Etocrilene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Etocrilene

EC Number:

226-029-0

EC Name:

Etocrilene

Cas Number:

5232-99-5

Molecular formula:

C18H15NO2

IUPAC Name:

ethyl 2-cyano-3,3-diphenylprop-2-enoate

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ordered from Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at study initiation: 10-12 weeks (time-mated females)
- Body weight of pregnant animals on Day 0: 166.9 – 219.4 g

- Housing: individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²). Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). As nesting material, compacted fibers of softwood (Typ SAFE® compact nesting large, supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) and cellulose wadding was offered toward the end of gestation in all pregnant females. For enrichment, wooden gnawing blocks (Typ SAFE® block large, supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) and play tunnel (Play tunnel large, Art. 14153, supplied by PLEXX B.V., Elst, The Netherlands) were offered.

- Diet (e.g. ad libitum): mouse and rat maintenance diet “GLP”, meal (Granovit AG, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): drinking, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared at the beginning of the administration period
and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% CMC suspension in deionized water in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.5% CMC suspension in deionized water
- Amount of vehicle (if gavage): 10 ml/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in 0.5% CMC suspension in deionized water over a period of 7 days at room temperature had been verified prior to the start of the study.
Samples of the test substance preparations were sent twice (at the beginning of administration and during the administration period) to the analytical laboratory for verification of the concentrations. The samples were also used to verify the homogeneity of the low- and high-concentrations (100 and 1000 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.

Details on mating procedure:

- The animals were paired by the breeder (“time-mated”)
- Proof of pregnancy: detection of vaginal plug/sperm referred to as Day 0 of gestation.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

once a day

Duration of test:

on GD 20, all surviving females were sacrificed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Test group 0 (vehicle)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Test group 1

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Test group 2

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Test group 3

No. of animals per sex per dose:

25 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose level rationale: In a combined repeated dose toxicity study with the reproductive/developmental screening test (OECD 422) in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose. Based on these data the dose level for this study was set to 100, 300 and 1000 mg/kg bw/d.


- The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- The rat was the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines. The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).
A cage-side examination was conducted at least once daily before and after treatment period
(GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and between 2 and 5 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 18 and 20.
- Corrected body weight gain: calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13,
13-15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined (weight, fixation, histopathology): thyroid glands (all dams) (see also Table 1 in section "any other information on materials and methods incl. tables")

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: live fetuses, dead fetuses

Blood sampling:

- Blood: Yes
- Serum: Yes
- Volume collected: 1 mL

Fetal examinations:

- Weight of each fetus: Yes
- Sex: Yes
- Gross evaluation of placentae: Yes
- External examinations: Yes: all per litter
- Soft tissue examinations (fixation with Harrison's fluid): Yes: half of the fetuses
- Skeletal examinations (fixation in ethanol): Yes: half of the fetuses
- Anogenital distance (AGD): Yes: all liveborn fetuses

Statistics:

Please refer to section "any other information on materials and methods incl. tables"

Indices:

Conception rate % = no. of pregnant animals/ number of fertilized animals x 100

Pre-implantation loss % = no. ofcorpora lutea − no. of implantations/ no. of corpora lutea x 100

Post-implantation loss % = no. of implantations−no. of live foetuses/ no. of implantations x 100

Anogenital Index = anogenital distance (mm)/ cubic root of fetal weight (g)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- The mean body weights of test group 1-3 dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

- The body weight change of the dams in test groups 2 and 3 (300 and 1000 mg/kg bw/d) were
statistically significantly reduced during GD 6-8 (up to 32% and 42% below control) but recovered
afterwards, respectively, and was comparable to the concurrent control at the end of the treatment period. If calculated for the entire treatment period (GD 6-19), the mid- and highdose dams, each, gained 4% less body weight than the control (without attaining statistical significance). The impairments of body weights in the mid- and high-dose groups are considered to be treatment-related but not adverse. The mean body weights and the average body weight gain of the low-dose dams (100 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
- The corrected body weight gain of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects 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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No necropsy findings which could be attributed to the test substance were seen in any dam. There occurred two spontaneous findings: a dilated renal pelvis in low-dose female No. 39 (both kidneys), in high-dose females No. 82 (both kidneys) and No. 84 (right kidney). Additionally, a cyst in the medulla of the left kidney in mid-dose female No. 54 was observed. These findings were detected in single females of different test groups and, therefore, were assessed as incidental.
- No macroscopic findings were noted in thyroid glands of control and all treatment groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A 100% post-implantation loss was observed in one female of test group 3 (1000 mg/kg bw/d).

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

In high-dose female No. 87 only 10 early resorptions were recorded which were visible without staining (no viable fetuses). Since only one dam was affected, this isolated finding was
regarded as incidental and not treatment-related.

Dead fetuses:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

In test group 2, the mean% value of live fetuses (mean 89.8* [p ≤ 0.05 Dunnett-test]) was statistically significantly decreased. The mean number of live fetuses (10.8) in this test group was well within the historical control range (HCD: mean 10.6 [10.1 - 11.2]). The mean number of resorptions was very low in the concurrent control group (0.4), therefore, the mean% value of live fetuses (96.3) was exceptionally high. Furthermore, there was no dose-response relationship visible (mean% live fetuses: 96.3 / 92.5 / 89.8* / 91.4). Therefore, the statistically significantly decrease in test group 2 is assessed as incidental and biologically not relevant.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in one fetus, each, of the control and the low-dose group (Table 6). All findings are assessed as not treatment-related since they occurred in single fetuses without a relation to dose.
The total incidences of skeletal malformations (Table 7) did not differ significantly from control and were comparable to the historical control data.

Details on Table 6 and 7 can be found in section "Any other information on results incl. tables".

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Three soft tissue variations were detected: short innominate in test groups 0 and 1, dilated renal pelvis in all test groups and dilated ureter in test group 3. The incidences of these variations did not differ significantly from the concurrent control group. All of them can be found in the historical control data at comparable incidences. Therefore, they are assessed as not treatment-related. The incidence of the finding ‘dilated ureter’ in two fetuses of two litters of test group 3 (affected fetuses/litter: 1.4%) was in the range of the historical control data (HCD: mean% 2.4 [0.8 - 4.0]) and, therefore, considered not biologically relevant.
For more details please refere to Table 5 in section "Any other information on results incl. tables".

Other effects:

effects observed, treatment-related

Description (incidence and severity):

FETAL SKELETAL VARIATIONS: EFFECTS OBSERVED, NOT TREATMENT-RELATED
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose (Table 8). The overall incidences of skeletal variations were comparable to the historical control data.
All skeletal variations with statistically significant differences between the control and any treated group were compiled in the table below (Table 9). All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside the historical control range were marked in italicized bold types. Beside the treatment-related effect regarding the affected fetuses/litter rate of ‘unilateral ossification of sternebra (unchanged cartilage), the other increased incidences of skeletal variations were either not related to dose or they were clearly inside the historical control ranges. Therefore, these findings are assessed as not treatment-related.

FETAL SKELETAL VARIATIONS: EFFECTS OBSERVED, TREATMENT-RELATED
As can be seen from table 9, the affected fetuses/litter rate of ‘unilateral ossification of sternebra (unchanged cartilage)’ was statistically significantly increased in test group 3 (1000 mg/kg bw/d). The value was outside the historical control range and therefore a treatment-related effect cannot be ruled out.

FETAL SKELETAL UNCLASSIFIED CARTILAGE OBSERVATIONS: EFFECTS OBSERVED, NOT TREATMENT-RELATED
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (Tab. 10). The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing.

For more details on Table 8, 9, 10 refer to section "Any other information on results incl. tables".

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 5 Total soft tissue variations

		Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day
Litter Fetuses	N N	25 126	25 127	25 130	24 126
Fetal incidence	N(%)	2(2.4)	3(2.4)	4(3.1)	5(4.0)
Litter incidence	N(%)	2(8.0)	3(12)	3(12)	3(13)
Affected fetuses/litter	Mean%	2.4	2.6	3.7	3.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 6 Individual fetal skeletal malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	19-01 M	cleft sternum
1 (100 mg/kg bw/d)	41-07 M	malpositioned and bipartite sternebra
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	none

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Table 7 Total skeletal malformations

		Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day
Litter Fetuses	N N	25 137	25 142	25 140	24 136
Fetal incidence	N(%)	1(0.7)	1(0.7)	0.0	0.0
Litter incidence	N(%)	1(4.0)	1(4.0)	0.0	0.0
Affected fetuses/litter	Mean%	0.8	0.7	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 8 Total fetal skeletal variations

		Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day
Litter Fetuses	N N	25 137	25 142	25 140	24 136
Fetal incidence	N(%)	129 (94)	138 (97)	137 (98)	132 (97)
Litter incidence	N(%)	25 (100)	25 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	94.9	97.1	97.2	96.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 9 Occurence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding	Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day	HCD Mean % (range)
Incomplete ossification of interparietal; unchanged cartilage	13.8	27.6**	15.1	24.7*	18.0 (11.8-26.6)
Dumbbell ossification of thoracic centrum; unchanged cartilage	0.8	1.4	3.7*	3.1	1.9 (0.0-5.5)
Bipartite ossification of thoracic centrum; dumbbell-shaped cartilage of centrum	0.0	2.2*	0.0	0.0	0.1 (0.0-1.3)
Incomplete ossification of sternebra; unchanged cartilage	74.6	82.5	80.9*	75.5	77.1 (67.6-84.2)
Unilateral ossification of sternebra; unchanged cartilage	0.0	0.0	0.0	3.1*	0.8 (0.0-2.2)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent
* = p =< 0.05 (Wilcoxon-test [one-sided])
** = p =< 0.01 (Wilcoxon-test [one-sided])

Table 10 Total unclassified cartilage observations

		Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day
Litter Fetuses	N N	25 137	25 142	25 140	24 136
Fetal incidence	N(%)	94 (69)	91 (64)	106 (76)	92 (68)
Litter incidence	N(%)	24 (96)	24 (96)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	67.9	63.9	73.0	67.3

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.

Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at dose levels of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 25 females per group had implantation sites.

The following test substance-related adverse effects/findings were noted:
Test group 3 (1000 mg/kg bw/d):
• No test substance-related adverse effects on dams, gestational parameters or fetuses
Test group 2 (300 mg/kg bw/d):
• No test substance-related adverse effects on dams, gestational parameters or fetuses
Test group 1 (100 mg/kg bw/d):
• No test substance-related adverse effects on dams, gestational parameters or fetuses