Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

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

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

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

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

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

Short description of key information:

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

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

Justification for selection of Effect on fertility via oral route:

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

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

Effects on developmental toxicity

Description of key information

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

- Gravid uterus weight: Yes 

- Number of corpora lutea: Yes 

- Number of implantations: Yes 

- Number of early resorptions: Yes 

- Number of late resorptions: Yes

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

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

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

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

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

Table 2. Summarized Details of Necropsy

Parameters 

GD 6 to 28 (total 23 days)

Group No. &

G1

G2

G3

G4

Dose (mg/kg/day)

0

15

30

60

Total No. of rabbits / group

23

23

23

23

Caesarean section

(day of presumed gestation)

29

Number of rabbits aborted

1

1

1

1

Number of rabbits sacrificed at caesarean section

22

22

22

22

Number of rabbits non-pregnant at caesarean section

1

3

2

2

Number of pregnant rabbits examined at caesarean section.

21

19

20

20

Number of litters examined

21

19

20

20

Total number of fetuses

123

110

125

100

Number fetuses evaluated

External, visceral and skeletal

123

110

125

100

Number of fetus for decapitation

57

51

57

45

Number of Entire fetuses

66

59

68

55

Table 3. Summary of Clinical Signs and Mortality

Observation from Day 0- 29

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

Normal

23

23

23

23

Killed - humane

1

1

1

1

Killed – terminal kill

22

22

22

22

Abortion

1

1

1

1

Table 4. Mean Maternal Body Weight of Pregnant Rabbits

Day(s) Relative to Mating

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

0

3.1839

3.2551

3.2157

3.1727

3

3.2278

3.2565

3.2214

3.1932

6

3.2397

3.2609

3.2450

3.2031

9

3.2364

3.2367

3.2246

3.1645

12

3.2203

3.2613

3.2137

3.1712

15

3.2463

3.2613

3.2394

3.1870

18

3.2881

3.2702

3.2400

3.2124

21

3.2652

3.2697

3.2312

3.2279

24

3.2688

3.2923

3.2361

3.2464

27

3.2667

3.2967

3.2399

3.2464

29

3.2898

3.3183

3.2701

3.2703

Table 5. Mean Maternal Body Weight Gain of Pregnant Rabbits

Day(s) Relative to Mating

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

Absolute Weight Gain (kg)

0-3[a]

0.0604

0.0655

0.0461

0.0577

3-6[a]

0.0120

0.0044

0.0236

0.0577

6®9[a]

-0.0033

-0.0243

-0.0204

-0.0386

9-12[a]

-0.0161

0.0246

-0.0109

 0.0386

12-15[a]

0.0260

0.0008

0.0258

0.0159

15-18[a]

0.0418

0.0081

0.0005

0.0253

18-21[a]

-0.0229

-0.0005

-0.0088

0.01155

21-24[a]

0.0036

0.0226

0.0049

0.0185

24-27[a]

-0.0021

0.0044

0.0038

0.0063

27-29[a]

0.0231

0.0216

0.0302

0.0176

Absolute Weight Gain (kg)

0-6[a]

0.0559

0.0058

0.0293

0.0304

6-29[a]

0.0501

0.0573

0.0251

0.0672

0-29[a]

0.1060

0.0632

0.0544

0.0976

Adjusted Bodyweight (kg)

[a]

2.9490

2.9851

2.9145

2.9708

Adjusted Weight Gain (kg)

[a]

-0.2908

-0.2759

-0.3304

-0.2323

Table 6. Summary of Total Food Consumption of Pregnant Rabbits

Day(s) Relative to Mating

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

0-3

352

339.12

325.11

339.43

3-6

359.95

343.88

363.06

349.58

6-9

343.10

284.01

261.62*

219.70*

9-12

313.12

301.31

267.23

209.51*

12-15

289.60

286.43

253.20

195.73*

15-18

277.97

242.42

241.84

222.53

18 -21

269.25

269.20

254.87

212.28*

21-24

244.97

236.44

214.13

210.16

24 - 27

223.53

250.10

194.14

173.13*

27- 29

183.45

193.56

176.08

130.10*

0 - 3[a]

0.0604

0.0655

0.0461

0.0577

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

Table 7. Mean Food Consumption of Pregnant Rabbits

Day(s) Relative to Mating

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

0 - 3

117.49

113.04

108.37

113.14

3 - 6

119.98

114.63

121.02

116.53

6 - 9

114.37

94.67

87.20*

73.23*

9 - 12

104.37

100.44

89.08

69.84*

12 -15

96.53

95.48

84.40

65.24*

15 - 18

92.66

80.81

80.61

74.18

18 - 21

89.75

89.73

84.96

70.76*

21 - 24

81.66

78.81

71.38

70.13

24 - 27

74.51

83.37

64.71

57.71*

27 - 29

74.51

83.37

88.04

65.05*

0 - 6

118.74

113.83

114.69

114.83

6 - 29

93.26

89.72

81.00*

68.41*

0 - 29

98.53

94.71

87.97

78.01*

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

Table 8. Summary of Gross Necropsy Findings

Parameter

Group No. &

G1

G2

G3

G4

Dose (mg/kg/day)

0

15

30

60

Total No. of rabbits / group

23

23

23

23

Number of dead during treatment

0

0

0

0

Number of moribund sacrifice

0

0

0

0

Number of terminally sacrificed

23

23

23

23

Number of examined for gross pathology.

23

23

23

23

Number showing gross pathology

Showing external pathology

0

0

0

0

Showing visceral organ pathology

0

0

0

0

Table 9. Summary of Maternal Data

Parameter

GD

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

Pregnant

GD29

21

19

20

20

Gravid Uterus (g)

GD29 [a]

340.9

333.2

355.6

299.5

Number of Corpora Lutea

GD29 [k]

7.4

7.7

8.3

7.5

No. of Implantation

GD29 [k]

6.3

6.2

6.7

5.9

No. of Early Deaths

GD29 [k]

0.1

0.3

0.2

0.8

No. of Late Deaths

GD29 [k]

0.3

0.2

0.2

0.4

Pre-implantation loss/Animal

GD29 [k]

1.14

1.53

1.65

1.60

%

Pre-implantation loss

GD29 [k]

15.7

20.3

19.8

22.7

Post-implantation loss/Animal

GD29 [k]

0.43

0.42

0.40

0.90

%

Post-implantation loss

GD29 [k]

6.2

6.5

5.5

17.7

Resorptions

GD29 [f]

21

19

20

20

[a] - Anova & Dunnett(Log)


[k] - Kruskal-Wallis & Wilcoxon

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

Table 10. Summary of Litter Data

Parameter

GD

G1

G2

G3

G4

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

Pregnant

GD29

21

19

20

20

Total No. of Fetuses

GD29 [a]

5.9

5.8

6.3

5.0

Dead

 [a]

0

0

0

0

Live Male

sum

67

58

59

50

Mean Fetal Weight (M)

(g)

GD29 [c]

38.64

41.16

38.59

39.02

Live Female

sum

56

52

66

50

Mean Fetal Weight (F)

(g)

GD29 [c1]

38.95

40.32

37.86

40.85

Live both

sum

123

110

125

100

Mean Fetal Weight (both)

(g)

GD29 [c2]

38.96

40.37

38.34

40.09

[a] ‑ Anova & Dunnett(Log)

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

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

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

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

Group

G1

G2

G3

G4

Dose

0mg/kg/day

15mg/kg/day

30mg/kg/day

60mg/kg/day

No. of Fetuses examined

123

110

125

100

No. of Litters Evaluated

21

19

20

20

Exam Type: External

 

 

 

 

Body

 

Umbilicus, Hernia - Anomaly

Fetuses N(%)

 

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

 

0(0.0)

0(0.0)

 

 

0(0.0)

Exam Type: Fresh Visceral-Body Only

 

 

 

 

Abdomen

 

Gallbladder, Hyperplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

1(1.0)

 

1(5.0)

Gallbladder, Bilobed gallbladder – Anomaly

Fetuses N(%)

 

 

Litters N(%)

0(0.0)

 

0(0.0)

2(1.8)

 

2(10.5)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

Gallbladder, Hypoplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

9(7.3)

 

5(23.8)

8(7.3)

 

6(31.6)

4(3.2)

 

 

3(15.0)

1(1.0)

 

 

1(5.0)

Exam Type: Skeletal - Body

 

 

 

 

Hindlimbs:

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

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

2(3.9)

 

1(5.3)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

Lumbar vertebrae

 

8th lumbar centrum and arch, Extra - Anomaly

Fetuses N(%)

 

Litters N(%)

8(14.0)

 

4(19.0)

3(5.9)

 

3(15.8)

4(7.0)

 

4(20.0)

4(8.9)

 

2(11.1)

Ribs

 

14th rib, Left, Rudimentary - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Right, Accessory - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

2(3.9)

 

2(10.5)

1(1.8)

 

1(5.0)

1(2.2)

 

1(5.6)

13th rib, Right, Rudimentary - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

1(2.0)

 

1(5.3)

1(1.8)

 

1(5.0)

1(2.2)

 

1(5.6)

13th rib, Right, Rudimentary Float - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

1(2.0)

 

1(5.3)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Right, Extra - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

1(5.6)

13th rib, Left, Accessory - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Left, Accessory Float - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

2(10.5)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Left, Rudimentary - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Left, Rudimentary Float - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

1(2.0)

 

1(5.3)

1(1.8)

 

1(5.0)

0(0.0)

 

0(0.0)

13th rib, Left, Extra - Anomaly

Fetuses N(%)

 

Litters N(%)

5(8.8)

 

3(14.3)

3(5.9)

 

3(15.8)

1(1.8)

 

1(5.0)

1(2.2)

 

1(5.6)

13th rib, Bilateral, Accessory Float - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

1(2.2)

 

1(5.6)

13th rib, Bilateral, Rudimentary - Anomaly

Fetuses N(%)

 

Litters N(%)

2(3.5)

 

2(9.5)

0(0.0)

 

0(0.0)

2(3.5)

 

2(10.0)

1(2.2)

 

1(5.6)

13th rib, Bilateral, Rudimentary Float - Anomaly

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

1(2.0)

 

1(5.3)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

13th rib, Bilateral, Extra - Anomaly

Fetuses N(%)

 

Litters N(%)

9(15.8)

 

5(23.8)

3(5.9)

 

3(15.8)

2(3.5)

 

2(10.0)

4(8.9)

 

3(16.7)

Sternebrae

 

4th & 5th sternebrae, Fused - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

6th sternebra, Hypoplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.8)

 

1(4.8)

3(5.9)

 

3(15.8)

1(1.8)

 

1(5.0)

4(8.9)

 

3(16.7)

6th sternebra, Incomplete ossification - Variation

Fetuses N(%)

 

Litters N(%)

7(12.3)

 

6(28.6)

4(7.8)

 

3(15.8)

6(10.5)

 

4(20.0)

1(2.2)

 

1(5.6)

6th sternebra, Poor ossification - Variation

Fetuses N(%)

 

Litters N(%)

6(28.6)

 

0(0.0)

3(15.8)

 

 

2(3.9)

4(20.0)

 

 

0(0.0)

0(0.0)

 

1(5.6)

6th sternebra, Delayed skeletal ossification - Variation

Fetuses N(%)

 

Litters N(%)

1(1.5)

 

1(4.8)

0(0.0)

 

0(0.0)

1(1.5)

 

1(5)

0(0.0)

 

0(0.0)

6th sternebra, Incomplete ossification - Variation

Fetuses N(%)

 

Litters N(%)

1(4.8)

 

3(4.5)

0(0.0)

 

5(8.5)

1(5.0)

 

4(5.9)

0(0.0)

 

2(3.6)

5th sternebra, Hypoplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

3(14.3)

 

14(21.2)

4(21.1)

 

15(25.4)

3(15.0)

 

17(25.0)

2(10.0)

 

8(14.5)

5th sternebra, Delayed skeletal ossification - Variation

Fetuses N(%)

 

Litters N(%)

14(21.2)

 

9(42.9)

15(25.4)

 

12(63.2)

17(25.0)

 

11(55.0)

8(21.1)

 

15(25.4)

5th sternebra, Poor ossification - Variation

Fetuses N(%)

 

Litters N(%)

6(28.6)

 

2(3.0)

9(15.3)*

 

 

7(36.8)

3(4.4)

 

3(15.0)

0(0.0)

 

0(0.0)

5th sternebra, Incomplete ossification - Variation

Fetuses N(%)

 

Litters N(%)

16(24.2)

 

9(42.9)

15(25.4)

 

12(63.2)

19(27.9)

 

11(55.0)

18(32.7)

 

15(75.0)

4th sternebra, Delayed skeletal ossification - Variation

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

1(1.8)

 

1(1.5)

2nd sternebra, Hypoplastic - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.5)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

2nd sternebra, Incomplete ossification - Variation

Fetuses N(%)

 

Litters N(%)

4(6.1)

 

3(14.3)

4(6.8)

 

2(10.5)

3(4.4)

 

3(15.0)

2(3.6)

 

2(10)

Forelimbs

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

Fetuses N(%)

 

Litters N(%)

5(7.6)

 

 

3(14.3)

4(6.8)

 

 

2(21.1)

3(4.4)

 

 

3(15.0)

3(5.5)

 

 

2(10.0)

Carpal bone, Right, Bent, Slight - Anomaly

Fetuses N(%)

 

Litters N(%)

1(1.5)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

Skull

Parietal, Extra suture - Variation

Fetuses N(%)

 

Litters N(%)

1(1.5)

 

1(4.8)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

Fontanelle, Enlarged - Variation

Fetuses N(%)

 

Litters N(%)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

0(0.0)

 

0(0.0)

1(1.8)

 

1(5.0)

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


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

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

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


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


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


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


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


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


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


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

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


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

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

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

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

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


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

Mode of Action Analysis / Human Relevance Framework

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

Justification for classification or non-classification

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