Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 421 (2010): No Observed Adverse Effect Level (NOAEL) of = 100 mg/kg bw/day for reproductive toxicity (a reduction of maternal body weight gain and feed consumption).

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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
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
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
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;
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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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.
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
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
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
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Dose formulation analysis showed they were within the acceptable limits.

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.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2009-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

Read-across is proposed for this endpoint, using data available for the analogue substance hexyl cinnamic aldehyde. A detailed justification for the proposed read-across approach is attached.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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
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
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
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;
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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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.
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
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
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
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Dose formulation analysis showed they were within the acceptable limits.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Adequate information for classification without conducting further studies.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The 14-day repeated-dose toxicity study showed marked toxicity (both systemic and local effects) at 1000 mg/kg bw/d. Furthermore, the findings observed in the reproduction screening test (similar to OECD TG 421) showed that the highest dose level of 100 mg/kg bw/day can be considered as the maximum tolerated dose (MTD) for the dams.

Although the exposure duration of this study may not be sufficient to detect all effects on the spermatogenic cycle, it was assumed that in practice the 2-week exposure period would be sufficient to detect the majority of testicular toxicants.

The absence of adverse effects on reproduction or on reproductive organs up to 100 mg/kg bw/d HCA (considered as MTD in this study) in the reproduction screening test together with the toxicity of HCA observed at higher doses in the 14-day repeated dose toxicity study were sufficient to meet the information needs for non-classification for toxicity to reproduction. It was concluded that the conduct of a 2 -generation reproductive toxicity study should be waived on the basis of this information since the study would not contribute significant additional information.

Supplementary evidence to support this conclusion, comes from a multi-generation database on 50 substances which was recently analysed for second generation parental (P1) / offspring (F2) compared to parental (P0) and first generation offspring (F1) with regard to type of effects as well as incidence, magnitude and severity, at different dose levels. It was demonstrated that P1/F2 generation findings did not play a crucial role in the classification decisions for any substances except one which already provided abundant alerts for endocrine activity and developmental neurotoxicity. The classification of cinnamic aldehydes including hexyl and amyl forms would not be changed by conducting further reproductive/fertility investigations

Short description of key information:

The 14-day repeated-dose toxicity studies showed marked toxicity (both systemic and local effects) at 1000 mg/kg bw/d.

Furthermore, the findings observed in the reproduction screening test (similar to OECD TG 421) showed that the highest dose level of 100 mg/kg bw/day can be considered as the maximum tolerated dose (MTD) for the dams.

Justification for selection of Effect on fertility via oral route:

A position paper was prepared to justify waiving the multi-generation reproductive toxicity study.  The position paper is based on findings from a single generation study with hexylcinnamic aldehyde.  The 14-day repeated-dose toxicity studies showed marked toxicity (both systemic and local effects) at 1000 mg/kg bw/d.

Furthermore, the findings observed in the reproduction screening test (similar to OECD TG 421) showed that the highest dose level of 100 mg/kg bw/day can be considered as the maximum tolerated dose (MTD) for the dams.

Effects on developmental toxicity

Description of key information

OECD 414 (2019): No Observed Adverse Effect Level (NOAEL) of 60 mg/kg bw/day for developmental toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 December 2018 - April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted to according to GLP and followed international guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
yes
Remarks:
The observation for clinical signs was delayed by approximately 40 minutes.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Tennants Fine Chemicals Ltd, Batch no;51757 / 45562
- Expiration date of the lot/batch:15th October 2020
- Purity test date: 98.43%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +15 to +25ºC purged with nitrogen
- Stability of formulation under test conditions: stable - confirmed at 0.8 and 250 mg/mL.
- Solubility and stability of the test substance in the diet: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Based on results of validation study (Study No. G17456), test item formulations prepared at 0.8 and 250 mg/mL in the vehicle (Corn oil) were stable and resuspendable in the vehicle for 48 hours when stored at room temperature at both the concentrations.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) N/A

OTHER SPECIFICS: N/A
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
. TEST ANIMALS
- Source: Liveon Biolabs Pvt. Ltd, Plot No.46 and 47, Water Tank Road, II Phase, KIADB Industrial Area, Antharasanahalli Kasaba Hobli, Tumkur District, Pin-572 106, Karnataka, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 months
- Weight at study initiation: Females: 3.207 - 3.244 kg.
- Fasting period before study: no
- Housing: The rabbits were housed individually, except during cohabitation, when the females rabbits were cohabited with the males in 1:1 ratio in rabbit cages (approximate size: Length 65 cm x Width 65 cm x Height 45 cm) with shallow cage body and facilities for providing pelleted food (Stainless steel feed hopper) and drinking water in bottle fitted with sipper tube. The waste collection tray was changed daily (except on Sundays and public holidays). .
- Water (e.g. ad libitum): ad libitum in polycarbonate bottles with stainless steel sipper tubes
- Acclimation period: 5 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Rabbit feed manufactured by Special Diets Services, P.O Box 705, Witham, Essex, CM8 3AD, England (Batch No.3439) was provided ad libitum in stainless steel feed hoppers to rabbitse. Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter-cum-purifier (Eureka Forbes Ltd, Mumbai, India) was provided ad libitum in polycarbonate bottles with stainless steel sipper tubes to rabbits.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21 ºC
- Humidity (%): 64 to 65 %
- Air changes (per hr): 12 - 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
IN-LIFE DATES: From: 29 December 2018 To 12 February 2019
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared prior to treatment and used within the stability period of 48 hours. 



VEHICLE
- Justification for use and choice of vehicle (if other than water): Cornoil
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 1 mL/kg body weight
- Lot/batch no. (if required): MKCF8882
- Purity: not stated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The method for determination of the test item concentration in dose formulation with the vehicle (corn oil) was successfully validated for specificity, linearity and range, LOD, LOQ, accuracy and precision at a claimed concentration of 0.833 and 250.50 mg/mL. The test item was observed to form homogenous suspension in the vehicle and found to be stable and re-suspendable in the vehicle at 0.833 and 250.50 mg/mL up to 48 hours, when stored at room temperature
Details on mating procedure:
During the mating period, females were cohabited randomly with males in a 1:1 ratio. in rabbit cages (approximate size: Length 65 cm x Width 65 cm x Height 45 cm) with shallow cage body and facilities for providing pelleted food (Stainless steel feed hopper) and drinking water in bottle fitted with sipper tube. The waste collection tray was changed daily (except on Sundays and public holidays). After confirmation of mating by visual examination, the Gestation Day 0 (GD 0) was recorded for each individual rabbit.
Duration of treatment / exposure:
GD 6 to GD 28
Frequency of treatment:
Daily
Duration of test:
23 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle Control
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
23
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out using 6 rabbits per group with the test item by oral gavage at a dose volume of 2 mL/kg body weight at 50, 150 and 300 mg/kg bw/day along with the concurrent vehicle control.
Based on the findings, 0, 15, 30 and 60 mg/kg/day dose levels at a dose volume of 1 mL/kg body weight were selected for the definitive study.

- Rationale for animal assignment (if not random): N/A
 - Other:
The test item formulations or vehicle was administered daily by oral gavage approximately the same time each day (varying by ± 2 hours). The dosing was performed using a suction catheter attached to a plastic disposable syringe from GD 6 to GD 28 of presumed gestation. Dose formulation was administered at 1 mL/kg body weight, with actual volume administered based on the most recent body weight.Dose formulations were continuously stirred during dose administrations. The animals in the vehicle control (G1) group were handled and administered vehicle in an identical manner to the treatment groups.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Not Stated
 - Time schedule: 
 - Cage side observations checked in table [No.?] were included. 
 
 DETAILED CLINICAL OBSERVATIONS: Yes 
 - Time schedule: All rabbits were observed for morbidity and mortality twice daily, i.e. once in the morning and once in the afternoon. Rabbits were observed for clinical signs during the treatment period of the study: pre-dose and post dose (within 1- 2 hours of administration).

 BODY WEIGHT: Yes 
 - Time schedule for examinations: All the females included in the study (G1 to G4) were weighed on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29.

 FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes 
 - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
About 600 g of food (food input) was provided on GD 0. The left-over food was recorded and replenished to about 600 g on GD 3, 6, 9, 12, 15, 18, 21, 24 and 27. Prior to terminal sacrifice, left over food was recorded on GD 29 of presumed gestation.

 WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not Stated
- Time schedule for examinations: Not Stated
 
 POST-MORTEM EXAMINATIONS: Yes
 - Sacrifice on gestation day # GD 29
- Organs examined: Yes, external and visceral organs

 OTHER: The following data were recorded.
* Pregnancy status
* Gravid Uterine weight (from all rabbits subjected to caesarean section)
* Number of corpora lutea
* Number of implantation sites
* Number of early resorptions
* Number of late resorptions
* Gross evaluation of placenta
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:

- Gravid uterus weight: Yes 

- Number of corpora lutea: Yes 

- Number of implantations: Yes 

- Number of early resorptions: Yes 

- Number of late resorptions: Yes

 - Other: Yes, gross evaluation of placenta, pregnancy status
Fetal examinations:
- External examinations: Yes:
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes:
- Other: The following litter data were recorded:
* Total number of fetuses
* Number of live fetuses
* Number of dead fetuses
* Individual fetal body weight
* Fetus sex
Statistics:
The data on maternal body weight and food consumption, interval body weight changes, gravid uterine weight, body weight change corrected to gravid uterine weight were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions/deaths, pre-implantation and post-implantation loss, external, soft tissue and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon test pair wise comparisons of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with and without resorptions was tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05) were designated as *
Indices:
n/a
Historical control data:
Yes
Clinical signs:
no effects observed
Description (incidence and severity):
There was a total of four abortions observed – one at each dose group. viz, one each in vehicle control and at 15 mg/kg/day on GD 26, one at 30 mg/kg/day on GD 27 and one at 60 mg/kg/day on GD 23. Rabbits being sensitive animals, occurance of abortions are considered incidental.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean maternal body weights, body weight gain and adjusted body weights during the different periods of gestation (GD 0-6; GD 6-29; GD 0-29) of the treated rabbits were statistically comparable to the vehicle control group at all the doses tested.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Compared to the vehicle control group, there was no change in food consumption of rabbits dosed at 15 mg/kg/day. There was a significant decrease (23 to 33%) in food consumption during initial stage of gestation (GD 6-9) at 30 mg/kg/day. The significant reduction (21 to 33%) in food consumption was observed during GD 6-9, 9-12, 12-15, 18-21, 24-27, 27-29, 6-29 and 0-29 at 60 mg/kg/day.
The reduction in food consumption at 30 mg/kg/day was observed during initial stage and hence was not considered adverse as the food consumption during treatment period was comparable to control group. Also the reduction at 60 mg/kg/day was not of any toxicological significance as there was no concomittent reduction in maternal body weights.
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
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological findings in any of the treated groups
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental abortions were observed in each of the, vehicle control, low, mid and high dose groups. There was a total of four abortions observed – one at each dose group. viz, one each in vehicle control and at 15 mg/kg/day on GD 26, one at 30 mg/kg/day on GD 27 and one at 60 mg/kg/day on GD 23. Rabbits being sensitive animals, occurance of abortions are considered incidental.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 2. Summarized Details of Necropsy

Parameters 

GD 6 to 28 (total 23 days)

Group No. &

G1

G2

G3

G4

Dose (mg/kg/day)

0

15

30

60

Total No. of rabbits / group

23

23

23

23

Caesarean section

(day of presumed gestation)

29

Number of rabbits aborted

1

1

1

1

Number of rabbits sacrificed at caesarean section

22

22

22

22

Number of rabbits non-pregnant at caesarean section

1

3

2

2

Number of pregnant rabbits examined at caesarean section.

21

19

20

20

Number of litters examined

21

19

20

20

Total number of fetuses

123

110

125

100

Number fetuses evaluated

External, visceral and skeletal

123

110

125

100

Number of fetus for decapitation

57

51

57

45

Number of Entire fetuses

66

59

68

55

Table 3. Summary of Clinical Signs and Mortality

Observation from Day 0- 29

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

Normal

23

23

23

23

Killed - humane

1

1

1

1

Killed – terminal kill

22

22

22

22

Abortion

1

1

1

1

Table 4. Mean Maternal Body Weight of Pregnant Rabbits

Day(s) Relative to Mating

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

0

3.1839

3.2551

3.2157

3.1727

3

3.2278

3.2565

3.2214

3.1932

6

3.2397

3.2609

3.2450

3.2031

9

3.2364

3.2367

3.2246

3.1645

12

3.2203

3.2613

3.2137

3.1712

15

3.2463

3.2613

3.2394

3.1870

18

3.2881

3.2702

3.2400

3.2124

21

3.2652

3.2697

3.2312

3.2279

24

3.2688

3.2923

3.2361

3.2464

27

3.2667

3.2967

3.2399

3.2464

29

3.2898

3.3183

3.2701

3.2703

Table 5. Mean Maternal Body Weight Gain of Pregnant Rabbits

Day(s) Relative to Mating

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

Absolute Weight Gain (kg)

0-3[a]

0.0604

0.0655

0.0461

0.0577

3-6[a]

0.0120

0.0044

0.0236

0.0577

6®9[a]

-0.0033

-0.0243

-0.0204

-0.0386

9-12[a]

-0.0161

0.0246

-0.0109

 0.0386

12-15[a]

0.0260

0.0008

0.0258

0.0159

15-18[a]

0.0418

0.0081

0.0005

0.0253

18-21[a]

-0.0229

-0.0005

-0.0088

0.01155

21-24[a]

0.0036

0.0226

0.0049

0.0185

24-27[a]

-0.0021

0.0044

0.0038

0.0063

27-29[a]

0.0231

0.0216

0.0302

0.0176

Absolute Weight Gain (kg)

0-6[a]

0.0559

0.0058

0.0293

0.0304

6-29[a]

0.0501

0.0573

0.0251

0.0672

0-29[a]

0.1060

0.0632

0.0544

0.0976

Adjusted Bodyweight (kg)

[a]

2.9490

2.9851

2.9145

2.9708

Adjusted Weight Gain (kg)

[a]

-0.2908

-0.2759

-0.3304

-0.2323

Table 6. Summary of Total Food Consumption of Pregnant Rabbits

Day(s) Relative to Mating

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

0-3

352

339.12

325.11

339.43

3-6

359.95

343.88

363.06

349.58

6-9

343.10

284.01

261.62*

219.70*

9-12

313.12

301.31

267.23

209.51*

12-15

289.60

286.43

253.20

195.73*

15-18

277.97

242.42

241.84

222.53

18 -21

269.25

269.20

254.87

212.28*

21-24

244.97

236.44

214.13

210.16

24 - 27

223.53

250.10

194.14

173.13*

27- 29

183.45

193.56

176.08

130.10*

0 - 3[a]

0.0604

0.0655

0.0461

0.0577

Anova & Dunnett(Log): * = p < 0.05

Table 7. Mean Food Consumption of Pregnant Rabbits

Day(s) Relative to Mating

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

0 - 3

117.49

113.04

108.37

113.14

3 - 6

119.98

114.63

121.02

116.53

6 - 9

114.37

94.67

87.20*

73.23*

9 - 12

104.37

100.44

89.08

69.84*

12 -15

96.53

95.48

84.40

65.24*

15 - 18

92.66

80.81

80.61

74.18

18 - 21

89.75

89.73

84.96

70.76*

21 - 24

81.66

78.81

71.38

70.13

24 - 27

74.51

83.37

64.71

57.71*

27 - 29

74.51

83.37

88.04

65.05*

0 - 6

118.74

113.83

114.69

114.83

6 - 29

93.26

89.72

81.00*

68.41*

0 - 29

98.53

94.71

87.97

78.01*

Anova & Dunnett(Log): * = p < 0.05

Table 8. Summary of Gross Necropsy Findings

Parameter

Group No. &

G1

G2

G3

G4

Dose (mg/kg/day)

0

15

30

60

Total No. of rabbits / group

23

23

23

23

Number of dead during treatment

0

0

0

0

Number of moribund sacrifice

0

0

0

0

Number of terminally sacrificed

23

23

23

23

Number of examined for gross pathology.

23

23

23

23

Number showing gross pathology

Showing external pathology

0

0

0

0

Showing visceral organ pathology

0

0

0

0

Table 9. Summary of Maternal Data

Parameter

GD

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

Pregnant

GD29

21

19

20

20

Gravid Uterus (g)

GD29 [a]

340.9

333.2

355.6

299.5

Number of Corpora Lutea

GD29 [k]

7.4

7.7

8.3

7.5

No. of Implantation

GD29 [k]

6.3

6.2

6.7

5.9

No. of Early Deaths

GD29 [k]

0.1

0.3

0.2

0.8

No. of Late Deaths

GD29 [k]

0.3

0.2

0.2

0.4

Pre-implantation loss/Animal

GD29 [k]

1.14

1.53

1.65

1.60

%

Pre-implantation loss

GD29 [k]

15.7

20.3

19.8

22.7

Post-implantation loss/Animal

GD29 [k]

0.43

0.42

0.40

0.90

%

Post-implantation loss

GD29 [k]

6.2

6.5

5.5

17.7

Resorptions

GD29 [f]

21

19

20

20

[a] - Anova & Dunnett(Log)


[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

Table 10. Summary of Litter Data

Parameter

GD

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

Pregnant

GD29

21

19

20

20

Total No. of Fetuses

GD29 [a]

5.9

5.8

6.3

5.0

Dead

 [a]

0

0

0

0

Live Male

sum

67

58

59

50

Mean Fetal Weight (M)

(g)

GD29 [c]

38.64

41.16

38.59

39.02

Live Female

sum

56

52

66

50

Mean Fetal Weight (F)

(g)

GD29 [c1]

38.95

40.32

37.86

40.85

Live both

sum

123

110

125

100

Mean Fetal Weight (both)

(g)

GD29 [c2]

38.96

40.37

38.34

40.09

[a] ‑ Anova & Dunnett(Log)

[c] ‑ Ancova/Anova & Dunnett; {Covariate(s): Number of Live Male Fetuses}

[c1] ‑ Ancova/Anova & Dunnett; Covariate(s): Number of Live Female Fetuses}

[c2] ‑ Ancova/Anova & Dunnett; {Covariate(s): Number of Live Fetuses}

Table 11. Summary of Fetal External, Visceral and Skeletal Observations

Group

G1

G2

G3

G4

Dose

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

No. of Fetuses examined

123

110

125

100

No. of Litters Evaluated

21

19

20

20

Exam Type: External

 

 

 

 

Body

 

Umbilicus, Hernia - Anomaly

Fetuses N(%)

 

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

 

0(0.0)

0(0.0)

 

 

0(0.0)

Exam Type: Fresh Visceral-Body Only

 

 

 

 

Abdomen

 

Gallbladder, Hyperplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

1(1.0)

 

1(5.0)

Gallbladder, Bilobed gallbladder – Anomaly

Fetuses N(%)

 

 

Litters N(%)

0(0.0)

 

0(0.0)

2(1.8)

 

2(10.5)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

Gallbladder, Hypoplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

9(7.3)

 

5(23.8)

8(7.3)

 

6(31.6)

4(3.2)

 

 

3(15.0)

1(1.0)

 

 

1(5.0)

Exam Type: Skeletal - Body

 

 

 

 

Hindlimbs:

Metatarsal, middle phalange-4, Delayed skeletal ossification - Variation

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

2(3.9)

 

1(5.3)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

Lumbar vertebrae

 

8th lumbar centrum and arch, Extra - Anomaly

Fetuses N(%)

 

Litters N(%)

8(14.0)

 

4(19.0)

3(5.9)

 

3(15.8)

4(7.0)

 

4(20.0)

4(8.9)

 

2(11.1)

Ribs

 

14th rib, Left, Rudimentary - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Right, Accessory - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

2(3.9)

 

2(10.5)

1(1.8)

 

1(5.0)

1(2.2)

 

1(5.6)

13th rib, Right, Rudimentary - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

1(2.0)

 

1(5.3)

1(1.8)

 

1(5.0)

1(2.2)

 

1(5.6)

13th rib, Right, Rudimentary Float - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

1(2.0)

 

1(5.3)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Right, Extra - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

1(5.6)

13th rib, Left, Accessory - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Left, Accessory Float - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

2(10.5)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Left, Rudimentary - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Left, Rudimentary Float - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

1(2.0)

 

1(5.3)

1(1.8)

 

1(5.0)

0(0.0)

 

0(0.0)

13th rib, Left, Extra - Anomaly

Fetuses N(%)

 

Litters N(%)

5(8.8)

 

3(14.3)

3(5.9)

 

3(15.8)

1(1.8)

 

1(5.0)

1(2.2)

 

1(5.6)

13th rib, Bilateral, Accessory Float - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

1(2.2)

 

1(5.6)

13th rib, Bilateral, Rudimentary - Anomaly

Fetuses N(%)

 

Litters N(%)

2(3.5)

 

2(9.5)

0(0.0)

 

0(0.0)

2(3.5)

 

2(10.0)

1(2.2)

 

1(5.6)

13th rib, Bilateral, Rudimentary Float - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

1(2.0)

 

1(5.3)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Bilateral, Extra - Anomaly

Fetuses N(%)

 

Litters N(%)

9(15.8)

 

5(23.8)

3(5.9)

 

3(15.8)

2(3.5)

 

2(10.0)

4(8.9)

 

3(16.7)

Sternebrae

 

4th & 5th sternebrae, Fused - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

6th sternebra, Hypoplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

3(5.9)

 

3(15.8)

1(1.8)

 

1(5.0)

4(8.9)

 

3(16.7)

6th sternebra, Incomplete ossification - Variation

Fetuses N(%)

 

Litters N(%)

7(12.3)

 

6(28.6)

4(7.8)

 

3(15.8)

6(10.5)

 

4(20.0)

1(2.2)

 

1(5.6)

6th sternebra, Poor ossification - Variation

Fetuses N(%)

 

Litters N(%)

6(28.6)

 

0(0.0)

3(15.8)

 

 

2(3.9)

4(20.0)

 

 

0(0.0)

0(0.0)

 

1(5.6)

6th sternebra, Delayed skeletal ossification - Variation

Fetuses N(%)

 

Litters N(%)

1(1.5)

 

1(4.8)

0(0.0)

 

0(0.0)

1(1.5)

 

1(5)

0(0.0)

 

0(0.0)

6th sternebra, Incomplete ossification - Variation

Fetuses N(%)

 

Litters N(%)

1(4.8)

 

3(4.5)

0(0.0)

 

5(8.5)

1(5.0)

 

4(5.9)

0(0.0)

 

2(3.6)

5th sternebra, Hypoplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

3(14.3)

 

14(21.2)

4(21.1)

 

15(25.4)

3(15.0)

 

17(25.0)

2(10.0)

 

8(14.5)

5th sternebra, Delayed skeletal ossification - Variation

Fetuses N(%)

 

Litters N(%)

14(21.2)

 

9(42.9)

15(25.4)

 

12(63.2)

17(25.0)

 

11(55.0)

8(21.1)

 

15(25.4)

5th sternebra, Poor ossification - Variation

Fetuses N(%)

 

Litters N(%)

6(28.6)

 

2(3.0)

9(15.3)*

 

 

7(36.8)

3(4.4)

 

3(15.0)

0(0.0)

 

0(0.0)

5th sternebra, Incomplete ossification - Variation

Fetuses N(%)

 

Litters N(%)

16(24.2)

 

9(42.9)

15(25.4)

 

12(63.2)

19(27.9)

 

11(55.0)

18(32.7)

 

15(75.0)

4th sternebra, Delayed skeletal ossification - Variation

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

1(1.8)

 

1(1.5)

2nd sternebra, Hypoplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.5)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

2nd sternebra, Incomplete ossification - Variation

Fetuses N(%)

 

Litters N(%)

4(6.1)

 

3(14.3)

4(6.8)

 

2(10.5)

3(4.4)

 

3(15.0)

2(3.6)

 

2(10)

Forelimbs

Metacarpal, middle phalange-5, Delayed skeletal ossification - Variation

Fetuses N(%)

 

Litters N(%)

5(7.6)

 

 

3(14.3)

4(6.8)

 

 

2(21.1)

3(4.4)

 

 

3(15.0)

3(5.5)

 

 

2(10.0)

Carpal bone, Right, Bent, Slight - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.5)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

Skull

Parietal, Extra suture - Variation

Fetuses N(%)

 

Litters N(%)

1(1.5)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

Fontanelle, Enlarged - Variation

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

1(1.8)

 

1(5.0)

Conclusions:
Exposure to the Amyl Cinnamic Aldehyde up to 60 mg/kg bw/day caused no evident toxicity related to clinical signs, body weights, food consumption or gross necropsy findings at any of the doses tested except for one incidental abortion in all treatment groups including vehicle control. The maternal and litter data parameters were unaffected, fetal external, visceral and skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 60 mg/kg/day. 


In conclusion, based on the above findings, under the test conditions employed in the study, it was concluded that No - Observed - Effect Level (NOEL) for: Maternal toxicity and fetal developmental toxicity is 60 mg/kg/day. Therefore, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP).
Executive summary:

OECD 414 - (2019): Amyl Cinnamic Aldehyde when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits. This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred. Test item in vehicle (Corn oil) was administered at 0, 15, 30 and 60 mg/kg/day at the dose volume of 1mL/kg body weight. The control group received the vehicle only.

The data of maternal and litter parameters were analysed and presented only for pregnant rabbits at caesarean section. There were no clinical signs or mortalities observed at any of the doses tested during the study period. However, there was a total of four abortions observed – one at each dose group viz, one each in vehicle control and at 15 mg/kg/day on GD 26, one at 30 mg/kg/day on GD 27 and one at 60 mg/kg/day on GD 24. Rabbits being sensitive animals, occurance of abortions are considered incidental. 


The mean maternal body weights, body weight gain and adjusted body weights during the different periods of gestation (GD 0-6; GD 6-29; GD 0-29) of the treated rabbits were statistically comparable to the vehicle control group at all the doses tested. 


Compared to the vehicle control group, there was no change in food consumption of rabbits dosed at 15 mg/kg/day. There was a significant decrease (24%) in food consumption during initial stage of gestation (GD 6-9) at 30 mg/kg/day. The significant reduction (21 to 36%) in food consumption was observed during GD 6-9, 9-12, 12-15, 18-21, 24-27, 27-29, 6-29 and 0-29 at 60 mg/kg/day.  The reduction in food consumption at 30 mg/kg/day was observed during initial stage and hence was not considered adverse as the food consumption during treatment period was comparable to control group. Also the reduction at 60 mg/kg/day was not of any toxicological significance as there was no concomittent reduction in maternal body weights. 


The maternal parameters comprising of gravid uterine weight and mean number of corpora lutea, implantations, early and late resorptions, pre and post implantation loss and dams with resorptions were statistically comparable between the vehicle control and rabbits treated at 15, 30 and 60 mg/kg/day. Gross evaluation of placenta did not reveal any findings in any dams at any tested dose levels. 


The litter parameters comprising total number of fetuses, mean fetal weight were comparable between the vehicle control group and rabbits treated at all the doses tested. 


No test item related major abnormality was observed during external observation of the fetuses at any of the doses. Anomaly of umbilical hernia was observed in one fetus of a litter with 2 fetuses (RBa5031) at 60 mg/kg/day. This was considered as an incidental finding. 


There were no test item related major visceral malformations observed in fetuses of dams treated up to 60 mg/kg/day. Anomalies such as gall bladder hypoplastic in vehicle and test item treated groups and hyperplastic gall bladder in control and high dose group, bilobed gall bladder in low dose group were observed. These findings were not considered adverse as these observations commonly occur in animals of this test model and the incidence of occurrence was consistent with concurrent or historical controls. 


There were no test item related skeletal malformations observed in fetuses of dams treated up to 60 mg/kg/day. Variants and anomalies observed across vehicle and test item treated groups were comparable and are consistent with concurrent and historical data.

There were no gross pathological changes at any dose level. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity.


In conclusion, based on the above findings, under the test conditions employed in the study, it was concluded that No - Observed - Effect Level (NOEL) for: Maternal toxicity and fetal developmental toxicity is 60 mg/kg/day. Therefore, the  substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP).

criteria for classification according to Regulation (EC) No 1272/2008 (CLP). criteria for classification according to Regulation (EC) No 1272/2008 (CLP).

Endpoint:
developmental toxicity
Remarks:
Dose-range finding test
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
02 November 2018 to 26 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Dose range finding test
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Liveon Biolabs Pvt. Ltd, Plot No. 46 & 47. Water Tank Road, II Phase, KIADB Industrial Area, Anthrarasanahalli Kasaba Hobli, Tumkur District, Pin-572-106, Karnataka, India
- Age at study initiation: 8-9 months
- Weight at study initiation:
Mean body weight of day 0 pregnant rabbits:
G1 - 3.440 +/- 0.19 kg
G2 - 3.422 +/- 0.20 kg
G3 - 3.475 +/- 0.24 kg
G4 - 3.460 +/- 0.24 kg
- Fasting period before study:
- Housing: The rabbits were housed individually (except during cohabitation when the female rabbits will be cohabited with males in 1:1 ratio) in rabbit cages (approx. size: Length 65 x Width 65 X Height 45 cm) with shallow cage body and perforated raised shelf and facilities for providing pelleted food (stainless steel feed hopper) and drinking water in bottle fitted with a sipper tube. The waste collection tray was changed daily (except on Sundays and general holidays).
- Diet (e.g. ad libitum): ad libitum (Rabbit feed manufactured by Special Diets Services, P.O BOX 705, Witham, Essex, CM8 3AD, England)
- Water (e.g. ad libitum): ad libitum (Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter-cum-purifier manufactured by Eureka Forbes Ltd, Mumbai, India)
- Acclimation period: After physical examination for good health and suitability for the study, 35 female and 30 male rabbits were acclimatized for five days before initiation of mating. During the acclimatization period, all rabbits will be observed once daily.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 (± 3) °C
- Humidity (%): 30 – 70 %,
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12:12 h light:dark

IN-LIFE DATES: From: To: 02 November 2018 to 06 December 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dose formulations were prepared prior to treatment and used within the stability period of 48 hours. 


- Mixing appropriate amounts with (Type of food): Based on the dose required quantities of the test item were weighed accurately (in the respective pre-calibrated beakers) and a small volume of vehicle was added and mixed thoroughly using a glass rod. The final volume was made up to the mark with the vehicle to get the required intended volume.
The homogeneity of the dose formulation during sampling/treatment was maintained by constant stirring using magnetic stirrer.
- Storage temperature of food: Not reported

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 50 mg/kg bw/day = 25 mg/mL; 150 mg/kg bw/day = 75 mg/mL; 300 mg/kg bw/day = 150 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg body weight
- Lot/batch no. (if required): MKCF8882
- Purity: not stated
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: Cohabitation
- If cohoused: Female rabbits were cohabited with males in 1:1 ratio; naïve males untreated with the test item were used for mating purpose only.
- M/F ratio per cage: 1:1
- Length of cohabitation: Not reported
- Further matings after two unsuccessful attempts: No
- Verification of same strain and source of both sexes: Not reported
- Proof of pregnancy: Not reported
- Any other deviations from standard protocol: N/A
Duration of treatment / exposure:
From gestation day (GD) 6 to 28.
Frequency of treatment:
Daily
Duration of test:
23 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle Control (G1)
Dose / conc.:
50 mg/kg bw/day
Remarks:
(G2)
Dose / conc.:
150 mg/kg bw/day
Remarks:
(G3)
Dose / conc.:
300 mg/kg bw/day
Remarks:
(G4)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on existing toxicity data on substance
- Rationale for animal assignment (if not random): Day 0 pregnant rabbits (GD 0 for each individual female rabbit in the study will be considered the day on which mating has occurred) obtained on each day were randomly distributed to different groups by body weight stratification method using ProvantisTM software.
- Other: N/A
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the rabbits were observed twice daily for clinical signs during the in-life phase of the study, once prior to treatment and once after post dose and observed for morbidity and mortality twice daily.

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Days 3, 6, 9, 12, 15, 18, 21, 24 and 27 and food output on Day 29 of presumed gestation was recorded.
- The intermittent food consumption i.e. gestation Day 0–3, 3–6, 6–9, 9–12, 12–15, 15–18, 18–21, 21–24, 24–27 and 27 to 29 was calculated. Further, food consumption for pre-treatment (GD 0 to 6), treatment (GD 6 to 28) and for entire gestation period (GD 0 to 29) was calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Gross necropsy, which involves an external observation and thoracic and abdominal viscera including uterine contents, was be performed on all animals in the study including dead, moribund and those sacrificed pre-term or at term (caesarean section).
Ovaries and uterine content:
- Examination of uterine contents:
Caesarean section: The gravid uterus including cervix was excised, weighed and immediately examined. The following maternal data were recorded: Pregnancy status, Gravid uterine weight (only for females killed at termination, not obtained from animals found dead or sacrificed moribund during the study), Number of corpora lutea, Number of implantation sites, Number of early resorptions, Number of late resorptions, Gross evaluation of placenta.
Fetal examinations:
- Fetal Data (Examination of Fetuses): The individual fetuses were removed sequentially, all the fetuses were identified by serial numbers. The fetuses were euthanized by sodium thiopentone injection.
The following litter data were recorded: Total number of fetuses, Number of live fetuses, Number of dead fetuses, Individual fetal body weight (g), Fetus sex. All the fetuses were also examined for external malformations.
Statistics:
The data on maternal body weight, body weight change in interval, gravid uterine weight, and body weight change corrected to gravid uterine weight, maternal food consumption were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances are heterogeneous, ANOVA were performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean were performed, when the group differences are found significant.
Fetal weight for male and female were analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.
Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external observations for variations were analyzed using Kruskal Wallis test for group comparison. Mann-Whitney pairwise comparison of the treated groups with the control group were performed, when the group differences are significant.
The incidence of with and without resorptions in dams were tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.
Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated.
Historical control data:
Not reported
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was treatment related clinical signs at the tested doses of 150 and 300 mg/kg/day. At 50 mg/kg there were transient clinical signs of slight to severe salivation and nasal discharge in one rabbit (RBa4840) during (GD16 to 19) which recovered from GD 20 onwards. The remaining rabbits in this group were normal.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
At the dose of 150 mg/kg/day there were 2/6 mortalities and two rabbits were moribund sacrificed. Hence this group was terminated on GD 19 and the surviving two rabbits were euthanized. At the dose of 300 mg/kg/day there were 3/6 mortalities and one rabbit was moribund sacrificed. Hence this group was terminated on GD 12 and the surviving two rabbits were euthanized.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights (134%) of rabbits at 50 mg/kg, was reduced during treatment period as compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption (17%) of rabbits at 50 mg/kg, was reduced during treatment period as compared to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
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:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Mean or individual values of maternal parameters comprising of uterine weight, number of corpora lutea, implantations and early and late resorptions were comparable to the vehicle control group at the dose of 50 mg/kg/day. Gross evaluation of placenta revealed no findings.
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The litter data parameters comprising of total number of fetuses, number of live fetuses, male and female fetal weights were statistically comparable to vehicle control group.
No abnormality was noticed on external observations of fetuses at any of the doses tested.

Table 1. Details of Necropsy Findings

Parameters           

 

Group No.

Dose (mg/kg/day)

G1

G2

G3

G4

0

50

150

300

Total number of mated rabbits / group

6

6

6

6

Duration of treatment (days)

Gestation Days 6 to 28 (23 days)

Day of cesarean section

(day of presumed gestation)

29

No. of rabbits dead before cesarean section

0

0

 

 

No. of rabbits aborted

0

0

 

 

No. of rabbits sacrificed at cesarean section

6

6

 

 

No. of non-pregnant rabbits at cesarean section

1

1

 

 

No. of rabbits pregnant at cesarean section

5

5

 

 

No. of litters examined

5

5

 

 

 

 

 

 

 

Total no. of fetuses

36

29

 

 

Number of fetuses evaluated

 

 

 

 

a. External

36

29

 

 

b. Abnormal fetus

0

0

 

 

c. Dead fetus

0

0

 

 

NOTE: Dose groups of 150 and 300 mg/kg/day were terminated due to treatment related clinical signs and mortalities.

Table 2. Summary of Clinical Signs and Mortality

 Observation Types (Day 0 - 29)  Female G1 (0 mg/kg bw/day)  Female G2 (50 mg/kg bw/day)  Female G3 (150 mg/kg bw/day)  Female G4 (300 mg/kg bw/day)
 Normal  6  6  6  6
 Dyspnoea  0  0  1  0
 Emaciation  0  0  1
 Faecal Consistency, Semi solid faeces  0  0  1  1
 Dead  0  0  2  3
 Killed - humane  0  0  2  2
 Killed - moribund  0  0  2  1
 Killed - terminal kill  6  6  0  0
 Salivation, slight  0  1  0  0
 Salivation, moderate  0  1  2  0
 Salivation, severe  0  0  1  1
 Posture, recumbent  0  0  2  0
 Posture, curled up, often asleep or sit  0  0  1  1
 Discharge, nose/snout, moderate  0  1  1  0
 Discharge, nose/snout, severe  0  0  1  1
 Discharge, both eyes, moderate  0  0  1  0

Values = number of animals affected

Table 3. Summary of Maternal Body Weight of Pregnant Rabbits

 Sex: Female

Day(s) Relative to Mating

  G1 (0 mg/kg bw/day)   G2 (50 mg/kg bw/day)  G3 (150 mg/kg bw/day)  G4 (300 mg/kg bw/day)
 0

Mean

SD

N

3.3779

0.1280

5

3.4240

0.2227

3.4754

0.2354

6

3.4600

0.2356

 3

Mean

SD

N

3.3764

0.1347

5

3.4831

0.2274

5

3.5021

0.2788

6

3.5099

0.2322

6

 6

Mean

SD

N

3.3929

0.1528

3.5059

0.2566

5

3.5213

0.3071

6

3.5108

0.2128

6

 9

Mean

SD

N

3.3573

0.1097

5

3.4228

0.3068

3.3293

0.3157

6

3.2016

0.2277

6

 12

Mean

SD

N

3.3500

0.1649

5

3.4142

0.4083

5

3.2978

0.3809

6

2.8931

0.2065

5

 15

Mean

SD

N

3.4210

0.1915

5

3.3097

0.3066

5

3.1593

0.4668

 18

Mean

SD

N

3.4182

0.1666

5

3.3178

0.3154

5

3.3481

0.5288

2

 -

 21

Mean

SD

N

3.3984

0.1671

5

3.3791

0.2912

5

 -

 -

 24

Mean

SD

N

3.4366

0.1845

5

3.3651

0.2954

5

 -

 -

 27

Mean

SD

N

3.4493

0.1364

5

3.4244

0.3039

 -

 -

 29

Mean

SD

N

3.4809

0.1389

5

3.4758

0.3078

 -

 -

Anova & Dunnett(Log)

Table 4. Summary of Maternal Body Weight Gain of Pregnant Rabbits

ABSOLUTE WEIGHT GAIN (kg)

Sex: Female

Day(s) Relative to Mating

  G1 (0 mg/kg bw/day)   G2 (50 mg/kg bw/day)  G3 (150 mg/kg bw/day)  G4 (300 mg/kg bw/day)
 0 - 3 [a]

Mean

SD

N

0.0015

0.0518

5

0.0591

0.0483

5

0.0267

0.0544

6

0.0499

0.0409

6

 3 - 6 [a]

Mean

SD

N

0.0166

0.0347

5

0.0229

0.0360

5

0.0193

0.0584

6

0.0009

0.0380

6

 6 - 9 [a]

Mean

SD

N

0.0357

0.0596

5

0.0832

0.1007

5

0.1921

0.1181

6

0.3092

0.0567

6

 9 - 12 [a]

Mean

SD

N

0.0073

0.1032

5

0.0086

0.1302

5

0.0314

0.0902

6

0.2454

0.0628

5

 12 - 15 [a]

Mean

SD

N

0.0711

0.0382

5

0.1045

0.1430

5

0.0531

0.1206

3

-

 15 - 18 [a]

Mean

SD

N

0.0028

0.0403

5

0.0081

0.0791

5

0.0265

0.0019

2

 18 - 21 [a]

Mean

SD

N

0.0198

0.0495

5

0.0613

0.0442

5

-

 -

 21 - 24 [a]

Mean

SD

N

0.0383

0.0478

5

0.0140

0.0310

5

 -

 -

 24 - 27 [a]

Mean

SD

N

0.0127

0.0864

5

0.0593

0.0265

5

 -

 -

 27 - 29 [a]

Mean

SD

N

0.0316

0.0104

5

0.0513

0.0242

5

 -

 -

[a] ‑ Anova & Dunnett(Log)

Table 4. Cont'd Summary of Maternal Body Weight Gain of Pregnant Rabbits

ABSOLUTE WEIGHT GAIN (kg)

Sex: Female

Day(s) Relative to Mating

  G1 (0 mg/kg bw/day)   G2 (50 mg/kg bw/day)  G3 (150 mg/kg bw/day)  G4 (300 mg/kg bw/day)
 0 - 6 [a]

Mean

SD

N

0.0151

0.0692

5

0.0819

0.0692

5

0.0460

0.0919

6

0.0508

0.0721

6

 6 - 29 [a]

Mean

SD

N

0.0879

0.1513

5

0.0302

0.2346

5

-

-

 0 - 29 [a]

Mean

SD

N

0.1030

0.1672

5

0.0518

0.2692

5

-

-

 Adjusted Bodyweight (kg)

GD29 UTWT [a]

Mean

SD

N

3.1070

0.1183

5

3.1161

0.2952

5

-

-

 Adjusted BW Gain

Adj BwtD6 [a]

Mean

SD

N

0.2860

0.1397

5

0.3899

0.2052

5

-

-

[a] ‑ Anova & Dunnett(Log)

Table 5. Summary of Food Consumption of Pregnant Rabbits

Total Food Consumption (g)

Sex: Female

Day(s) Relative to Mating

  G1 (0 mg/kg bw/day)   G2 (50 mg/kg bw/day)  G3 (150 mg/kg bw/day)  G4 (300 mg/kg bw/day)
 0 - 3

Mean

SD

N

381.36

112.12

5

396.98

18.92

5

374.82

73.28

6

491.52*

47.98

6

 3 - 6

Mean

SD

N

424.70

86.84

5

447.54

43.30

5

397.87

77.19

6

468.98

72.30

6

 6 - 9

Mean

SD

N

255.10

134.85

5

248.50

129.72

5

100.62

101.65

6

24.53

34.12

6

 9 - 12

Mean

SD

N

297.34

146.48

5

196.98

78.62

5

156.68

123.98

6

12.62

15.44

5

 12 - 15

Mean

SD

N

257.72

130.28

5

100.18

56.19

5

130.63

171.27

3

-

 15 - 18

Mean

SD

N

268.88

81.16

5

158.92*

61.38

5

163.45

135.55

2

 18 - 21

Mean

SD

N

259.94

66.51

5

266.80

100.41

5

-

 -

 21 - 24

Mean

SD

N

245.24

110.32

5

226.02

62.26

5

 -

 -

 24 - 27

Mean

SD

N

232.44

26.50

5

255.40

42.23

5

 -

 -

 27 - 29

Mean

SD

N

154.44

68.96

5

181.32

63.23

5

 -

 -

Anova & Dunnett(Log): * = p < 0.05;

Table 5 Contd. Summary of Food Consumption of Pregnant Rabbits

FOOD MEAN DAILY CONSUMPTION 9g/day)

Sex: Female

Day(s) Relative to Mating

  G1 (0 mg/kg bw/day)   G2 (50 mg/kg bw/day)  G3 (150 mg/kg bw/day)  G4 (300 mg/kg bw/day)
 0 - 3

Mean

SD

N

127.12

37.37

5

132.33

6.31

5

124.94

24.43

6

163.84*

15.99

6

 3 - 6

Mean

SD

N

141.57

28.95

5

149.18

14.43

5

132.62

25.73

6

156.33

24.10

6

 6 - 9

Mean

SD

N

85.03

44.95

5

82.83

43.24

5

33.54

33.88

6

8.18

11.37

6

 9 - 12

Mean

SD

N

99.11

48.83

5

65.66

26.21

5

52.23

41.33

6

4.21

5.15

5

 12 - 15

Mean

SD

N

85.91

43.43

5

33.39

18.73

5

43.54

57.09

3

-

 15 - 18

Mean

SD

N

89.63

27.05

5

52.97*

20.46

5

54.48

45.18

2

 18 - 21

Mean

SD

N

86.65

22.17

5

88.93

33.47

5

-

 -

 21 - 24

Mean

SD

N

81.75

36.77

5

75.34

20.75

5

 -

 -

 24 - 27

Mean

SD

N

77.48

8.83

5

85.13

14.08

5

 -

 -

 27 - 29

Mean

SD

N

77.22

34.48

5

90.66

31.61

5

 -

 

Anova & Dunnett(Log): * = p < 0.05;

Table 5 Contd. Summary of Food Consumption of Pregnant Rabbits

TOTAL FOOD CONSUMPTION (g)

Sex: Female

Day(s) Relative to Mating

  G1 (0 mg/kg bw/day)   G2 (50 mg/kg bw/day)  G3 (150 mg/kg bw/day)  G4 (300 mg/kg bw/day)
 0 - 6

Mean

SD

N

806.06

194.67

5

844.52

46.68

5

772.68

142.31

6

960.50

118.65

6

 6 - 29

Mean

SD

N

1971.10

633.14

5

1634.12

434.46

5

-

-

 0 - 29

Mean

SD

N

2777.16

747.07

5

2478.64

476.09

5

-

-

Table 5 Contd. Summary of Food Consumption of Pregnant Rabbits

FOOD MEAN DAILY CONSUMPTION (g/day)

Sex: Female

Day(s) Relative to Mating

  G1 (0 mg/kg bw/day)   G2 (50 mg/kg bw/day)  G3 (150 mg/kg bw/day)  G4 (300 mg/kg bw/day)
 0 - 6

Mean

SD

N

134.34

32.44

5

140.75

7.78

5

128.78

23.72

6

160.08

19.77

6

 6 - 29

Mean

SD

N

85.70

27.53

5

71.05

18.89

5

-

-

 0 - 29

Mean

SD

N

95.76

25.76

5

85.47

16.42

5

-

-

Table 6. Summary of Maternal Data

 Sex: Female      G1 (0 mg/kg bw/day)  G2 (50 mg/kg bw/day)
 Group Size      6  6
 Pregnant      5  5
 Gravid Uterus (g)  [a]

Mean

SD

N

 373.9

39.4

5

359.7

52.6

 Number of Corpora Lutea  [k]

Mean

SD

N

 9.0

1.0

5

8.8

1.3

 Number of Implantations

 [k]

Mean

SD

N

 8.0

1.4

5

 6.6

1.3

5

 Number of Early Deaths

 [k]

Mean

SD

N

 0.4

0.9

5

 0.6

0.5

5

 Number of Late Deaths

 [k]

Mean

SD

N

 0.4

0.5

5

 0.2

0.4

5

 Pre-implantation loss/animal

 [k]

Mean

SD

N

 1.00

1.00

5

 2.20

1.30

5

 Post-implantation loss/animal

 [k]

Mean

SD

N

 0.80

1.30

5

 0.80

0.45

5

 With late resorptions

 

 

 2

 1

 Total Resorptions

 

 

 0

 0

[a] Anova & Dunnett(Log)

[k] Kruskal Wallis & Wilcoxon

Table 7. Summary of Litter Data

 Sex: Female

     G1 (0 mg/kg bw/day)  G2 (50 mg/kg bw/day)
 Pregnant      5  5
 Total Number    Sum  36 29 
 Dead    Sum  0  0
 Live Male    Sum  23  16
 Mean Fetal Weight (M) (g)  [c]

Mean

SD

N

 38.54

6.44

5

41.53

5.45

 Live Female    Sum  13 13 
 Mean Fetal Weight (F) (g)  [c1]

Mean

SD

N

 38.19

6.36

5

38.72

6.83

 Total Live    Sum  36 29 
 Mean Fetal Weight (both sexes) (g)  [c2]

Mean

SD

N

 38.13

6.26

5

40.56

5.74

 % male in group      64 55 

[c] - Ancova/Anova & Dunnett


{Covariate(s): Number of Live Male Fetuses}

[c1] - Ancova/Anova & Dunnett


{Covariate(s): Number of Live Female Fetuses}

[c2] - Ancova/Anova & Dunnett {Covariate(s): Number of Live Fetuses}

Table 8. Summary of Necropsy Findings

 Group No. G1  G2   G3  G4
 Dose (mg/kg bw/day)  0  50  150 300 
 Number of Animals on Study  6  6
 Number of Animals Completed  (6) (6)  (6)  (6) 

 Kidneys:

Submitted

No visual lesions

Enlarged, unilaterla

Discoloration, red, bilateral

 

(6)

6

0

0

(6)

6

0

(6)

6

0

(6)

4

1

 Liver:

Submitted

No visible lesions

Pale

 

(6)

6

0

(6)

6

0

(6)

5

 

(6)

6

0

 Stomach:

Submitted

No visible lesions

Focus (i); multifocal

Foxus (i); red, multifocal

 

(6)

6

0

0

 

(6)

6

0

0

(6)

6

0

 (6)

4

1

1

 Thymus:

Submitted

No visible lesions

Discoloration, red

(6)

6

(6)

6

(6)

5

(6)

6

Table 9. Summary of Fetal External Observations

 Group Number  G1  G2
 Dose (mg/kg bw/day)  0  50
 No. Litters Examined  5  5
 No. of Fetuses Examined 36  29 
 Normal Variant  None  None
 Minor Anomalies  None None 
 Major Malformations  None  None

Conclusions:
Based on the results of the dose range finding study the following doses are selected for the definitive study:
Vehicle control - 0 mg/kg/day
Low dose - 15 mg/kg/day
Mid dose - 30 mg/kg/day
High dose - 60 mg/kg/day
Executive summary:

The objective of this dose range finding study was to select the doses for the definitive prenatal developmental toxicity study in New Zealand white rabbits. This study evaluated the maternal and developmental toxicity of the test item Amyl Cinnamic Aldehyde when administered orally to pregnant New Zealand white rabbits from gestation days (GD) 6 to 28.

The test item, Amyl cinnamic aldehyde was suspended in vehicle (corn oil) and administered orally from gestation Day (GD) 6 to 28 at the doses of 50 (G2), 150 (G3) and 300 (G4) mg/kg/day, along with the concurrent vehicle control (G1). The animals in the vehicle control group were handled in an identical manner as in the treatment groups and treated with vehicle.

In this dose range finding study, 24 mated female rabbits were assigned to four groups. Each group consisted of 6 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred.

All the rabbits were observed twice daily for clinical signs during the in-life phase of the study, once prior to treatment and once after post dose and observed for morbidity and mortality twice daily. Body weights changes and food consumption was recorded. Caesarean section was performed for all the rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses. The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations.

The results from the study are as follows:

- There was treatment related clinical signs at the tested doses of 150 and 300 mg/kg/day. At the dose of 150 mg/kg/day there were 2/6 mortalities and two rabbits were moribund sacrificed. Hence this group was terminated on GD 19 and the surviving two rabbits were euthanized. At the dose of 300 mg/kg/day there were 3/6 mortalities and one rabbit was moribund sacrificed. Hence this group was terminated on GD 12 and the surviving two rabbits were euthanized.

- At 50 mg/kg there were transient clinical signs of slight to severe salivation and nasal discharge in one rabbit (RBa4840) during (GD16 to 19) which recovered from GD 20 onwards. The remaining rabbits in this group were normal.

- Body weight and food consumption: The mean body weights (134%) and food consumption (17%) of rabbits at 50 mg/kg, were reduced during treatment period as compared to the control group.

- Mean or individual values of maternal parameters comprising of uterine weight, number of corpora lutea, implantations and early and late resorptions were comparable to the vehicle control group at the dose of 50 mg/kg/day. Gross evaluation of placenta revealed no findings.

- The litter data parameters comprising of total number of fetuses, number of live fetuses, male and female fetal weights were statistically comparable to vehicle control group.

- No abnormality was noticed on external observations of fetuses at any of the doses tested.


Based on the results of the dose range finding study the following doses are selected for the definitive study:

Vehicle control - 0 mg/kg/day

Low dose - 15 mg/kg/day

Mid dose - 30 mg/kg/day

High dose - 60 mg/kg/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The endpoint is concluded based on a single key study with a Klimish rating of 1. Adequate for classification purposes.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 414 - (2019): Amyl Cinnamic Aldehyde when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits. This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred. Test item in vehicle (Corn oil) was administered at 0, 15, 30 and 60 mg/kg/day at the dose volume of 1mL/kg body weight. The control group received the vehicle only.

Exposure to the Amyl Cinnamic Aldehyde  up to 60 mg/kg bw/day caused no evident toxicity related to clinical signs, body weights, food consumption or gross necropsy findings at any of the doses tested except for one incidental abortion in all treatment groups including vehicle control.  The maternal and litter data parameters were unaffected, fetal external, visceral and skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 60 mg/kg/day. 


In conclusion, based on the above findings, under the test conditions employed in the study, it was concluded that No - Observed - Effect Level (NOEL) for: Maternal toxicity and fetal developmental toxicity is 60 mg/kg/day.

Mode of Action Analysis / Human Relevance Framework

The test item did not induced any effects on the exposured animals and as such, no mode of action could be deduced.

Justification for classification or non-classification

Based on the available read-across data for alpha-hexylcinnamic aldehyde as well as the target sustance, there are no indications of concerns regarding reproductive toxicity or developmental effects for amyl cinnamic aldehyde. Therefore, the  substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP).