diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

EC Number:

603-923-2

Cas Number:

135590-91-9

Molecular formula:

C16H18Cl2N2O4

IUPAC Name:

diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Test animals

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst breeding colony
- Age at study initiation: about 6 - 10 months
- Weight at study initiation: mean body weight of 2639 ± 152 g
- Housing: individual metal-barred cages on wire-mesh floors
- Diet (e.g. ad libitum): ERKA 6000 standard diet in the form of pellets 4 mm in diameter (manufactured by Robert Koch OHG, Hamm/Westphalia), ad libitum; also 40-50 g autoclaved hay daily.
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-23.5
- Humidity (%): 43-62
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: starch mucilage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspended freshly each day in starch mucilage (20 g potato starch per litre distilled water)

VEHICLE
- Justification for use and choice of vehicle (if other than water): handling at dosing
- Concentration in vehicle: 8, 20, 50 g/L
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the suspensions were guaranteed for a period of at least 4 hours (statements from the Analytical Laboratory, dated 24 August 1990 and 06 February 1991).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: during the morning hours, not further specified
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: Those females with sperm in the vaginal smear were mated again after about 6 hours in case the first mating had not been successful.

Duration of treatment / exposure:

GD 6-18

Frequency of treatment:

once daily

Duration of test:

until GD29

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

15 - 16 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Preliminary range finder study with groups of 2-4 mated Himalayan rabbits administered doses of 200, 320 or 500 mg/kg once daily from the 6th to the 18th day after mating.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: behaviour, general health condition


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: prior to first treatment, once weekly (body weight gains) in the first 3 weeks, GD 19 and GD 29


FOOD CONSUMPTION: Yes, same intervals as body weight
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes


WATER CONSUMPTION: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: gall-bladder, heart, intestines, kidney, liver, spleen, ovary, uterus; (heart, liver, kidneys and spleen were weighed)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead foetuses, placentae, placental weights, diameter of conceptuses under resorption, crown/rump length, foetal weight, sex, survival rate after 24h incubator

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter (brain, eyes, heart and both kidneys)
- Skeletal examinations: Yes: all per litter
- Head examinations: No

Statistics:

Comparisons with the control group of body weights and organ weights: standard MANOVA with sequentially rejective multiple comparisons, and relative food consumption by means of the nonparametric linear model of PURI & SEN (1985) with sequentially rejective multiple comparisons.
The Mantel-Haenszel test with sequentially rejective multiple comparisons (MANTEL & HAENSZEL, 1959) was used for analysing implants, corpora lutea, survival rates, ratios of live and dead foetuses, and conceptuses under resorption. The foetal weights, crown-rump lengths and placental weights were evaluated by means of a multivariate variance analysis corrected for litter effects. With each of these methods the probability of error for each group of parameters was 5%.
The findings at autopsy and at the examinations of organ cross-sections and skeletons were evaluated for foetuses and litters separately by the exact Fisher test. This tested whether the relative incidences of these findings in the dose groups differed from those in the control. For comparing the data with historical controls, ranges of normal variation for groups were calculated. These were drawn up in such a way as to include with a probability of 95 % at least 75 of the values for a group of control animals. The parallelipiped method of WALD (1943) was employed for body weight gains and food consumption among the dams and for mean foetal weights, crown-rump lengths and placental weights per litter. Comparisons of corpora lutea, implants and survival rates were performed with univariate normal ranges by the method of WILKS (1942). For the ratios of live and dead foetuses and of conceptuses under resorption, normal ranges were determined by the triangular method of ROSENKRANZ (1988).

Historical control data:

A group of animals of a given size is normal with respect to a parameter, if at least 75 % of the values lie within the ranges of the historical control groups.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One dam in the 250 mg/kg group aborted during the night from days 22 to 23 of gravidity. Four dams had vaginal haemorrhages, which pointed to an abortion - the first on day 16, the second on day 20, the third on day 23, and the fourth during the night from days 16 to 17 of gravidity. One
other dam delivered prematurely on day 27 of gravidity. In addition to these findings, a decrease or absence of excrement was observed on one or several days between days 11 and 27, involving one dam from the 40 mg/kg group, two dams in the 100 mg/kg group and about half of the dams in the 250 mg/kg group. One dam in the 250 mg/kg group had soft excrement on days 19 and 23 of gravidity. The dams in this group which were killed intercurrently due to abortions had shown marked reductions in food consumption during the one or two weeks prior to killing. The considerably lower
food consumption was matched by reduced quantities of excrement. Lower food consumption was accompanied by reduced water consumption. In the 250 mg/kg group almost all the dams showed generally slight, but in several cases also more marked, body weight losses during the 1st week of treatment.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed

2

Key result

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

pre and post implantation loss

water consumption and compound intake

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
One dam in the 250 mg/kg group had only empty implantation sites in the uterus. There were also five dams in the 250 mg/kg group which had aborted with clinical signs. The animal killed on day 16 of gravidity had 3 empty implantation sites in addition to 6 conceptuses under resorption. Two dams had only conceptuses under resorption in the uterus: 7 in the one which was killed on day 17 of gravidity (having aborted in the night from days 16 to 17) , and 8 in the one which was killed on day 23. The animal killed on day 20 of gravidity had 3 conceptuses under resorption and also 5 dead foetuses, 3 of them severely stunted.
There was no evidence for teratogenic effects.

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

2

Key result

Dose descriptor:

LOAEL

Remarks:

embryotoxicity

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

changes in litter size and weights

3

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

>= 250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other:

Remarks:

highest dose tested

Overall developmental toxicity

Key result

Developmental effects observed:

not specified

Any other information on results incl. tables

Selected maternal parameters:

	Dose (mg/kg bw)
Parameter	0	40	100	250
Dams with live foetuses	13	14	15	8
Number of live foetuses (total)	86	82	84	46

Conclusion:

Based on the results of this study, the NOAEL for maternal toxicity and embryotoxicity in the rabbit was 100 mg/kg bw/day Hoe 107892. No teratogenic effect was observed at any dose level tested.

5/6 abortions observed in the high dose group occurred in dams which demonstrated the most severe characteristics of reduced food and water consumption, body weight loss, and reduced defecation in this group.

It is widely known that food deprivation of does during the phase of organogenesis has significant effects on the development of their offspring and the dams themselves, manifesting as reduced body weight gain or even loss, reduced water consumption and increased number of abortions. Publications are available demonstrating this phenomenon: Petrere, J.A. et al. (1993). Fundam. Appl. Toxicol. 21: 517-522; Matsuzawa, T. et al. (1981). Toxicology 22: 255-259.

Therefore, abortions and embryotoxicity observed in this study have to be considered as secondary effects due to maternal toxicity, induced by reduced food uptake and body weight loss of the dams during the phase of organogenesis.