ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Oct - 09 Nov 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Several parameters according to the current version of OECD TG 414 (2018) are missing.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Current version adopted in 2018

Deviations:

yes

Remarks:

Animals were dosed from Gestation Day (GD) 7 - 16 (not GD 5 - 20). Measurement of thyroid weight and histopathological examination of thyroid were not done. T4, T3 and TSH blood levels were not measured. Anogenital distance was not measured.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Guideline in place during study conduct: adopted in 1981

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted in 1987

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted in 1984

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: JMAFF (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted in 1985

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-870-0

EC Name:

-

Cas Number:

163520-33-0

Molecular formula:

C18H17NO3

IUPAC Name:

ethyl 5,5-diphenyl-4,5-dihydro-1,2-oxazole-3-carboxylate

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hoe: WISKf(SPF71)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, Germany; SPF breeding colony
- Age at study initiation: approx. 8 - 10 weeks
- Mean body weight at Day 1: 221.5 g
- Housing: individually, in Makrolon cages (type III) on soft wood granulate
- Diet: Ssniff R-Z (V1324), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 45 - 68
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Oct 1995 To: 09 Nov 1995

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% (w/v) aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance dosing formulation was prepared daily, immediately before dosing.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each concentration, samples were taken towards the start, middle and end of the dosing period (Day 7 - 16). Only the samples from the start were analysed. The archieved concentration, stability and the homogeneous distribution of the test substance in the vehicle over 4 h were acceptable.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: detection of sperm in vaginal smears. The day of sperm detection was defined as Day 1 of gestation, and the day of mating was defined as Day 0 of pregnancy. Pregnancy was confirmed at necropsy by the detection of implantation sites or normally developed corpora lutea.

Duration of treatment / exposure:

Day 7 - 16 of pregnancy

Frequency of treatment:

Daily

Duration of test:

21 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

120 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

23 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose-range finding study groups of mated female Wistar rats received the test substance orally at the dose levels of 500 mg/kg bw/day (2 females) or 1000 mg/kg bw/day (4 females) from Day 7 to 16 of pregnancy and were killed on Day 21. One animal at 1000 mg/kg bw/day was found dead on Day 17 (after showing clinical signs of toxicity on Day 16). No clinical signs were observed in the other animals of the 1000 mg/kg bw/day dose group or in the animals treated with 500 mg/kg bw/day. Body weight change, food consumption and litter data did not reveal any compound-related effect. Based on the results of this study and on request of the sponsor, the dose levels of 0, 15, 120 and 1000 mg/kg bw/day were selected. 1000 mg/kg bw/day was included because it is the recommended limit dose according to guideline. The lowest dose level of 15 mg/kg bw /day was selected based on a 90-day oral repeated dose toxicity study with rats, where the NOEL was set at 15.3 mg/kg bw/day. The mid dose level, 120 mg/kg bw/day, was approximately the log mid-point between the high and low dose level.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Several times daily (on weekends and public holidays once daily).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 1, 4, 7, 10, 14, 17, 19 and 21 of pregnancy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Between Days 1 - 7, 7 - 10, 10 - 14, 14 - 19 and 19 - 21 of pregnancy.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes, externally and internally
- Sacrifice on Gestation Day 21
- Organs examined: thoracic and abdominal contents, Uterus

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
- Number of live and dead fetuses: Yes
- Other: The fetuses, the placentae and conceptuses undergoing resorption were removed from the uterus, weighed or measured and examined for gross external abnormalities.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No
- Other: crown-rump length was recorded

Statistics:

All data were recorded on-line and compiled by a data processing system (ARTEMIS). The statistical evaluation is based on the assumption of a monotone dose-response relationship.
Statistical comparisons of the low dose groups with the simultaneous control group were only carried out if significant effects were detectable in the high dose group. Two-sided questions were generally tested as follows: a two-sided comparison with the high dose group was followed by a one-sided test for the low-dose group. In case of the caesarean section data of the multivariate statistics were first of all calculated and used in selecting relevant dose groups. For the individual parameters, sequential comparisons with the high dose group and sequential tests at the 5% level for the low dose were then conducted.

For the Wilcoxon test the exact distribution of the meaned ranks was calculated.

Food consumption: mean consumption/100 g bw was always calculated between two successive measurement times and evaluated by the rank sum test after Wilcoxon.
Body weight gain: the change in weight was determined in comparison to the initial weight. The univariate evaluation was carried out using t-tests.

The caesarean section data of the foetuses were used to calculate litter mean values. Multivariate evaluation was carried out using the test statistics of Wilks. In the univariate analysis,t-tests were used.

The number of corpora lutea, implantation sites and live foetuses, and quotas of dead embryonic primordia undergoing resorption in the animals were likewise analysed using one-sided Wilcoxon tests.

The findings obtained at autopsy, during visceral examination and skeletal examination of the foetuses were evaluated separately for the foetuses and for the litters by exact Fisher test at significance levels of 5% and 1% and compared with actual and previous control group data.

Indices:

% of pre- and post-implantation loss, % of implantation

Historical control data:

Historical data used for comparison are not given in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in any of the animals.

Mortality:

mortality observed, treatment-related

Description (incidence):

One high dose group animal died on Day 21 of pregnancy. The mortality was considered to be treatment-related since 1000 mg/kg bw/day was about half of the acute oral LD50 value for rats and one mortality occurred on Day 17 in the dose range-finding study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, body weight gain was slightly but statistically significantly reduced from Day 10 until the end of the study (Table 1 under "Any other information on results incl. tables"). Body weight changes before beginning of treatment (Days 4 and 7) were not considered to be treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, food consumption was slightly but statistically significantly decreased between Days 7 - 19 of pregnancy (Table 2 under "Any other information on results incl. tables"). Decreases in food consumption before beginning of treatment (Days 1 and 7) were not considered to be treatment-related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Gravid uterus weights were comparable in all groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day one female exhibited whitish fluid in the renal pelvis at necropsy. The animal found dead on Day 21 showed autolysis. The uterus contained 12 well-developed foetuses.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

No effects on relative pre-implantation loss values were observed (18.46, 13.37, 8.83, and 18.47% with ascending dose, respectively).
In the 1000 mg/kg bw/day dose group (high dose, inducing mortality of dams), the relative post-implantation loss values were increased (5.38, 4.69, 6.76, and 12.00% with ascending dose, respectively). However, as the high dose exceeds the maximum tolerable dose (MTD), this increase does not indicate an developmental toxicity potential of the test substance.
See also Table 3 under "Any other information on results incl. tables".

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Number of early and late conceptuses undergoing resorption based on per cent of implantations ('early intrauterine deaths') was slightly but statistically significantly increased in the animals dosed at 1000 mg/kg bw/day (high dose, inducing mortality of dams) (Table 3 under "Any other information on results incl. tables").

Dead fetuses:

no effects observed

Description (incidence and severity):

Because the number of early and late conceptuses undergoing resorption based on per cent of implantations ('early intrauterine deaths') was slightly but statistically significantly increased in the animals dosed at 1000 mg/kg bw/day, the number of live foetuses based on per cent of implantations was slightly, but statistically significantly decreased (Table 3 under "Any other information on results incl. tables"). Incidence of dead foetuses ('late intrauterine deaths') was not increased.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

With the exception of 1 female of the control group and 4 females of the 120 mg/kg bw/day mid dose group, respectively, all animals became pregnant.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no toxic effects observed

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

3

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no toxic effects observed

4

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Effects observed at high dose level exceeding the MTD.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The number of early and late conceptuses undergoing resorption, based on per cent of implantations ('early intrauterine deaths'), was slightly but statistically significantly increased in the animals dosed at 1000 mg/kg bw/day. In direct consequence, the number of live foetuses based on per cent of implantations was slightly, but statistically significantly decreased (Table 3 under "Any other information on results incl. tables"). Incidence of dead foetuses ('late intrauterine deaths') was not increased.
One dead foetus occurred in the control group, and two dead foetuses were observed in the intermediate dose group.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no test substance-related effects. Situs inversus viscerum occurred in 1 foetus of the high dose group. In the mid dose group, 1 foetus showed diaphragmatic hernia and undescended testes (retentio testis). Statistical evaluation did not reveal differences between the groups, and the incidences were within the historical range of the rat strain used.
External examination of dead foetuses did not reveal any abnormalities.

Skeletal malformations:

no effects observed

Description (incidence and severity):

In all skeletal examinations, no test substance-related effects were observed, no statistical differences of the findings between the groups combined with dose-dependency were found, and all incidences were within the historical control data range of the strain used.
No test substance-induced variations and retardations were observed.

Visceral malformations:

no effects observed

Description (incidence and severity):

In all visceral examinations, no test substance-related effects were observed, no statistical differences of the findings between the groups combined with dose-dependency were found, and all incidences were within the historical control data range of the strain used.
No test substance-induced variations and retardations were observed.

Other effects:

no effects observed

Description (incidence and severity):

Crown-rump lengths and placental weights remained unaffected by test substance administration.

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxic effects observed

2

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Remarks on result:

other: Effects observed at high dose level exceeding the MTD.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 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: Group mean body weights (g)

Day	Dose level (mg/kg bw/day)
	0	15	120	1000
1	221.0	223.6	220.7	220.8
4	239.9	239.8	240.0	236.7*
7	252.9	249.8	250.4	247.4*
10	264.2	260.0	262.7	254.1*
14	280.6	275.7	280.5	269.2*
17	299.4	296.3	303.5	286.5*
19	322.3	318.8	328.0	306.5*
21	348.8	344.5	354.4	328.7*

* Significantly less than control

 

Table 2: Group mean food consumption (g/100 g bw)

Day	Dose level (mg/kg bw/day)
	0	15	120	1000
1-7	8.3	7.9	8.0	7.7*
7-10	7.8	7.5	7.7	6.9*
10-14	7.8	7.5	7.7	7.2*
14-19	8.0	7.6	8.0	7.5*
19-21	7.4	7.1	7.2	7.3

* Significantly less than control

 

Table 3: Developmental toxic effects (Group means)

	Dose level (mg/kg bw/day)
	0	15	120	1000
Early intrauterine deaths
Total	14	14	13	30
% of implantations	5.03	4.69	5.60	12.00^#
Late intrauterine deaths
Total	1	0	2	0
% of implantations	0.35	0.00	1.16	0.00
Number of foetuses
Total	256	275	233	230
% of implantations	94.62	95.31	93.24	88.0*

^#Significantly higher than control

* Significantly less than control

Applicant's summary and conclusion

Conclusions:

In conclusion, daily administration of the test substance to rats at the regulatory limit dose level of 1000 mg/kg bw/day from Day 7 - 16 of pregnancy caused maternal, systemic toxicity and exceeded the maximum tolerable dose (as evidenced by mortality, decreased body weight gain and decreased food consumption) and a slightly increased incidence of embryonic deaths. However, there was no evidence of any test substance-induced effect on foetal morphology and no teratogenic effect. The effects reported were secondary to systemic toxicity and are not indicative for a developmental toxicity and teratogenic potential of the test substance.
Neither maternal nor embryo-foetal toxicity were observed after repeated administration of 120 mg/kg bw/day. The 'No Observed (Adverse) Effect Level' (NO(A)EL) for maternal toxicity and embryofetal effects was 120 mg/kg bw/day.