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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:
key study
Study period:
2009-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hexylcinnamic aldehyde; octanal, 2-(phenylmethylene)-
- Physical state: Yellow liquid
- Analytical purity:98.6%
- Lot/batch No.:2814692
- Storage condition of test material:Room temperature, protected from light
- Other: Source: International Flavors and Fragrances, Inc., Union Beach, NJ

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc.
- Age at study initiation: (P) Males: 85 days; Females: 79 days
- Weight at study initiation: (P) Males: 316-342 g; Females: 201-230 g
- Fasting period before study:no data
- Housing: P generation individually housed in steel wire-bottomed cages except during cohabitation and postpartum periods. From day 20 of presumed gestation female rats were housed individually in nesting boxes. Each dam and delivered litter were housed in a common nesting box during postpartum.
- Diet (e.g. ad libitum):ad libitum, Certified Rodent Diet #5002 (PMI Nutrition International)
- Water (e.g. ad libitum):ad libitum, mains sourced and filtered by reverse osmosis.
- Acclimation period:13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%):30-70
- Air changes (per hr):at least 10
- Photoperiod (hrs dark / hrs light):12/12


IN-LIFE DATES: From: 17 Aug 2009 To: 2 Oct 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Suspensions of test substance were prepared daily, stored at room temperature and protected from light.
VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 3.125, 6.25, 12.5, 25mg/ml
- Amount of vehicle (if gavage): 4ml/kg
- Lot/batch no. (if required):J-145
- Purity:no data
Details on mating procedure:
- M/F ratio per cage: 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 not mated females will be considered to be at day 0 of presumed gestation
- After successful mating each pregnant female was caged individually. Also those female rats in presumed gestation.
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
by HPLC-UV, validated internal method.
Duration of treatment / exposure:
Male rats: total 45 days - from 14days before cohabitation, through cohabitation (7 days) and continuing through the day before sacrifice after completion of the dosage period.
Female rats: total 44 or 45 days - from 14 days before cohabitation, through cohabitation (7 days), and continuing through the day before sacrifice on postpartum day 5, or study day 46 (for rats that did not become pregnant) or gestation day 25 (for rats that did not deliver). Dams with no surviving pups were sacrificed on postpartum day 1.
Pups were not administered the test substance directly but may have been exposed in utero or via maternal milk.
Frequency of treatment:
Once daily. Dosage volume was adjusted daily on the basis of individual body weights recorded before administration.
Details on study schedule:
- No selection of F1 parental animals took place
- Age at mating of the mated animals in the study: approximately 13 weeks for females and 14 weeks for males
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
12.5 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
8 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Results from two 90-day dermal studies.
- Rationale for animal assignment (if not random):random
- Other: none
Positive control:
none; not required for this study type

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:at least weekly during acclimation period; First 5 days of dosage: prior to dosage and at approx. hourly intervals for the first 4 hours after dosage administration, and at the end of day; From day 6 of treatment: prior to dosage administration, 1-2hrs after dosage administration and at the end of the day; Day of sacrifice: once.; Postdosage periods: once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Acclimation period: at least weekly; Dosage period: daily; On sacrifice day: Once

FOOD CONSUMPTION: Yes; at least weekly for males; For females, at least weekly during acclimation and precohabitation periods, on gestation days 0, 7, 8, 9, 10, 14, 18, 21 and 25, and on postpartum days 1 and 5.

WATER CONSUMPTION No

OTHER: Rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (from day 0 of gestation till the day the first pup was observed), litter sizes (all pups delivered) and pup viability at birth. Maternal behaviour was evaluated on pospartum days 1 and 5.
Oestrous cyclicity (parental animals):
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.
Sperm parameters (parental animals):
Epididymides, prostate, seminal vesicles and testes were weighed and microscopically evaluated.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, weight gain, clinical observations

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. Those that died before examination of the litter for pup viability were evaluated by immersing their lungs 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 on days 2-5 postpartum were preserved in Bouin's solution for possible future evaluation except when precluded by autolysis.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after completion of cohabitation period on day 46 of treatment.
- Maternal animals: All surviving animals on postpartum day 5, day study 46 or day gestation 25.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Uteri of apparently nonpregnant rats were examined, while being pressed between glass plates, to confirm the absence of implantation sites, and were retained in 10% NBF.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed: Epididymides, gross lesions/masses (only histology), ovaries, prostate, seminal vesicles, testes, uterus with cervix.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring at 4 days of age (postpartum day 5).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including 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.

Statistics:
Averages and percentages calculated
Reproductive indices:
% rats that mated; Fertility index (number of pregnancies/number of rats that mated); % rats mated with female on days 1-7; % Pregnant rats among those cohabited
Offspring viability indices:
% delivered litters; number implantation sites per delivered litter; % dams with stillborn pups; Gestation index (number of dams with one or more liveborn pups/number of pregnant rats); % dams with all pups dying days 1-5 postpartum; Viability index (number of live pups on day 5 postpartum/ number of liveborn pups on day 1 postpartum; Surviving pups/litter on day 1 and on day 5; % male pups per number of pups sexed on day 1 and 5; Live litter size at weighing on day 1 and 5; Pup weight/litter on day 1 and 5;

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
maternal body weight gains in the 100 mg/kg/day group were reduced on days 1-5 of lactation, in comparison with control group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
maternal food consumption in the 100 mg/kg/day group was reduced on days 1-5 of lactation, in comparison with control group
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:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no mortalities. All clinical observations were considered unrelated to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: body weights and body weight gains, and absolute (g/day) and relative (g/kg/day) feed consumption were generally comparable among the dosage groups.
Females:
Precohabitation - body weights increased during second week compared to control group and overall for the entire premating period (14% greater than controls). These increases were not considered adverse since they didn't persist and there was not corresponding effect on feed consumption. Gestation - Body weights and feed consumption were unaffected by treatment.
Lactation - maternal body weight gains in the 100mg/kg/day were reduced on days 1-5 of lactation, in comparison with control group. Corresponding to reduced body weight gains during the lactation period, feed consumption values in the 100mg/kg/day were reduced in comparison to the vehicle group.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The number of estrous stages per 14 days was comparable among the five dosage groups during the precohabitation period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All mating and fertility parameters were unaffected at all dosages (fertility index, rats with confirmed mating dates during cohabitation and number of pregnancies per number of rats in cohabitation).
There was an apparent increase in the average number of days to mating and a reduction in % of rats that mated that occurred in the highest dose group. This was attributed to two male rats that did not mate with their cohort rats. These effects were not attributed to treatment for the following reasons: 1) there was no apparent effect on male or female reproductive organ weights; 2) the remaining rats in the 100mg/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.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related gross lesions at any dosage level tested.

HISTOPATHOLOGY (PARENTAL ANIMALS)
None of the microscopic findings that occurred were considered related to the test substance. They were all considered to be incidental or spontaneous changes.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Natural delivery and litter observations were unaffected by treatment. Values for the numbers of dams delivering litters, the duration of gestation, averages for implantation sites per delivered litter, the gestation index, 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.
No treatment related clinical observations occurred or tretment related necropsy observations. All pups that died early or survived to scheduled sacrificed appeared normal at necropsy.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed.
Remarks on result:
not determinable due to absence of adverse toxic effects

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 analysis showed they were within the acceptable limits.

Applicant's summary and conclusion

Conclusions:
Thie highest dose level of 100 mg/kg bw/d was found to cause mild maternal toxicity and no effects on reproduction or prenatal development.
Executive summary:

In an OECD 421 screening study, groups of male and female rats were administered hexylcinnamaldehyde in corn oil by gavage daily for 14 days before mating. The administered doses were 0, 12.5, 25, 50 and 100 mg/kg bw/d. The cohabitation lasted 7 days. The day when the presence of spermatozoa and/or vaginal plug was detected was assigned as Gestation Day 0. Dams were allowed to deliver their litters. Animals continued to be dosed through cohabitation and continuing through the day before sacrifice. For males this was on Day 45 of the study, and for females was on postpartum day 5. F1 generation were sacrificed on Lactation Day 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. The only effects observed were a reduction of maternal body weight gain and feed consumption in the 100 mg/kg bw/d group on days 1 and 5 of lactation in comparison to the vehicle control group. The NOAEL for reproduction and developmental toxicity was 100 mg/kg bw/d, in the absence of any treatment-related findings at the highest dose level.