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

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test 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:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

800-696-3

Cas Number:

78605-96-6

Molecular formula:

C14 H18 O

IUPAC Name:

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

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle Control

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Mid dose

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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).