Cinnamyl alcohol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from a study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Principles of method if other than guideline:

According to OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cinnamaldehyde

EC Number:

203-213-9

EC Name:

Cinnamaldehyde

Cas Number:

104-55-2

Molecular formula:

C9H8O

IUPAC Name:

(2E)-3-PHENYLPROP-2-ENAL

Test material form:

liquid

Remarks:

Clear yellow coloured liquid

Details on test material:

Name: Cinnamaldehyde
CAS No: 104-55-2
Molecular Weight: 132.1612 g/mol
Molecular Formula: C9-H8-O
SMILES: O=C\C=C\c1ccccc1
InChi: InChI=1S/C9H8O/c10-8-4-7-9-5-2-1-3-6-9/h1-8H/b7-4+

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were procured from CPCSEA approved vendor
- Age at study initiation: 10-12 weeks at the time of receipt
- Weight at study initiation: 213.94 g to 215.56 g
- Fasting period before study: No Data Available
- Housing: One to three rats were housed in each polycarbonate cage (length 37 cm X breadth 21 cm X height 20 cm). During mating, one male and two female rats were housed in a single cage. Pregnant females were housed individually. Sterilized corn-cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
- Diet (e.g. ad libitum): A conventional laboratory pellet diet from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum.
- Water (e.g. ad libitum): Aquaguard™ filtered drinking water was offered ad libitum in regularly cleaned bottles.
- Acclimation period: 2-3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.20 and 23.90 °C
- Humidity (%): 46.20 and 66.50 %.
- Air changes (per hr): at least 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test chemical was weighed and dissolved in corn oil to achieve desired concentration of test item, at each dose level. Formulations were prepared one day prior or every day and stored at room temperature until usage due to the proved stability of up to 24 hours. At the time of dosing, dose formulations were kept on a magnetic stirrer for maintaining homogeneity of test formulation.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test chemical was dissolved in corn oil. Corn oil was selected as a vehicle, since it is widely used as vehicle in oral toxicity study and it is well tolerated at the selected dose volume.
- Concentration in vehicle: 0 mg/ml (control), 31.25 mg/ml (Low Dose), 62.5 mg/ml (Mid Dose) and 125 mg/ml (High Dose)
- Amount of vehicle (if gavage): 4 ml/kg bw
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulation samples [of doses viz; 0 mg/kg (Control), 125 mg/kg (Low dose), 250 mg/kg (Mid dose), and 500 mg/kg (High dose)] were analyzed. Two replicates of approximately 2 mL samples from each upper, middle and lower layer were analyzed for homogeneity and active ingredient analysis. The concentration were calculated and reported. The dose formulation analysis was carried out in the first week of treatment and in the last week of treatment, using a validated analytical method.

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: two females were kept with a single male (2:1 pairing)
- Length of cohabitation: Until evidence of copulation was observed.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data Available
- Further matings after two unsuccessful attempts: No Data Available
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: N/A

Duration of treatment / exposure:

Pregnant females were exposed to test chemical from gestation day 5 to gestation day 19.

Frequency of treatment:

Once Daily from gestation day 5 to gestation day 19.

Duration of test:

Up to GD 20

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control Group

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Remarks:

Low Dose Group

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Mid Dose Group

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

High Dose Group

No. of animals per sex per dose:

25 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Experimental design: Pregnant females were exposed from GD 5 to 19. On gestation day 20 females were sacrificed, the uterine content was examined, and the foetuses were evaluated for gross, visceral, and skeletal malformations and variations/anomalies.
Dose selection rationale: dose levels were selected based on published data.
The rationale for animal assignment (if not random): Randomization was done based on the body weight of the pregnant females on GD 0. The animals were assigned in an unbiased manner to the control and treatment groups. Females inseminated with the same male were evenly distributed across the groups. Individual body weights were within ± 20% of the respective groups mean after randomiz

Examinations

Maternal examinations:

MORTALITY/MORBIDITY: Yes
All animals were observed twice daily (once before and once after the day’s activities) for morbidity and/or mortality.
DETAILED CLINICAL OBSERVATIONS: Yes
All animals were observed daily for clinical signs and symptoms after approximately 1 hour after dose administration.
BODY WEIGHT: Yes
Animals were weighed at the time of receipt, on gestation day 0, on the first day of dosing (GD 5), and on gestation days 8, 11, 14, 17 and 20.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes,
The weight of feed provided, and that leftover was recorded on gestation day 0, on the first day of dosing (GD 5), and on gestation days 8, 11, 14, 17 and 20. The mean feed consumption was calculated on gestation day 5, 8, 11, 14, 17 and 20.
Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not data.
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not examined.
POST-MORTEM EXAMINATIONS: Yes
Sacrifice on gestation day (GD) 20. All animals including those found dead or sacrificed in moribund condition were subjected to complete gross necropsy.
BLOOD COLLECTION & HORMONE ANALYSIS: Yes
Blood was collected at termination (on GD 20) from all females; serum was separated and stored. Blood samples were assessed for serum levels of thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH).
HISTOPATHOLOGY: Yes
Histopathological analysis was performed on females from the control and high-dose (500 mg/kg bw/day) groups. The thyroid and parathyroid glands were collected from all females, but the histopathological evaluation was conduced on samples from the control and high-dose (500 mg/kg bw/day) groups. All gross lesions found during the necropsy examination were collected and subjected to microscopic examination.

Ovaries and uterine content:

EXAMINATION OF UTERINE CONTENT: Yes
After the termination (GD 20), uteri were removed, and the pregnancy status of the animals was evaluated. Uteri that appear non-gravid were further examined using ammonium sulphide staining to confirm non-pregnant status. The total weight of the gravid or non-gravid uteri including cervix were recorded for all animals except for the animals found dead during the study. The ovarian and placental weight, the number of corpora lutea, number of implantation sites, number of live/viable foetuses, number of dead foetuses and number of resorptions were recorded.

Fetal examinations:

All foetuses were weighed, sexed and examined for external abnormalities, crown to rump length, anogenital distances (AGD) and skeletal and soft tissue abnormalities with special emphasis to reproductive organs. Male foetuses were evaluated for incomplete testicular descent/cryptorchidism.
External examinations: Yes: [all per litter]
- Soft tissue examinations (visceral alterations & head razor sectioning: Yes: [half per litter]
- Skeletal examination including (growth retardation, delayed ossification, etc): Yes: [half per litter]

Statistics:

The mean and standard deviation were calculated using the software and all data were summarized in tabular form. All continuous data (body weight, feed consumption, hormone estimation, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks. P values of ≤ 0.05 were deemed to be statistically significant.

Indices:

Pregnancy rate (%), Live foetuses (%)
Pre-implantation loss (%): [(no. of corpora lutea − no. of implantations)/ no. of corpora lutea] x 100;
Post-implantation loss (%): [(no. of implantations - no. of live foetuses)/ no. of total implantations] x 100

Historical control data:

In-house historical control data was used.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs were observed in all females dosed with 500 mg/kg bw/day. These clinical signs included hypothermia, lethargy, prostration and excessive salivation, they were observed from the first day of dosing and continued till termination. No clinical signs or symptoms were observed in animals dosed up to 250 mg/kg body weight/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

No morbidities or mortality were recorded in animals up to the dose level of 250 mg/kg bw/day throughout of the experiment.
At 500 mg/kg, one animal was found dead on GD 9.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant changes in body weight or body weight gain were observed at 125 or 250 mg/kg. At 500 mg/kg, a significant decrease in maternal body weight (by 6.4%) was found on GD 17 as compared to the control group. On GD 20, a trend of lower maternal body weight (by 6.9%) was found at 500 mg/kg as compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in maternal food intake were observed at 500 mg/kg on GD 8 (by 31.2%) and on GD 11 (by 13.9%) as compared to the 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:

no effects observed

Description (incidence and severity):

No significant changes in T3, T4 or TSH levels were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant changes were observed in the absolute and relative weights of ovary, uterus and thyroid-parathyroid gland.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No gross findings were observed at 0 or 125 mg/kg. At 250 mg/kg, two non-pregnant animals showed pathological alterations in stomach which included white spots of muscular portion, increased size/swelling of muscular portions, and swelling of glandular portion. At 500 mg/kg, a total of 15 pregnant/non-pregnant animals showed pathological alterations in stomach which included white spots of muscular tissue, increased size of muscular portion, and increased size/swelling of glandular portion and red discolouration of the lung.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings at 250 mg/kg included focal hyperkeratosis in stomach in one animal (minimal in severity). At 500 mg/kg, microscopic findings included focal/multifocal hyperkeratosis in stomach; focal, erosion and oedema of muscular tissue in stomach, leukocyte infiltration in muscular tissue in stomach, diffuse degeneration of glandular tissue in stomach and focal alveolar haemorrhage in lung in 7 animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No signs of abortion was observed in any dose group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of implantation sites and the pre-and post-implantation loss (%) remained unchanged in treatment groups when compared to control.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No significant changes in the number of resorptions were observed in any dose groups.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The early and late resorptions were found to be comparable among all the treatment groups and control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were observed in any dose groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

At termination 18/25, 24/25, 17/25 and 13/25 females were found to be pregnant from the control 125 mg/kg bw, 250 mg/kg bw, and 500 mg/kg bw dose groups, respectively. Pregnancy rates were calculated as 72%, 96%, 68% and 52% for the control, 125 mg/kg bw, 250 mg/kg bw/day, and 500 mg/kg dose groups, respectively. The reduction in pregnancy rate at 500 mg/kg bw/day was considered a non-treatment related effect as no other maternal developmental toxicity parameters examined i.e. number of corpora lutea, implantation sites, pre-and post-implantation loss, resorption demonstrated significant changes when compared to the control. As per historical control data from the test facility, pregnancy rate in previosly conducted OECD 414 studies range from 72 to 100%.

Other effects:

not specified

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

organ weights and organ / body weight ratios

2

Dose descriptor:

LOAEL

Remarks:

Systemic toxicity

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

dead fetuses

early or late resorptions

maternal abnormalities

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg, a significant decrease in female foetal weight (by 8.2%) and a significant decrease in sex-combined foetal weight (by 6.4%) were observed as compared to the control group. No other significant changes in foetal weight were observed.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No reduction in number of live offspring was observed in any dose group when compared to control.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratios were comparable in all groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No significant differences in litter size were observed in any groups.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Gross external observation of foetuses revealed that 2.09, 3.79, 5.00 and 6.60 percentage of foetuses from G1 (control), G5 (125 mg/kg), G2 (250 mg/kg) and G3 (500 mg/kg) respectively showed malformations/variations. The variation included haemorrhage:1.57 (G1), 2.07 (G5), 3.89 (G2), 4.72 (G3) percent of foetuses and the malformations include: retarded growth (Runt): 0.52 (G1), 1.38 (G5), 0.56 (G2) and 1.89 (G3) percent of foetuses; dome-shaped head: 1.03 (G5) and 0.94 (G3) percent of foetuses; Anury (absence of tail): 0.56 (G2) percent of foetuses.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A number of skeletal variations/malformations were observed, however none of them were attributed to the test chemical since they lacked dose dependency and (or) were considered common to developing foetuses.
The following skeletal malformations/variations were observed in the skull: incomplete ossification of Frontal and Parietal observed in 1 /57 foetus at 500 mg/kg, dome-shaped Frontal and Parietal found in 1/150 foetus at 125 mg/kg, 1/57 at 500 mg/kg, flattened Parietal observed in 1/150 at 125 mg/kg and 1/57 at 500 mg/kg, fused Nasal, Premaxilla and Maxilla found in 1/150 at 250 mg/kg, elongated and bipartite Interparietal in 1/94 foetus at 250 mg/kg, small triangular-shaped Interparietal observed in 1/150 at 125 mg/kg, absence of Zygomatic in 2/94 at 250 mg/kg, absence of Hyoid in 2/100 foetuses at 0 mg/kg. Fused ribs were observed in 1/94 foetuses at 250 mg/kg, branched ribs in 1/57 foetuses at 500 mg/kg, misaligned ribs at 1/94 at 250 mg/kg and 2/57 foetuses at 500 mg/kg. Misaligned ribs were seen in 2/57 foetuses at 500 mg/kg, absent Thoracic, Lumbar, Sacral and caudal Vertebrae in 1/94 foetuses at 250 mg/kg.

Visceral malformations:

no effects observed

Description (incidence and severity):

No variations/malformations were observed during visceral and head razor examinations in any of the groups.

Other effects:

no effects observed

Description (incidence and severity):

No significant differences in crown to rump length or anogenital distance were observed in any of the groups.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

other: crown to rump length, AGD

2

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

Table 1. Table for Maternal Evaluation

Groups	Control	Low Dose	Mid Dose	High Dose
Dose (mg/kg body weight)	0	125	250	500
Initial animals per group	25	25	25	25
Confirmed Pregnancy at necropsy	18	24	17	13
Pregnancy rate (%)	72	96	68	52
No. of Corpora lutea	14.50 ± 2.62	13.33 ±1.97	13.94 ± 2.73	13.15 ± 2.34
No. of implantation sites	12.44 ± 3.17	12.92 ± 1.84	12.24 ± 3.17	11.08 ± 3.80
No. of resorptions	1.83 ± 2.81	0.83 ± 1.01	1.65 ± 2.26	1.92 ± 3.48
Pre-implantation loss (%)	13.15 ± 20.98	2.91 ± 5.41	12.82 ±15.23	16.66 ± 25.53
Post-implantation loss (%)	14.11 ± 22.04	6.19 ± 6.77	15.23 ± 22.21	18.66 ± 34.90
Litter size	10.61 ± 3.81	12.08 ± 1.69	10.59 ± 4.18	8.83 ± 4.95
No of live foetuses	10.61 ± 3.81	12.08 ± 1.69	10.59 ± 4.18	8.83 ± 4.95
No of dead foetuses	0	0	0	0
Live Foetuses (%)	100	100	100	100

Values are represented as Mean± SD.

Pre-implantations loss = [(no. of corpora lutea − no. of implantations)/ no. of corpora lutea] x 100

Post-implantation loss = [(no. of resorptions)/ no. of total implantations] x 100

Table 2. Table for Body Weight

Pregnant Females
Group	Dose (mg/kg)		Day of gestation and body weight in grams
			0	5	8	11	14	17	20
Control	0	Mean	215.56	236.39	245.11	257.50	272.56	298.06	332.00
		SD	13.26	13.14	13.53	14.36	14.97	17.06	25.65
		N	18	18	18	18	18	18	18
Low Dose	125	Mean	214.25	234.92	243.00	256.83	273.13	298.08	334.58
		SD	12.81	9.66	10.44	10.87	12.16	14.10	15.37
		N	24	24	24	24	24	24	24
Mid Dose	250	Mean	213.94	237.00	244.94	258.06	272.41	294.35	330.18
		SD	12.23	14.83	16.37	17.83	21.72	25.19	32.90
		N	17	17	17	17	17	17	17
High Dose	500	Mean	215.54	237.54	241.69	247.67	263.83	279.08↓	309.00
		SD	11.64	15.23	13.93	10.80	13.03	21.94	30.36
		N	13	13	13	12	12	12	12

↓: Decreased as compared to control (P<0.05)

Non-Pregnant Female
Group	Dose (mg/kg)		Day of gestation and body weight in grams
			0	5	8	11	14	17	20
Control	0	Mean	215.14	233.57	240.29	244.29	244.14	246.14	250.14
		SD	11.96	12.22	10.27	17.04	16.93	17.14	20.45
		N	7	7	7	7	7	7	7
Low Dose	125	Mean	211.00	219.00	219.00	223.00	225.00	230.00	238.00
		SD	./.	./.	./.	./.	./.	./.	./.
		N	1	1	1	1	1	1	1
Mid Dose	250	Mean	222.00	240.38	242.25	249.88	247.75	250.25	253.00
		SD	6.72	16.10	10.65	11.21	9.38	9.91	11.30
		N	8	8	8	8	8	8	8
High Dose	500	Mean	223.33	242.58	242.50	245.33	242.83	242.25	248.33
		SD	15.20	22.44	19.64	20.83	15.80	15.78	16.74
		N	12	12	12	12	12	12	12

Table 3. Table for Body Weight Change

Pregnant Females
Group	Dose (mg/kg)		Gestation Day and body weight change (%)
			5	8	11	14	17	20
Control	0	Mean	9.74	13.80	19.61	26.62	38.48	54.15
		SD	2.76	3.32	5.27	5.92	6.98	9.50
		N	18	18	18	18	18	18
Low Dose	125	Mean	9.86	13.64	20.12	27.74	39.43	56.56
		SD	5.12	5.43	6.03	6.64	7.85	9.72
		N	24	24	24	24	24	24
Mid Dose	250	Mean	10.80	14.51	20.64	27.32	37.55	54.29
		SD	3.85	4.48	5.23	7.07	8.19	12.13
		N	17	17	17	17	17	17
High Dose	500	Mean	10.19	12.14	15.92	23.48	30.60↓	44.72
		SD	3.27	2.70	2.86	4.04	8.72	14.17
		N	13	13	12	12	12	12

↓: Decreased as compared to control (P<0.05)

Non-Pregnant Females
Group	Dose  (mg/kg)		Gestation Day and body weight change (%)
			5	8	11	14	17	20
Control	0	Mean	8.66	11.85	13.66	13.51	14.43	16.27
		SD	4.35	5.23	7.35	5.65	5.52	7.20
		N	7	7	7	7	7	7
Low Dose	125	Mean	3.79	3.79	5.69	6.64	9.00	12.80
		SD	./.	./.	./.	./.	./.	./.
		N	1	1	1	1	1	1
Mid Dose	250	Mean	8.19	9.12	12.55	11.60	12.73	13.95
		SD	4.42	3.38	3.52	2.32	3.06	3.15
		N	8	8	8	8	8	8
High Dose	500	Mean	8.88	8.92	10.15	9.00	8.65	11.34
		SD	9.71	9.58	9.78	7.38	5.89	5.80
		N	12	12	12	12	12	12

Table 4. Table for External Gross Evaluation of Foetuses

Group	Control	Low Dose	Mid Dose	High Dose
Dose level (mg/kg body weight)	0	125	250	500
Total No. of Litters with live foetuses	18	24	17	11
Total No. of live Foetuses	191	290	180	106
Total No. of Male Foetuses	98	133	89	61
Total No. of Female Foetuses	93	157	91	45
Weight of foetuses (g)	3.74 ± 0.59	3.69 ± 0.35	3.57 ± 0.38	3.50 ± 0.37↓
Weight of male foetuses (g)	3.82 ± 0.63	3.82 ± 0.30	3.64 ± 0.35	3.62 ± 0.32
Weight of female foetuses (g)	3.65± 0.54	3.57 ± 0.35	3.51 ± 0.39	3.35 ± 0.40↓
Weight of Placenta (g)	0.47 ± 0.09	0.49 ±0.07	0.50 ± 0.09	0.45 ± 0.09
Crown to rump length (cm)	3.68 ± 0.23	3.41 ± 0.31↓↓↓	3.68 ± 0.28	3.67 ± 0.28
AGD of Male foetuses (mm)	3.20 ± 0.33	3.10 ± 0.25	3.17 ± 0.26	3.10 ± 0.28
AGD of Female foetuses (mm)	1.30 ± 0.21	1.28 ± 0.16	1.30 ± 0.35	1.27 ± 0.14
Normalised AGD of Male foetuses	2.06 ± 0.23	1.98 ± 0.18	2.06 ± 0.16	2.02 ± 0.16
Normalised AGD of Female foetuses	0.85 ± 0.14	0.84 ± 0.10	0.86 ± 0.27	0.85 ± 0.10
Male/Female sex ratio (per dam)	1.31 ± 1.28	0.94 ± 0.45	1.25 ± 1.03	1.53 ± 1.28
External observations
Total no. of Foetuses with No Abnormality Detected	187	279	171	99
Total no. of Foetuses with abnormalities	4	11	9	7
Foetuses with abnormalities (%)	2.09	3.79	5.00	6.60 ↑
Litters with abnormalities (%)	5.56	33.33	35.29	27.27
	Abnormalities
Haemorrhage (V)	3(1)	6(5)	7(5)	5(3)
Haemorrhage (%)	1.57(5.56)	2.07(20.83)	3.89(29.41)	4.72(27,27) ↑
Runt (M)	1(1)	4(4)	1(1)	2(2)
Runt (%)	0.52(5.56)	1.38(16.67)	0.56(5.88)	1.89(18.18)
Anury (M)	0(0)	0(0)	1(1)	0(0)
Anury (%)	0(0)	0(0)	0.56(5.88)	0(0)
Dome-shaped Head (M)	0(0)	3(3)	0(0)	1(1)
Dome-shaped Head (%)	0(0)	1.03(12.50)	0(0)	0.94(9.09)

Values are represented as Mean± SD; AGD: Anogenital distance; Normalised AGD: AGD/ Body weight^(1/3); the incidence of the individual defect is presented as a number/ Percent of foetuses (number/ percent of litters); Abnormalities: includes malformations and variations; M: Malformations; V: Variations ↓:Decreased as compared to control (P<0.05); ↓↓↓:Decreased as compared to control (P<0.001); ↑:Increased as compared to control (P<0.05)

Table 5. Table for Skeletal Anomalies of the Foetuses

Groups	Control		Low Dose		Mid Dose		High Dose
Dose (mg/kg body weight)	0		125		250		500
	No.	%	No.	%	No.	%	No.	%
Foetuses examined	100		150		94		57
No abnormality detected	83		134		86		41
Total and % Foetuses with abnormalities	17	17.00	16	10.67	8	8.51	16	28.07
	Skull
Incomplete ossification of Frontal & Parietal (V)	0	0	0	0	0	0	1	1.75
Dome-shaped frontal and Parietal (M)	0	0	1	0.67	0	0	1	1.75
Flattened Parietal (M)	0	0	1	0.67	0	0	0	0
Fused Nasal, Premaxilla & Maxilla (M)	0	0	1	0.67	0	0	0	0
Elongated, Bipartite Interparietal (M)	0	0	0	0	1	1.06	0	0
Small Triangular-shaped Interparietal (M)	0	0	1	0.67	0	0	0	0
Absence of Zygomatic (M)	0	0	0	0	2	2.13	0	0
Absence of Hyoid (M)	2	2	0	0	0	0	0	0
	Ribs
Fused (M)	0	0	0	0	1	1.06	0	0
Branched (M)	0	0	0	0	0	0	1	1.75
Misaligned (M)	0	0	0	0	1	1.06	2	3.51
Absent (M)	0	0	0	0	0	0	0	0
	Sternebrae
Unossified/Incompletely ossified Sternebrae (1 or more) (V)	13	13.0	12	8.0	5	5.32	13	22.81
Misaligned (M)	1	1	0	0	0	0	2	3.51
	Vertebrae
Unossified Caudal vertebrae (V)	1	1	0	0	0	0	0	0
Thoracic, Lumbar, Sacral and caudal Vertebrae absent (M)	0	0	0	0	1	1.06	0	0

The incidence of the individual defect is presented as a number and percentages of litters

Abnormalities: includes malformations and variations; M: Malformations; V: Variations

Table 6. Table for Skeletal Anomalies of the Foetuses(with respect to Litters)

Groups	Control		Low Dose		Mid Dose		High Dose
Dose (mg/kg body weight)	0		125		250		500
	No.	%	No.	%	No.	%	No.	%
Litters examined	18		24		17		11
No abnormality detected	7		11		13		2
Total and % Litters with abnormalities	11	61.11	13	54.17	4	23.53	9	81.82
	Skull
Incomplete ossification of Frontal & Parietal (V)	0	0	0	0	0	0	1	9.09
Dome-shaped frontal and Parietal (M)	0	0	1	4.17	0	0	1	9.09
Flattened Parietal (M)	0	0	1	4.17	0	0	0	0
Fused Nasal, Premaxilla & Maxilla (M)	0	0	1	4.17	0	0	0	0
Elongated, Bipartite Interparietal (M)	0	0	0	0	1	5.88	0	0
Small Triangular-shaped Interparietal (M)	0	0	1	4.17	0	0	0	0
Absence of Zygomatic (M)	0	0	0	0	2	11.76	0	0
Absence of Hyoid (M)	2	11.11	0	0	0	0	0	0
	Ribs
Fused (M)	0	0	0	0	1	5.88	0	0
Branched (M)	0	0	0	0	0	0	1	9.09
Misaligned (M)	0	0	0	0	1	5.88	2	18.18
	Sternebrae
Unossified/Incompletely ossified Sternebrae (1 or more) (V)	9	50.0	11	45.83	3	17.65	6	54.55
Misaligned (M)	1	5.56	0	0	0	0	2	18.18
	Vertebrae
Unossified Caudal vertebrae (V)	1	5.56	0	0	0	0	0	0
Thoracic, Lumbar, Sacral and caudal Vertebrae absent (M)	0	0	0	0	1	5.88	0	0

The incidence of the individual defect is presented as a number and percentages of litters

Abnormalities: includes malformations and variations; M: Malformations; V: Variations

Table 7. Table for Absolute Organ Weight

Pregnant
Group	Dose (mg/ Kg)		Body weight	Uterus	Ovaries
					Right	Left
Control	0	Mean	332.00	61.73	0.0878	0.0818
		SD	25.65	20.64	0.0152	0.0208
		N	18	18	18	18
Low dose	125	Mean	334.58	66.61	0.0791	0.0828
		SD	15.37	8.57	0.0197	0.0145
		N	24	24	24	24
Mid dose	250	Mean	330.18	62.60	0.0860	0.0854
		SD	32.90	25.27	0.0138	0.0183
		N	17	17	17	17
High dose	500	Mean	309.00	49.70	0.0861	0.0753
		SD	30.36	24.55	0.0202	0.0112
		N	12	12	12	12

Non-Pregnant
Group	Dose (mg/ Kg)		Body weight	Uterus	Ovaries
					Right	Left
Control	0	Mean	250.14	0.87	0.0634	0.0706
		SD	20.45	0.14	0.0053	0.0083
		N	7	7	7	7
Low dose	125	Mean	238.00	0.82	0.0655	0.0539
		SD	./.	./.	./.	./.
		N	1	1	1	1
Mid dose	250	Mean	253.00	0.88	0.1388	0.0712
		SD	11.30	0.14	0.2047	0.0074
		N	8	8	8	8
High dose	500	Mean	248.33	1.10	0.07	0.08
		SD	16.74	0.41	0.0102	0.0100
		N	12	12	12	12

Table 8. Table for Relative Organ weight to Body weight

Pregnant
Group	Dose (mg/ Kg)		Body weight	Uterus	Ovaries
					Right	Left
Control	0	Mean	332.00	18.41	0.0264	0.0249
		SD	25.65	5.63	0.0042	0.0073
		N	18	18	18	18
Low dose	125	Mean	334.58	19.88	0.0237	0.0248
		SD	15.37	2.13	0.0059	0.0043
		N	24	24	24	24
Mid dose	250	Mean	330.18	18.68	0.0261	0.0259
		SD	32.90	7.16	0.0040	0.0050
		N	17	17	17	17
High dose	500	Mean	309.00	15.57	0.0278	0.0245
		SD	30.36	7.21	0.0053	0.0035
		N	12	12	12	12

Non Pregnant
Group	Dose (mg/ Kg)		Body weight	Uterus	Ovaries
					Right	Left
Control	0	Mean	250.14	0.35	0.0255	0.0283
		SD	20.45	0.07	0.0027	0.0033
		N	7	7	7	7
Low dose	125	Mean	238.00	0.35	0.0275	0.0226
		SD	./.	./.	./.	./.
		N	1	1	1	1
Mid dose	250	Mean	253.00	0.35	0.0534	0.0282
		SD	11.30	0.06	0.0761	0.0030
		N	8	8	8	8
High dose	500	Mean	248.33	0.44	0.0277	0.0317
		SD	16.74	0.16	0.0045	0.0048
		N	12	12	12	12

Table 9. Table for Gross Pathology Observation

Pregnant Females
Group	Control	Low dose	Mid dose	High dose
Dose (mg/kg)	0	125	250	500
Total No. of TS Animals Observed	18	24	17	13
Organ & Lesion
No. of animals with abnormalities	0	0	0	7
No. of animals with no abnormalities	18	24	17	6
Stomach: Muscular portion	X	X	X
White Spot	X	X	X
Minimal	X	X	X	1
Mild	X	X	X	1
Moderate	X	X	X	1
Severe	X	X	X
Increased size / swollen	X	X	X
Minimal	X	X	X	1
Mild	X	X	X	1
Moderate	X	X	X	1
Glandular portion	X	X	X
Increased size / Swollen	X	X	X
Moderate	X	X	X	1
Red Spot	X	X	X
Minimal	X	X	X	1
Lung: Red discoloration	X	X	X
Minimal	X	X	X	1
Mild	X	X	X
Moderate	X	X	X

Note:All organs/tissues as plan were observed for study

Non-Pregnant Females
Group	Control	Low dose	Mid dose	High dose
Dose (mg/kg)	0	125	250	500
Total No. of TS Animals Observed	7	1	8	12
Organ & Lesion
Abnormality Detected	0	0	2	8
No Abnormality Detected	7	1	6	4
Stomach: Muscular portion	X	X
White Spot	X	X
Minimal	X	X
Mild	X	X	1	1
Moderate	X	X		4
Severe	X	X		1
Increased size / swollen	X	X
Minimal	X	X
Mild	X	X		1
Moderate	X	X	1
Glandular portion	X	X
Increased size / Swollen	X	X
Moderate	X	X	1	1
Red Spot	X	X
Minimal	X	X		1
Lung: Red discoloration	X	X
Minimal	X	X		1
Mild	X	X
Moderate	X	X

Note:All organs/tissues as plan were observed for study

Table 10. Table for Microscopic Observation

Pregnant Females
Group	Control	Low dose	Mid dose	High dose
Dose (mg/kg)	0	125	250	500
Total No. of TS Animals Observed	18	24	17	13
Organ & Lesion
Abnormality Detected	0	0	0	7
No Abnormality Detected	18	24	17	6
Stomach:Muscular Stomach
Hyperkeratosis
Focal Minimal	X	X	X	1
Focal Mild	X	X	X	2
Multifocal Mild	X	X	X	2
Diffuse Minimal	X	X	X	1
Erosion
Focal Mild	X	X	X	1
Focal Moderate	X	X	X	1
Edema
Diffuse minimal	X	X	X	1
Lymphocyte Infiltration
Focal Minimal	X	X	X	1
Glandular Stomach
Degeneration
Diffuse minimal	X	X	X	1
Lung: Haemorrhage Alveolar
Focal Minimal	X	X	X	1
Thyroid:Congenital Cyst
Unilateral	X	X	X	1

Non-Pregnant Females
Group	Control	Low dose	Mid dose	High dose
Dose (mg/kg)	0	125	250	500
Total No. of TS Animals Observed	7	1	8	12
Organ & Lesion
Abnormality Detected	0	0	1	8
No Abnormality Detected	7	1	1	4
Stomach:Muscular Stomach
Hyperkeratosis
Focal Minimal	X	X	1	1
Focal Mild	X	X		1
Focal moderate	X	X		1
Multifocal Minimal	X	X		1
Multifocal Mild	X	X		3
Diffuse Minimal	X	X		1
Glandular Stomach
Infiltration lymphocyte Mucosal
Focal Mild	X	X		1
Thyroid:Congenital Cyst
Bilateral	X	X		1
Unilateral	X	X		1

Table 11. Table for Head Razor and Visceral Observation Record of Foetuses

Group	Control	Low dose	Mid dose	High dose
Dose (mg/kg)	0	125	250	500
Total No. of Foetuses Observed	63	132	86	50
Abnormality Detected	00	00	00	00
No Abnormality Detected	63	132	86	50

Applicant's summary and conclusion

Conclusions:

NOAEL for maternal systemic toxicity was considered at 250 mg/kg bw/day. This effect level was based on mortality, clinical signs of toxicity, statistically/biologically significant decreases in body weight on GD 17 and 20, significant decreases in food intake on GD 8 and 11, and several gross/histopathology findings. NOAEL for developmental toxicity was considered at 250 mg/kg bw/day. This effect level was based on lower foetal body weights at 500 mg/kg bw/day.

Executive summary:

The substance was given by oral gavage to 25 pregnant Wistar rats at 0 (vehicle), 250, 500 and 1000 mg/kg bw/day from GD 5 to 19. Due to unexpected mortality at 1000 mg/kg, an additional group of 25 pregnant Wistar rats treated at 125 mg/kg bw/day from GD 5 to 19 was included. The study was performed according to OECD 414 (adopted in 2018) and GLP.Results (adults):All animals treated at 0, 125, and 250 mg/kg survived to planned death. At 500 mg/kg, one animal was found dead on GD 9. No clinical signs of toxicity were observed at 125 or 250 mg/kg. At 500 mg/kg, at least two of the following symptoms were observed in all animals from GD 7 and onwards: hypothermia, lethargy, prostration, and excessive salivation.No significant changes in body weight or body weight gain were observed at 125 or 250 mg/kg. At 500 mg/kg, a significant decrease in maternal body weight (by 6.4%) was found on GD 17 as compared to the control group. On GD 20, a trend of lower maternal body weight (by 6.9%) was found at 500 mg/kg as compared to the control group. These body weight effects were preceded bysignificant decreases in maternal food intake at 500 mg/kg on GD 8 (by 31.2%) and on GD 11 (by 13.9%) as compared to the control group.No significant changes in T3, T4 or TSH levels were observed.Pregnancy rates at termination were 72% (18/25), 96% (24/25), 68% (17/25), and 54% (13/24) at 0, 125, 250 and 500 mg/kg, respectively. For pregnant animals, no significant changes in the numbers of corpora lutea, implantation sites, resorptions, pre-implantation loss, or post-implantation loss were observed. No significant changes in absolute or relative weight of the uterus, ovary, or thyroid and parathyroid were observed. No remarkable effects on placenta weight were observed.No gross findings were made at 0 or 125 mg/kg. At 250 mg/kg, two non-pregnant animals showed pathological alterations in stomach which included white spots of muscular portion, increased size/swelling of muscular portions, and swelling of glandular portion. At 500 mg/kg, a total of 15 pregnant/non-pregnant animals showed pathological alterations in stomach which included white spots of muscular tissue, increased size of muscular portion, and increased size/swelling of glandular portion.Microscopic findings at 250 mg/kg included focal hyperkeratosis in stomach in one animal (minimal in severity). At 500 mg/kg, microscopic findings included focal/multifocal hyperkeratosis in stomach; focal alveolar haemorrhage; diffuse degeneration of glandular tissue in stomach; focal erosion of muscular tissue in stomach; leukocyte infiltration in muscular tissue in stomach; and oedema of muscular tissue in stomach.Results (foetuses):No significant changes in litter size or sex ratio were observed.At 500 mg/kg, a significant decrease in female foetal weight (by 8.2%) and a significant decrease in sex-combined foetal weight (by 6.4%) were observed as compared to the control group. No other significant changes in foetal weight were observed. No significant differences in anogenital distance were observed.Gross foetal findings occurred at low incidences at all dose levels and were not attributed to the test item. No variations or malformations were observed during visceral and head razor examinations. A number of skeletal variations/malformations were observed, however none of them were attributed to the test chemical since they lacked dose dependency and (or) were considered common to developing foetuses.Conclusion:NOAEL for maternal systemic toxicity was considered at 250 mg/kg bw/day. This effect level was based on mortality, clinical signs of toxicity, statistically/biologically significant decreases in body weight on GD 17 and 20, significant decreases in food intake on GD 8 and 11, and several gross/histopathology findings. NOAEL for developmental toxicity was considered at 250 mg/kg bw/day. This effect level was based on lower foetal body weights at 500 mg/kg bw/day.