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Heptanal, 2-(phenylmethylene)-, (2E)-

EC number: 800-696-3 | CAS number: 78605-96-6

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

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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

Reference 1

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
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 or test system 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
: 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
: 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
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.

Reference 2

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 for cross-reference:: read-across source
Qualifier:: according to guideline
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 or test system 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
: 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
: 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
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.

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 all close all

Reference 1

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

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. QA statement)

Limit test:

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 or test system 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:

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:

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
Observation from Day 0- 29	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
Day(s) Relative to Mating	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
Day(s) Relative to Mating		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
Day(s) Relative to Mating	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
Day(s) Relative to Mating	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
Number showing gross pathology	Showing visceral organ pathology	0	0	0	0

Table 9. Summary of Maternal Data

Parameter	GD	G1	G2	G3	G4
Parameter	GD	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
Parameter	GD	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

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

Exam Type: External

Body

Umbilicus, Hernia - Anomaly

Fetuses N(%)

Litters N(%)

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)

1(1.0)

1(5.0)

Gallbladder, Bilobed gallbladder – Anomaly

Fetuses N(%)

Litters N(%)

0(0.0)

2(1.8)

2(10.5)

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)

2(3.9)

1(5.3)

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)

13th rib, Right, Accessory - Anomaly

Fetuses N(%)

Litters N(%)

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)

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)

1(2.0)

1(5.3)

0(0.0)

13th rib, Right, Extra - Anomaly

Fetuses N(%)

Litters N(%)

1(1.8)

1(4.8)

0(0.0)

1(5.6)

13th rib, Left, Accessory - Anomaly

Fetuses N(%)

Litters N(%)

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)

13th rib, Left, Rudimentary - Anomaly

Fetuses N(%)

Litters N(%)

0(0.0)

13th rib, Left, Rudimentary Float - Anomaly

Fetuses N(%)

Litters N(%)

0(0.0)

1(2.0)

1(5.3)

1(1.8)

1(5.0)

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)

1(2.2)

1(5.6)

13th rib, Bilateral, Rudimentary - Anomaly

Fetuses N(%)

Litters N(%)

2(3.5)

2(9.5)

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)

1(2.0)

1(5.3)

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)

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)

1(5.6)

6th sternebra, Delayed skeletal ossification - Variation

Fetuses N(%)

Litters N(%)

1(1.5)

1(4.8)

0(0.0)

1(1.5)

1(5)

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)

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)

1(1.8)

1(1.5)

2nd sternebra, Hypoplastic - Anomaly

Fetuses N(%)

Litters N(%)

1(1.5)

1(4.8)

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)

Skull

Parietal, Extra suture - Variation

Fetuses N(%)

Litters N(%)

1(1.5)

1(4.8)

0(0.0)

Fontanelle, Enlarged - Variation

Fetuses N(%)

Litters N(%)

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.  

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).

Reference 2

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Dose range finding test

Deviations:

GLP compliance:

Limit test:

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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:

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:

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	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)

Mean

3.3779

0.1280

3.4240

0.2227

3.4754

0.2354

3.4600

0.2356

Mean

3.3764

0.1347

3.4831

0.2274

3.5021

0.2788

3.5099

0.2322

Mean

3.3929

0.1528

3.5059

0.2566

3.5213

0.3071

3.5108

0.2128

Mean

3.3573

0.1097

3.4228

0.3068

3.3293

0.3157

3.2016

0.2277

Mean

3.3500

0.1649

3.4142

0.4083

3.2978

0.3809

2.8931

0.2065

Mean

3.4210

0.1915

3.3097

0.3066

3.1593

0.4668

Mean

3.4182

0.1666

3.3178

0.3154

3.3481

0.5288

Mean

3.3984

0.1671

3.3791

0.2912

Mean

3.4366

0.1845

3.3651

0.2954

Mean

3.4493

0.1364

3.4244

0.3039

Mean

3.4809

0.1389

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

0.0015

0.0518

0.0591

0.0483

0.0267

0.0544

0.0499

0.0409

3 - 6 [a]

Mean

0.0166

0.0347

0.0229

0.0360

0.0193

0.0584

0.0009

0.0380

6 - 9 [a]

Mean

0.0357

0.0596

0.0832

0.1007

0.1921

0.1181

0.3092

0.0567

9 - 12 [a]

Mean

0.0073

0.1032

0.0086

0.1302

0.0314

0.0902

0.2454

0.0628

12 - 15 [a]

Mean

0.0711

0.0382

0.1045

0.1430

0.0531

0.1206

15 - 18 [a]

Mean

0.0028

0.0403

0.0081

0.0791

0.0265

0.0019

18 - 21 [a]

Mean

0.0198

0.0495

0.0613

0.0442

21 - 24 [a]

Mean

0.0383

0.0478

0.0140

0.0310

24 - 27 [a]

Mean

0.0127

0.0864

0.0593

0.0265

27 - 29 [a]

Mean

0.0316

0.0104

0.0513

0.0242

[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

0.0151

0.0692

0.0819

0.0692

0.0460

0.0919

0.0508

0.0721

6 - 29 [a]

Mean

0.0879

0.1513

0.0302

0.2346

0 - 29 [a]

Mean

0.1030

0.1672

0.0518

0.2692

Adjusted Bodyweight (kg)

GD29 UTWT [a]

Mean

3.1070

0.1183

3.1161

0.2952

Adjusted BW Gain

Adj BwtD6 [a]

Mean

0.2860

0.1397

0.3899

0.2052

[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

381.36

112.12

396.98

18.92

374.82

73.28

491.52*

47.98

3 - 6

Mean

424.70

86.84

447.54

43.30

397.87

77.19

468.98

72.30

6 - 9

Mean

255.10

134.85

248.50

129.72

100.62

101.65

24.53

34.12

9 - 12

Mean

297.34

146.48

196.98

78.62

156.68

123.98

12.62

15.44

12 - 15

Mean

257.72

130.28

100.18

56.19

130.63

171.27

15 - 18

Mean

268.88

81.16

158.92*

61.38

163.45

135.55

18 - 21

Mean

259.94

66.51

266.80

100.41

21 - 24

Mean

245.24

110.32

226.02

62.26

24 - 27

Mean

232.44

26.50

255.40

42.23

27 - 29

Mean

154.44

68.96

181.32

63.23

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

127.12

37.37

132.33

6.31

124.94

24.43

163.84*

15.99

3 - 6

Mean

141.57

28.95

149.18

14.43

132.62

25.73

156.33

24.10

6 - 9

Mean

85.03

44.95

82.83

43.24

33.54

33.88

8.18

11.37

9 - 12

Mean

99.11

48.83

65.66

26.21

52.23

41.33

4.21

5.15

12 - 15

Mean

85.91

43.43

33.39

18.73

43.54

57.09

15 - 18

Mean

89.63

27.05

52.97*

20.46

54.48

45.18

18 - 21

Mean

86.65

22.17

88.93

33.47

21 - 24

Mean

81.75

36.77

75.34

20.75

24 - 27

Mean

77.48

8.83

85.13

14.08

27 - 29

Mean

77.22

34.48

90.66

31.61

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 5
Number of Corpora Lutea	[k]	Mean SD N	9.0 1.0 5	8.8 1.3 5
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 5
Live Female		Sum	13	13
Mean Fetal Weight (F) (g)	[c1]	Mean SD N	38.19 6.36 5	38.72 6.83 5
Total Live		Sum	36	29
Mean Fetal Weight (both sexes) (g)	[c2]	Mean SD N	38.13 6.26 5	40.56 5.74 5
% 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	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 0	(6) 6 0 0	(6) 4 1 1
Liver: Submitted No visible lesions Pale	(6) 6 0	(6) 6 0	(6) 5 1	(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 0	(6) 4 1 1
Thymus: Submitted No visible lesions Discoloration, red	(6) 6 0	(6) 6 0	(6) 5 1	(6) 6 0

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

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).

Additional information

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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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Heptanal, 2-(phenylmethylene)-, (2E)-

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Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

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Effects on developmental toxicity

Description of key information

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Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Mode of Action Analysis / Human Relevance Framework

Justification for classification or non-classification

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