained in NBF for possible future evaluation.

The dam with no surviving pups was sacrificed after the last pup was found dead. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed; the number and distribution of implantation sites were recorded. The carcass was discarded without further evaluation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [2] were prepared for microscopic examination and weighed, respectively.

The tissues identified in the Tissue Collection and Preservation table (Table 2) were collected from all P generation rats and preserved in 10% neutral buffered formalin (unless otherwise indicated). Tissues were shipped (ambient conditions) for processing at Charles River Laboratories Pathology Associates - Maryland. Tissues identified for microscopic evaluation in the Tissue Collection and Preservation table (i.e., male and female reproductive organs and gross lesions) from all 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.Histopathological evaluation was performed by a board-certified veterinary pathologist. All gross lesions from rats in all dosage groups were evaluated microscopically.
Postmortem examinations (offspring):
F1 generation pups were sacrificed by an intraperitoneal injection of sodium pentobarbital.

All surviving pups were sacrificed on PPD 5 and examined for gross lesions. 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.

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 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, except when precluded by autolysis.
Statistics:
Data generated during the course of this study were recorded either by hand or using the Argus Automated Data Collection and Management System. All data were tabulated and/or summarized using the Argus Automated Data Collection and Management System, Microsoft® Excel (part of Microsoft® Office 97/2000/2003/XP) and Quattro Pro 8. Averages and percentages were calculated. Litter values were used where appropriate.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Males: clinical observations included ungroomed coat, sparse hair coat or localized alopecia on the limbs, light brown feces, chromorhinorrhea, soft or liquid feces, rales and vocalization to touch. No other clinical signs occurred in the treated male rats.
All clinical observations were considered unrelated to treatment with hexyl cinnamic aldehyde because:
1) most observations occurred sporadically during the dosage period;
2) the observations were independent of dose; and
3) the observations occurred in only one or three rats in a dosage group.

Females: clinical observations included localized alopecia on the limbs, slight excess salivation, soft or liquid feces, mild dehydration (based on skin turgor), sparse hair coat on the limbs, chromorhinorrhea and red perivaginal substance. No other clinical observations occurred. All clinical signs observed during the precohabitation, gestation and lactation periods were considered unrelated to treatment with hexyl cinnamic aldehyde because:
1) none of the observations occurred in more than one or two rats in any dosage group; and/or
2) the observations were independent of dose. These
Mortality:
no mortality observed
Description (incidence):
All P generation male and female rats survived until scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males: Body weights and body weight gains of the treated P generation male rats were generally comparable among the dosage groups. Body weight gains in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 95%, 104%, 85% and 92% of the vehicle control group value, respectively, for the first two weeks of the dosage period (DSs 1 to 14). Overall, for the entire dosage period (DSs 1 to 47), body weight gains in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 85%, 97%, 98% and 88% of the vehicle control group value, respectively. The average body weight for male rats in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 97%, 101%, 101% and 97% of the vehicle control group value, respectively, on DS 47.

Females:
Precohabitation: Body weight gains were increased during the second week of the precohabitation dosage period in the 100 mg/kg/day dosage group, as compared to the vehicle control group value. As a result, body weight gains in the 100 mg/kg/day were increased by 14% for the entire premating period (DSs 1 to 14), in comparison to the vehicle control group value. These increases in body weight gain were not considered adverse because there was no corresponding effect on feed consumption, and the increase in weight gain did not persist during the gestation period. Body weight gains in the 12.5, 25 and 50 mg/kg/day dosage groups were 95%, 105% and 100% of the vehicle control group value, respectively, on DSs 1 to 14. The average body weight on DS 14 was 99%, 101%, 99% and 100% of the vehicle control group value in the four respective hexyl cinnamic aldehyde-treated groups.

Gestation: Body weights and body weight gains of the female rats were unaffected by treatment with Hexyl cinnamic aldehyde during the gestation period. Body weight gains in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 93%, 97%, 89% and 101% of the vehicle control group value, respectively, on DGs 0 to 21. The average body weight on DG 21 was 99%, 103%, 98% and 104% of the vehicle control group value in the four respective hexyl cinnamic aldehyde-treated groups.

Lactation: Maternal body weight gains in the 100 mg/kg/day dosage group were reduced on days 1 to 5 of lactation (DLs 1 to 5), in comparison to the vehicle control group value. Body weight gains in the 100 mg/kg/day dosage group were 78% lower than the vehicle control group value.There were no other apparent trends in body weight gains during the five-day lactation period. Body weight gains in the 12.5, 25 and 50 mg/kg/day dosage groups were 148%, 176% and 167% of the vehicle control group value, respectively, on DLs 1 to 5. The average body weight on DL 5 was 98%, 105%, 100% and 99% of the vehicle control group value in the four respective hexyl cinnamic aldehyde-treated groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males: Absolute (g/day) and relative (g/kg/day) feed consumption values were comparable among the dosage groups for the first two weeks of the dosage period (DSs 1 to 14) and overall for the entire dosage period (DSs 1 to 47), in comparison to the vehicle control group values. Absolute feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 102%, 100%, 93% and 97% of the vehicle control group value, respectively, for the first two weeks of the dosage period (DSs 1 to 14). Overall, for the entire dosage period (DSs 1 to 47), absolute feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 101%, 99%, 100% and 99% of the vehicle control group value, respectively.

Females:
Precohabitation: Absolute and relative feed consumption values were unaffected by treatment with Hexyl cinnamic aldehyde during the precohabitation period. Absolute feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 100%, 100%, 94% and 102% of the vehicle control group value, respectively, on DSs 1 to 14. Relative feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 99%, 100%, 94% and 103% of the vehicle control group value, respectively, on DSs 1 to 14.

Gestation: Absolute and relative feed consumption values were unaffected by treatment with hexyl cinnamic aldehyde during the gestation period. Absolute feed consumption values in the 12.5, 25, 50 and 100mg/kg/day dosage groups were 93%, 101%, 95% and 114% of the vehicle control group value, respectively, on DGs 0 to 21. Relative feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 93%, 97%, 95% and 109% of the vehicle control group value, respectively, on DGs 0 to 21.

Lactation: Absolute and relative feed consumption values in the 100 mg/kg/day dosage group were reduced on DLs 1 to 5, in comparison to the vehicle control group values. Absolute and relative feed consumption values in the 100 mg/kg/day dosage group were 84% of the vehicle control group value on DLs 1 to 5. Absolute and relative feed consumption values were unaffected by dosages of hexyl cinnamic aldehyde as high as 50 mg/kg/day during the lactation period. Absolute feed consumption values in the 12.5, 25 and 50 mg/kg/day dosage groups were 95%, 107% and 94% of the vehicle control group value, respectively, on DLs 1 to 5. Relative feed consumption values in the 12.5, 25 and 50 mg/kg/day dosage groups were 98%, 104% and 98% of the vehicle control group value, respectively, on DLs 1 to 5.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
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:
no effects observed
Description (incidence and severity):
Males: Terminal body weights were comparable among the five dosage groups. The weights of the epididymides, testes, seminal vesicles (with and without fluid) and prostate and the ratios of these organ weights to terminal body weight were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 mg/kg/day.

Females: Terminal body weights were comparable among the five dosage groups. The weights of the paired ovaries and non-gravid uterus (with cervix) and the ratios of these organ weights to terminal body weight were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: There were no treatment-related gross lesions at any dosage level tested. One rat in each of the 0 (Vehicle) and 12.5 mg/kg/day dosage groups had slight or moderate dilation of the pelvis in the right kidney. One rat in the 100 mg/kg/day dosage group had two firm areas on the caudal portion of the prostate. No other gross lesions occurred.

Females: There were no treatment-related gross lesions at any dosage level tested. One rat in the vehicle control group had a bursal cyst on the right ovary. One rat in the 50 mg/kg/day dosage group had fetal tissue present in the stomach, a finding commonly associated with cannibalization. No other gross lesions occurred.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
None of the microscopic findings that occurred were considered related to hexyl cinnamic aldehyde. All changes observed in the P generation rats were considered to be incidental or spontaneous changes that are commonly observed in control Crl:CD(SD) rats
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
The number of estrous stages per 14 days was comparable among the five dosage groups during the precohabitation period.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating and Fertilty:

Males:
All mating and fertility parameters [the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 mg/kg/day.

The apparent increase in the average number of days to mating and the reduction in the percentage of rats that mated that occurred in the 100 mg/kg/day dosage group was attributed to two male rats (no. 3433 and 3436) that did not mate with their cohort female
rats (no. 3473 and 3476, respectively). These effects on mating and fertility were not attributed to treatment with hexyl cinnamic aldehyde because:
1) there was no apparent effect on male or female reproductive organ weights;
2) the remaining rats in the 100 mg/kg/day dosage group (N=6) mated and had viable litters;
3) there were not apparent effects on natural delivery in this dosage group; and
4) the average value (3.4 ± 2.3 days) was within the historical range of the Testing Facility

Females:
All mating and fertility parameters [the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages of hexyl
cinnamic aldehyde as high as 100 mg/kg/day.

The apparent increase in the average number of days to mating and the reduction in the percentage of rats that mated that occurred in the 100 mg/kg/day dosage group was attributed to two female rats (no. 3473 and 3476) that did not mate with their cohort male
rats (no. 3433 and 3436, respectively). These effects on mating and fertility were not attributed to treatment with hexyl cinnamic aldehyde because:
1) there was no apparent effect on male or female reproductive organ weights;
2) the remaining rats in the 100 mg/kg/day dosage group (N=6) mated and had viable litters;
3) there were not apparent effects on natural delivery in this dosage group; and
4) the average value (3.4 ± 2.3 days) was within the historical range of the Testing Facility
Natural Delivery Observations:
All pregnant rats (7, 8, 7, 8 and 6 rats in the five respective dosage groups) delivered a litter.

Natural delivery and litter observations were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 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, viability index, surviving pups per litter, percent male pups per number of pups sexed per litter, live litter size at weighing and pup weight per litter were comparable among the five dosage groups. One dam (no. 3468) in the 50 mg/kg/day dosage group had no liveborn pups. However, it could not be determined whether the single pup was liveborn or not because partial cannibalization precluded determination of viability.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment-related effects observed at the highest dose tested.
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical observations occurred in the F1 generation pups. Transient clinical signs that occurred in the F1 generation pups included mild dehydration (based on skin turgor), gasping, a pale appearance to the entire body and discoloration in one or
more areas of the body.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related necropsy observations occurred in the F1 generation pups. All pups that died early or survived to scheduled sacrificed appeared normal at necropsy examination.
No treatment-related clinical observations occurred in the F1 generation pups. Transient clinical signs that occurred in the F1 generation pups included mild dehydration (based on skin turgor), gasping, a pale appearance to the entire body and discoloration in one or more areas of the body. In addition, no treatment-related necropsy observations occurred in the F1 generation pups. All pups that died early or survived to scheduled sacrificed appeared normal at necropsy examination
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Dose Formulation Analyses

Mean measured hexyl cinnamic aldehyde concentrations for all dose formulations were within the acceptable limits (±15% of nominal concentration). Homogeneity was acceptable (≤ 5% RSD) for all concentrations prepared at the start of the study. The values obtained were 0.2%, 0.6%, 0.4% and 0.6% RSD for the 3.125, 6.25, 12.5 and 25 mg/mL concentrations, respectively. Stability of Hexyl cinnamic aldehyde formulations at concentrations of 3.125 mg/mL and 25 mg/mL was evaluated for storage at room temperature, protected from light for 14 days. Mean measured concentrations for stored samples for stability were within the acceptable limits (±10% of the initial mean measured concentration).

Conclusions:
Based on a lack of treatment-related effects observed, the NOAEL for systemic toxicity and the NOEL for reproductive toxicity were determined to be 100 mg/Kg/day.
Executive summary:

In a key read across one-generation reproductive toxicity study, the test material (hexyl cinnamic aldehyde; CAS# 101-86-0) was administered via oral gavage to Crl:CD(SD) rats (8/sex/dose) at doses of 0, 12.5, 25, 50 and 100 mg/Kg/day in a corn oil vehicle. Male rats were administered the test material and/or the vehicle once daily beginning 14 days before cohabitation, through cohabitation, and continuing through the day before sacrifice. Female rats were given hexyl cinnamic aldehyde and/or the vehicle once daily beginning two weeks before cohabitation, through cohabitation, and continuing through the day before sacrifice. Male rats were sacrificed after completion of the dosage period. Female rats were allowed to deliver their litters and were sacrificed on PPD 5. F1 generation pups were sacrificed on PPD 5.

 

The following parameters were evaluated: viability, clinical observations, body weights, feed weights, mating and fertility, delivery and litter observations, organ weights, necropsy observations and histopathology.

 

All parental (P) generation male and female rats survived to scheduled sacrifice. There were no treatment-related clinical observations or gross lesions in the P generation male and female rats at any dosage level tested. In addition, none of the microscopic findings that occurred were considered related to treatment with hexyl cinnamic aldehyde.

 

Body weights and body weight gains of the treated P generation male rats were generally comparable among the dosage groups. In treated P generation female rats, body weight gains were increased during the second week of the precohabitation dosage period in the 100 mg/Kg/day dosage group, and overall for the entire premating period (14% greater than controls). These increases in body weight gain were not considered adverse because there was no corresponding effect on feed consumption, and the increase in weight gain did not persist during the gestation period.

 

Body weights and body weight gains of the female rats were unaffected by treatment with the test material during the gestation period. During the lactation period, maternal body weight gains in the 100 mg/Kg/day dosage group were reduced on days 1 to 5 of lactation (DLs 1 to 5), in comparison to the vehicle control group value. Body weight gains in the 100 mg/Kg/day dosage group were 78% lower than the vehicle control group value.

 

Absolute and relative feed consumption values in male rats were unaffected by treatment with hexyl cinnamic aldehyde during the entire dosage period. In treated P generation female rats, absolute and relative feed consumption values were unaffected by treatment with hexyl cinnamic aldehyde during the precohabitation and gestation periods. Corresponding to reduced body weight gains during the lactation period, feed consumption values (g/day and g/Kg/day) in the 100 mg/Kg/day dosage group were reduced on DLs 1 to 5, in comparison to the vehicle control group values (84% of controls).

 

Terminal body weights and male and female reproductive organ weights were comparable among the five dosage groups. There was no effect of hexyl cinnamic aldehyde on estrous cycling or mating and fertility at any dosage level tested. All pregnant rats (7, 8, 7, 8 and 6 rats in the five respective dosage groups) delivered a litter. Natural delivery and litter observations were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 mg/Kg/day. No treatment-related clinical or necropsy observations occurred in the F1 generation pups.

 

Based on the results observed, the NOAEL for systemic toxicity and the NOEL for reproductive toxicity were determined to be 100 mg/Kg/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 (One-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Deviations did not affect the outcome or interpretation of this study
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: International Flavors and Fragnances, Inc. (Union Beach, NJ); Lot# 2814692
- Expiration date of the lot/batch: August 2010
- Purity test date: August - October 2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, Protected from light
- Stability under test conditions: Stable at room temperature, protected from light

FORM AS APPLIED IN THE TEST (if different from that of starting material): Yellow liquid

Test animals

Species:
rat
Strain:
Crj: 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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, MI)
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: (P): Males: 72 days, Females: 66 days
- Weight at study initiation: (P) Males: 316-342 g; Females: 201-230 g;
- Fasting period before study: Not specified
- Housing: Upon arrival, P generation rats were assigned to individual housing on the basis of computer-generated random units. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals.
- Diet (e.g. ad libitum): Rats were given ad libitum access to Certified Rodent Diet® #5002 meal (PMI® Nutrition International, Inc., St. Louis, MO) in individual feeders.
- Water (e.g. ad libitum): Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to 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: 7 days
- Sanitization: Cage pan liners were changed at least three times weekly. Cages were changed approximately every other week. Bedding was changed as often as necessary to keep the rats dry and clean.
- Bedding Material: Bed-o'cobs® bedding (The Andersons Industrial Products Group, Maumee, OH) was used as the nesting material.
- Enrichment: Chewable Nylabones® (Nylabone®Products, Neptune, NJ, USA) were supplied to all rats during the course of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64°F to 79°F (18°C to 26°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): minimum of ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters
- Photoperiod (hrs dark / hrs light): An automatically controlled 12-hours light:12-hours dark fluorescent light cycle was maintained. Each dark period began at 1900 (± 30 minutes).

IN-LIFE DATES: From: 2009-08-11 To: 2009-10-02

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
25% active in Corn Oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Suspensions of the test substance were prepared daily at the Testing Facility. Prepared formulations were stored at room temperature, protected from light. Each aliquot was stirred continuously during dosage administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn Oil (Charkit Chemical Corporation (South Norwalk, CT)).
- Concentration in vehicle: 0, 3.125, 6.25, 12.5, and 25 mg/mL
- Amount of vehicle (if gavage): 4 mL/Kg
- Lot/batch no. (if required): J-145
- Purity: Not specified
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 7 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing female rats not mated were considered to be at DG 0 on the last day of cohabitation and assigned to individual housing.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): assigned to individual housing
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration and Homogeneity:
Concentration and homogeneity of the prepared formulations were verified during the course of this study; concentration and homogeneity were assessed for all test article groups (Groups II through V), while only concentration was verified for the vehicle group (Group I). Quadruplicate samples (1 mL each) were taken from the top, middle and bottom of each concentration on the first day prepared. Two samples from each quadruplicate set were shipped for analysis; the remaining samples were retained at the Testing Facility as backup samples. Quadruplicate samples (1 mL each) were taken from each concentration at the mid-point of the study period and on the last day of preparation. Two samples from each quadruplicate set were shipped for analysis; the remaining samples were retained as backup samples. Backup samples were stored and discarded at the Testing Facility following issue of the final report.

Stability:
Stability of the prepared formulations was documented during this study in conjunction with a concentration analysis. Stability was determined for the lowest and highest concentration after storage for at least 10 days.

For the stability condition and timepoint listed in the paragraph above, a quadruplicate set of samples (1 mL each) were taken from the lowest and highest concentrations. All stability samples were shipped for analysis and stored. At the scheduled timepoints after the initial concentration analysis, a duplicate set of samples was analyzed. The remaining stability samples served as backups to be analyzed if the analytical results were not accepted from one of the stability analyses. The backup samples were discarded after issue of the final report.
Duration of treatment / exposure:
P Generation Male Rats
Male rats were given the test substance and/or the vehicle once daily beginning 14 days before cohabitation, through cohabitation, and continuing through the day before sacrifice. The dosage volume was adjusted daily on the basis of individual body weights recorded before administration. Dosages were given at approximately the same time each day.

P Generation Female Rats
Female rats were given the test substance and/or the vehicle once daily beginning 14 days before cohabitation, through cohabitation, and continuing through PPD 4 (rats that delivered a litter) or DS 45. Any dam in the process of parturition was not given the test substance and/or vehicle until the following work day; no dams missed more than one dosagea. The dosage volume was adjusted daily on the basis of individual body weights recorded before administration. Dosages were given at approximately the same time each day.

F1 Generation Litters
Pups were not administered the test substance directly, but may have been exposed in utero during gestation or via maternal milk and maternal feed during the postpartum period.
Frequency of treatment:
Once daily
Details on study schedule:
P Generation Male and Female Rats
Within each dosage group, consecutive order was used to assign P generation rats to cohabitation, one male rat per female rat. The cohabitation period consisted of a maximum of seven days. 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 and assigned to individual housing. Female rats not mated after completion of the 7-day cohabitation period were considered to be at DG 0 on the last day of cohabitation and assigned to individual housing.

Rats were observed for viability at least twice each day of the study and for clinical observations and general appearance at least weekly during the acclimation period.

On the first five days of dosage, clinical observations were recorded prior to dosage administration and at approximately hourly intervals for the first four hours after dosage administration, and at the end of the normal working day. Beginning on the sixth day of dosage administration, clinical observations were recorded prior to dosage administration, between one and two hours after dosage administration and at the end of the normal working day as well as once on the day sacrifice occurred.a In addition to clinical observations, female rats were observed for abortions and premature deliveries. During the postdosage periods, clinical observations were recorded once daily.

Body weights for male and female rats were recorded at least weekly during the acclimation period, daily during the dosage period and on the day sacrifice occurred. Feed consumption values for male rats were recorded at least weekly during the acclimation and dosage periods, except during cohabitation, and on the day sacrifice occurred.a Feed consumption values for female rats were recorded at least weekly during the acclimation and precohabitation periods, on DGs 0, 7, 8, 9, 10, 14, 18, 21 and 25, and on PPDs 1 and 5.

Estrous cycling was evaluated by examination of vaginal cytology for 14 days before cohabitation, and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. Rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter sizes (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on PPDs 1 and 5.

F1 Generation Litters
Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam).

Each litter was evaluated for viability at least twice daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily. Pup body weights were recorded on PPDs 1 and 5.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control (vehicle)
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
8/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: There were no available data on developmental or reproductive effects of hexyl cinnamic aldehyde in rats. In a 14-day repeat-dose study, hexyl cinnamic aldehyde was administered orally by gavage to 2 rats/sex/dosage at 150, 375, 750, 1000, and 1500 mg/kg/day. One male died after three days of treatment at 1500 mg/kg/day. Profuse salivation occurred in the majority of animals following daily gavage. Body weights did not appear to be affected by the administration of hexyl cinnamic aldehyde at the dose levels investigated. Gross pathological observation revealed mild to moderate irritation of the gastric mucosa in all groups with the exception of three animals treated with 150 mg/kg/day.

In a 90-day subchronic dermal study in 5 rats/sex at 25 mg/kg/day hexyl cinnamic aldehyde, no effects were observed in the limited tissues examined. In a second 90-day subchronic dermal study, 15 rats/sex/dosage were administered 125, 250, 500, or 1000 mg/kg/day hexyl cinnamic aldehyde. Dose-dependent dermal irritation was observed. Mortality was observed at the 1000 mg/kg/day dosage. Body weights were significantly decreased at 500 and 1000 mg/kg/day. Relative food consumption was increased at all dosage levels. Gross examination at necropsy revealed dose-related irritation of the gastrointestinal tract mucosa. The liver and kidney weights of treated females were significantly increased at 250, 500, and 1000 mg/kg/day. Histopathological examination revealed morphological alterations at 1000 mg/kg/day.

Due to the gastric irritation observed, the dosages of 12.5, 25, 50, and 100 mg/kg/day were selected for this dosage-range finding study.

- Rationale for animal assignment (if not random):
P Generation Rats:
Upon arrival, P generation rats were assigned to individual housing on the basis of computer-generated random units. After acclimation, rats were selected for study on the basis of physical appearance and body weight. The rats were assigned to five dosage groups (Groups I through V), eight rats per sex per group, using a computer-generated (weight-ordered) randomization procedure.

F1 Generation Litters:
Litters were not culled during the lactation period, because random selection of pups for culling could have resulted in potential biases in pup viabilities and body weight gains during this period.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least weekly during the acclimation period. On the first five days of dosage, clinical observations were recorded prior to dosage administration and at approximately hourly intervals for the first four hours after dosage administration, and at the end of the normal working day. Beginning on the sixth day of dosage administration, clinical observations were recorded prior to dosage administration, between one and two hours after dosage administration and at the end of the normal working day as well as once on the day sacrifice occurred. In addition to clinical observations, female rats were observed for abortions and premature deliveries. During the postdosage periods, clinical observations were recorded once daily.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
Feed consumption values for male rats were recorded at least weekly during the acclimation and dosage periods, except during cohabitation, and on the day sacrifice occurred.a Feed consumption values for female rats were recorded at least weekly during the acclimation and precohabitation periods, on DGs 0, 7, 8, 9, 10, 14, 18, 21 and 25, and on PPDs 1 and 5.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Estrous cyclicity (parental animals):
Estrous cycling was evaluated by examination of vaginal cytology for 14 days before cohabitation, and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period Rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter sizes (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on PPDs 1 and 5.
Sperm parameters (parental animals):
Testes and epididymides were excised and individual organ weights were recorded. The epididymides were retained in NBF. The testes were fixed in modified Davidson’s fixative, rinsed per Testing Facility SOP, and then retained in ethyl alcohol.
Litter observations:
Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam). Each litter was evaluated for viability at least twice daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily. Pup body weights were recorded on PPDs 1 and 5.
Postmortem examinations (parental animals):
P generation rats were sacrificed by carbon dioxide asphyxiation.

After completion of the cohabitation period, all male rats were sacrificed and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Testes and epididymides were excised and individual organ weights were recorded. The epididymides were retained in NBF. The testes were fixed in modified Davidson’s fixative, rinsed per Testing Facility SOP, and then retained in ethyl alcohol.

Female rats that delivered a litter were sacrificed after completion of the 5-day postpartum period. The number and distribution of implantation sites were recorded. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed.

Female rats that did not deliver a litter were sacrificed on an estimated DG 25. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed, and the rats were examined for pregnancy status. Uteri of apparently nonpregnant rats were examined, while being pressed between glass plates, to confirm the absence of implantation sites; uteri and ovaries were retained in NBF for possible future evaluation.

The dam with no surviving pups was sacrificed after the last pup was found dead. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed; the number and distribution of implantation sites were recorded. The carcass was discarded without further evaluation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [2] were prepared for microscopic examination and weighed, respectively.

The tissues identified in the Tissue Collection and Preservation table (Table 2) were collected from all P generation rats and preserved in 10% neutral buffered formalin (unless otherwise indicated). Tissues were shipped (ambient conditions) for processing at Charles River Laboratories Pathology Associates - Maryland. Tissues identified for microscopic evaluation in the Tissue Collection and Preservation table (i.e., male and female reproductive organs and gross lesions) from all 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.Histopathological evaluation was performed by a board-certified veterinary pathologist. All gross lesions from rats in all dosage groups were evaluated microscopically.
Postmortem examinations (offspring):
F1 generation pups were sacrificed by an intraperitoneal injection of sodium pentobarbital.

All surviving pups were sacrificed on PPD 5 and examined for gross lesions. 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.

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 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, except when precluded by autolysis.
Statistics:
Data generated during the course of this study were recorded either by hand or using the Argus Automated Data Collection and Management System. All data were tabulated and/or summarized using the Argus Automated Data Collection and Management System, Microsoft® Excel (part of Microsoft® Office 97/2000/2003/XP) and Quattro Pro 8. Averages and percentages were calculated. Litter values were used where appropriate.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Males: clinical observations included ungroomed coat, sparse hair coat or localized alopecia on the limbs, light brown feces, chromorhinorrhea, soft or liquid feces, rales and vocalization to touch. No other clinical signs occurred in the treated male rats.
All clinical observations were considered unrelated to treatment with hexyl cinnamic aldehyde because:
1) most observations occurred sporadically during the dosage period;
2) the observations were independent of dose; and
3) the observations occurred in only one or three rats in a dosage group.

Females: clinical observations included localized alopecia on the limbs, slight excess salivation, soft or liquid feces, mild dehydration (based on skin turgor), sparse hair coat on the limbs, chromorhinorrhea and red perivaginal substance. No other clinical observations occurred. All clinical signs observed during the precohabitation, gestation and lactation periods were considered unrelated to treatment with hexyl cinnamic aldehyde because:
1) none of the observations occurred in more than one or two rats in any dosage group; and/or
2) the observations were independent of dose. These
Mortality:
no mortality observed
Description (incidence):
All P generation male and female rats survived until scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males: Body weights and body weight gains of the treated P generation male rats were generally comparable among the dosage groups. Body weight gains in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 95%, 104%, 85% and 92% of the vehicle control group value, respectively, for the first two weeks of the dosage period (DSs 1 to 14). Overall, for the entire dosage period (DSs 1 to 47), body weight gains in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 85%, 97%, 98% and 88% of the vehicle control group value, respectively. The average body weight for male rats in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 97%, 101%, 101% and 97% of the vehicle control group value, respectively, on DS 47.

Females:
Precohabitation: Body weight gains were increased during the second week of the precohabitation dosage period in the 100 mg/kg/day dosage group, as compared to the vehicle control group value. As a result, body weight gains in the 100 mg/kg/day were increased by 14% for the entire premating period (DSs 1 to 14), in comparison to the vehicle control group value. These increases in body weight gain were not considered adverse because there was no corresponding effect on feed consumption, and the increase in weight gain did not persist during the gestation period. Body weight gains in the 12.5, 25 and 50 mg/kg/day dosage groups were 95%, 105% and 100% of the vehicle control group value, respectively, on DSs 1 to 14. The average body weight on DS 14 was 99%, 101%, 99% and 100% of the vehicle control group value in the four respective hexyl cinnamic aldehyde-treated groups.

Gestation: Body weights and body weight gains of the female rats were unaffected by treatment with Hexyl cinnamic aldehyde during the gestation period. Body weight gains in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 93%, 97%, 89% and 101% of the vehicle control group value, respectively, on DGs 0 to 21. The average body weight on DG 21 was 99%, 103%, 98% and 104% of the vehicle control group value in the four respective hexyl cinnamic aldehyde-treated groups.

Lactation: Maternal body weight gains in the 100 mg/kg/day dosage group were reduced on days 1 to 5 of lactation (DLs 1 to 5), in comparison to the vehicle control group value. Body weight gains in the 100 mg/kg/day dosage group were 78% lower than the vehicle control group value.There were no other apparent trends in body weight gains during the five-day lactation period. Body weight gains in the 12.5, 25 and 50 mg/kg/day dosage groups were 148%, 176% and 167% of the vehicle control group value, respectively, on DLs 1 to 5. The average body weight on DL 5 was 98%, 105%, 100% and 99% of the vehicle control group value in the four respective hexyl cinnamic aldehyde-treated groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males: Absolute (g/day) and relative (g/kg/day) feed consumption values were comparable among the dosage groups for the first two weeks of the dosage period (DSs 1 to 14) and overall for the entire dosage period (DSs 1 to 47), in comparison to the vehicle control group values. Absolute feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 102%, 100%, 93% and 97% of the vehicle control group value, respectively, for the first two weeks of the dosage period (DSs 1 to 14). Overall, for the entire dosage period (DSs 1 to 47), absolute feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 101%, 99%, 100% and 99% of the vehicle control group value, respectively.

Females:
Precohabitation: Absolute and relative feed consumption values were unaffected by treatment with Hexyl cinnamic aldehyde during the precohabitation period. Absolute feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 100%, 100%, 94% and 102% of the vehicle control group value, respectively, on DSs 1 to 14. Relative feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 99%, 100%, 94% and 103% of the vehicle control group value, respectively, on DSs 1 to 14.

Gestation: Absolute and relative feed consumption values were unaffected by treatment with hexyl cinnamic aldehyde during the gestation period. Absolute feed consumption values in the 12.5, 25, 50 and 100mg/kg/day dosage groups were 93%, 101%, 95% and 114% of the vehicle control group value, respectively, on DGs 0 to 21. Relative feed consumption values in the 12.5, 25, 50 and 100 mg/kg/day dosage groups were 93%, 97%, 95% and 109% of the vehicle control group value, respectively, on DGs 0 to 21.

Lactation: Absolute and relative feed consumption values in the 100 mg/kg/day dosage group were reduced on DLs 1 to 5, in comparison to the vehicle control group values. Absolute and relative feed consumption values in the 100 mg/kg/day dosage group were 84% of the vehicle control group value on DLs 1 to 5. Absolute and relative feed consumption values were unaffected by dosages of hexyl cinnamic aldehyde as high as 50 mg/kg/day during the lactation period. Absolute feed consumption values in the 12.5, 25 and 50 mg/kg/day dosage groups were 95%, 107% and 94% of the vehicle control group value, respectively, on DLs 1 to 5. Relative feed consumption values in the 12.5, 25 and 50 mg/kg/day dosage groups were 98%, 104% and 98% of the vehicle control group value, respectively, on DLs 1 to 5.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
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:
no effects observed
Description (incidence and severity):
Males: Terminal body weights were comparable among the five dosage groups. The weights of the epididymides, testes, seminal vesicles (with and without fluid) and prostate and the ratios of these organ weights to terminal body weight were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 mg/kg/day.

Females: Terminal body weights were comparable among the five dosage groups. The weights of the paired ovaries and non-gravid uterus (with cervix) and the ratios of these organ weights to terminal body weight were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: There were no treatment-related gross lesions at any dosage level tested. One rat in each of the 0 (Vehicle) and 12.5 mg/kg/day dosage groups had slight or moderate dilation of the pelvis in the right kidney. One rat in the 100 mg/kg/day dosage group had two firm areas on the caudal portion of the prostate. No other gross lesions occurred.

Females: There were no treatment-related gross lesions at any dosage level tested. One rat in the vehicle control group had a bursal cyst on the right ovary. One rat in the 50 mg/kg/day dosage group had fetal tissue present in the stomach, a finding commonly associated with cannibalization. No other gross lesions occurred.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
None of the microscopic findings that occurred were considered related to hexyl cinnamic aldehyde. All changes observed in the P generation rats were considered to be incidental or spontaneous changes that are commonly observed in control Crl:CD(SD) rats
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
The number of estrous stages per 14 days was comparable among the five dosage groups during the precohabitation period.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating and Fertilty:

Males:
All mating and fertility parameters [the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 mg/kg/day.

The apparent increase in the average number of days to mating and the reduction in the percentage of rats that mated that occurred in the 100 mg/kg/day dosage group was attributed to two male rats (no. 3433 and 3436) that did not mate with their cohort female
rats (no. 3473 and 3476, respectively). These effects on mating and fertility were not attributed to treatment with hexyl cinnamic aldehyde because:
1) there was no apparent effect on male or female reproductive organ weights;
2) the remaining rats in the 100 mg/kg/day dosage group (N=6) mated and had viable litters;
3) there were not apparent effects on natural delivery in this dosage group; and
4) the average value (3.4 ± 2.3 days) was within the historical range of the Testing Facility

Females:
All mating and fertility parameters [the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages of hexyl
cinnamic aldehyde as high as 100 mg/kg/day.

The apparent increase in the average number of days to mating and the reduction in the percentage of rats that mated that occurred in the 100 mg/kg/day dosage group was attributed to two female rats (no. 3473 and 3476) that did not mate with their cohort male
rats (no. 3433 and 3436, respectively). These effects on mating and fertility were not attributed to treatment with hexyl cinnamic aldehyde because:
1) there was no apparent effect on male or female reproductive organ weights;
2) the remaining rats in the 100 mg/kg/day dosage group (N=6) mated and had viable litters;
3) there were not apparent effects on natural delivery in this dosage group; and
4) the average value (3.4 ± 2.3 days) was within the historical range of the Testing Facility

Details on results (P0)

Natural Delivery Observations:
All pregnant rats (7, 8, 7, 8 and 6 rats in the five respective dosage groups) delivered a litter.

Natural delivery and litter observations were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 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, viability index, surviving pups per litter, percent male pups per number of pups sexed per litter, live litter size at weighing and pup weight per litter were comparable among the five dosage groups. One dam (no. 3468) in the 50 mg/kg/day dosage group had no liveborn pups. However, it could not be determined whether the single pup was liveborn or not because partial cannibalization precluded determination of viability.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment-related effects observed at the highest dose tested.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical observations occurred in the F1 generation pups. Transient clinical signs that occurred in the F1 generation pups included mild dehydration (based on skin turgor), gasping, a pale appearance to the entire body and discoloration in one or
more areas of the body.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related necropsy observations occurred in the F1 generation pups. All pups that died early or survived to scheduled sacrificed appeared normal at necropsy examination.

Details on results (F1)

No treatment-related clinical observations occurred in the F1 generation pups. Transient clinical signs that occurred in the F1 generation pups included mild dehydration (based on skin turgor), gasping, a pale appearance to the entire body and discoloration in one or more areas of the body. In addition, no treatment-related necropsy observations occurred in the F1 generation pups. All pups that died early or survived to scheduled sacrificed appeared normal at necropsy examination

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Dose Formulation Analyses

Mean measured hexyl cinnamic aldehyde concentrations for all dose formulations were within the acceptable limits (±15% of nominal concentration). Homogeneity was acceptable (≤ 5% RSD) for all concentrations prepared at the start of the study. The values obtained were 0.2%, 0.6%, 0.4% and 0.6% RSD for the 3.125, 6.25, 12.5 and 25 mg/mL concentrations, respectively. Stability of Hexyl cinnamic aldehyde formulations at concentrations of 3.125 mg/mL and 25 mg/mL was evaluated for storage at room temperature, protected from light for 14 days. Mean measured concentrations for stored samples for stability were within the acceptable limits (±10% of the initial mean measured concentration).

Applicant's summary and conclusion

Conclusions:
Based on a lack of treatment-related effects observed, the NOAEL for systemic toxicity and the NOEL for reproductive toxicity were determined to be 100 mg/Kg/day.
Executive summary:

In a key read across one-generation reproductive toxicity study, the test material (hexyl cinnamic aldehyde; CAS# 101-86-0) was administered via oral gavage to Crl:CD(SD) rats (8/sex/dose) at doses of 0, 12.5, 25, 50 and 100 mg/Kg/day in a corn oil vehicle. Male rats were administered the test material and/or the vehicle once daily beginning 14 days before cohabitation, through cohabitation, and continuing through the day before sacrifice. Female rats were given hexyl cinnamic aldehyde and/or the vehicle once daily beginning two weeks before cohabitation, through cohabitation, and continuing through the day before sacrifice. Male rats were sacrificed after completion of the dosage period. Female rats were allowed to deliver their litters and were sacrificed on PPD 5. F1 generation pups were sacrificed on PPD 5.

 

The following parameters were evaluated: viability, clinical observations, body weights, feed weights, mating and fertility, delivery and litter observations, organ weights, necropsy observations and histopathology.

 

All parental (P) generation male and female rats survived to scheduled sacrifice. There were no treatment-related clinical observations or gross lesions in the P generation male and female rats at any dosage level tested. In addition, none of the microscopic findings that occurred were considered related to treatment with hexyl cinnamic aldehyde.

 

Body weights and body weight gains of the treated P generation male rats were generally comparable among the dosage groups. In treated P generation female rats, body weight gains were increased during the second week of the precohabitation dosage period in the 100 mg/Kg/day dosage group, and overall for the entire premating period (14% greater than controls). These increases in body weight gain were not considered adverse because there was no corresponding effect on feed consumption, and the increase in weight gain did not persist during the gestation period.

 

Body weights and body weight gains of the female rats were unaffected by treatment with the test material during the gestation period. During the lactation period, maternal body weight gains in the 100 mg/Kg/day dosage group were reduced on days 1 to 5 of lactation (DLs 1 to 5), in comparison to the vehicle control group value. Body weight gains in the 100 mg/Kg/day dosage group were 78% lower than the vehicle control group value.

 

Absolute and relative feed consumption values in male rats were unaffected by treatment with hexyl cinnamic aldehyde during the entire dosage period. In treated P generation female rats, absolute and relative feed consumption values were unaffected by treatment with hexyl cinnamic aldehyde during the precohabitation and gestation periods. Corresponding to reduced body weight gains during the lactation period, feed consumption values (g/day and g/Kg/day) in the 100 mg/Kg/day dosage group were reduced on DLs 1 to 5, in comparison to the vehicle control group values (84% of controls).

 

Terminal body weights and male and female reproductive organ weights were comparable among the five dosage groups. There was no effect of hexyl cinnamic aldehyde on estrous cycling or mating and fertility at any dosage level tested. All pregnant rats (7, 8, 7, 8 and 6 rats in the five respective dosage groups) delivered a litter. Natural delivery and litter observations were unaffected by dosages of hexyl cinnamic aldehyde as high as 100 mg/Kg/day. No treatment-related clinical or necropsy observations occurred in the F1 generation pups.

 

Based on the results observed, the NOAEL for systemic toxicity and the NOEL for reproductive toxicity were determined to be 100 mg/Kg/day.