1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Apr 1987 to 16 May 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

EC Number:

262-104-4

EC Name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Cas Number:

60207-90-1

Molecular formula:

C15H17Cl2N3O2

IUPAC Name:

1-{[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl}-1H-1,2,4-triazole

Test animals

Species:

rat

Strain:

other: Crl:COBS CD (SD)BR VAF/PLUS

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight (at mating): 210-300 g
- Fasting period before study: no
- Housing: Individually in solid bottom cages
- Diet: Certified Chow and water ad libitum
- Water: Ad libitum
- Acclimation period: Approximately 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 14/10

IN-LIFE DATES: 29 Apr 1987 to 16 May 1985

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 3 % aqueous cornstarch containing 0.50 % Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing preparations of test substance were prepared as a 0.30, 0.90 or 3.60% suspension in 3% aqueous cornstarch containing 0.50% Tween 80.

VEHICLE
- Concentration in vehicle: 0.30, 0.90 or 3.60% suspension in 3% aqueous cornstarch containing 0.50% Tween 80
- Amount of vehicle: 10 mL/kg bw/day (on the sixth day of dosing the dosing volume of the highest dose group was lowered to 8.3 mL/kg bw/day because of severe maternal toxicity)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each of the samples submitted was assayed for the test substance with HPLC method ASM-87 with modifications.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: not reported
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0

Duration of treatment / exposure:

From day 6 to 15 of gestation

Frequency of treatment:

Daily

Duration of test:

Until day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 1: low dose

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2: mid dose

Doses / concentrations 3

Dose / conc.:

360 mg/kg bw/day (nominal)

Remarks:

Group 3. High dose. At the sixth day of dosing the dose level was lowered to 300 mg/kg bw/day.

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: The 120 females were randomly distributed into 4 dose groups of 30 animals each and a maximum of 24 sperm positive animals were placed on study within each dose group.

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: body weights recorded on gestational days 0, 6, 8, 12, 16 and 20

FOOD CONSUMPTION:
- Food consumption for each animal determined: food consumptions were recorded for a weekly period from day 0 to day 6, and daily thereafter throughout gestation.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 20

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: number of dead and viable fetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes

Statistics:

Body weight, body weight gain and food consumption by One-Way Analysis of Variance (ANOVA) with Bartlett's Test for Homogeneity of Variance and Dunnett's Method of Multiple Comparisons between control and treatment groups. The outcome of the ANOVA are not discussed. The calculations derived from the ANOVA are employed in the subsequent Bartlett's and Dunnett's Tests. The focus of these analyses is on the results of the Dunnett's comparisons between the control and each of the treated groups. When Bartlett's Test results are highly significant (i.e., p<.001), the set of comparisons may be recalculated with the exclusion of outlier(s) or another statistical test may be used.
Foetal weight analysed by the Healy Analysis.
Number of corpora lutea, implantations, resorption sites, viable foetuses; % post-implantation loss, and foetal sex ratios were also analysed statistically.

Indices:

% post-implantation loss

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Severe signs of compound-related maternal toxicity were observed in dams of the high dose group during the first week of dosing, including a statistically significant increase in the incidence of lethargy, ataxia, and salivation when compared to the control group and biologically significant signs of rales, prostration, hypothermia and bradypnea. Due to the severity of these toxic signs, the high dose level was lowered from 360 mg/kg/day to 300 mg/kg/day on the sixth day of dosing. The incidence and severity of these toxic signs decreased immediately following the lowering of the dose level. There were no treatment-related clinical observations at 30 mg/kg/day and only a single observation of rales at 90 mg/kg/day.
Other incidental clinical signs that were observed in the study were scabs, blood around the mouth, and chromodacryorrhea. Alopecia was also observed in all dose groups

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One dam from the control group was found dead on day 20 of gestation in this study. The cause of death was thought to be complications resulting during early labor and delivery. Since this was a control animal, this finding was not compound-related. All other females survived to scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no significant differences in body weight or corrected gestational day 20 body weight between the controls and any treatment group during the course of the study. Statistically significant decreases in maternal weight gain were detected at 90 and 360/300 mg/kg/day for the interval of gestational days 6-8. This interval coincides with the initiation of dosing and also with the observation of severe maternal toxicity in the high dose group. The decrease in weight gain in the high dose group is further substantiated by a statistically significant decrease in weight gain in dams of this group for the interval of gestational days 6-16. There were no statistically significant differences in the corrected body weight change between the control animals and any treatment group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant reduction in food consumption, at 300/360 mg/kg/day from gestational days 7-10, and at 90 mg/kg/day during the intervals of gestational days 8-9 and days 10-11. These intervals correspond to the first few days of dosing, and can be correlated with the severe maternal toxicity observed in the high-dose group and the less pronounced maternal toxicity observed in the intermediate dose group during this same period.

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:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

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

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

30 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

Remarks on result:

other: Marked maternal toxicity at 360/300 mg/kg bw/day

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two out of a total of 1141 viable foetuses were grossly malformed. One female at 90 mg/kg/day had a cleft lip and palate, micromelia and a club foot and a female at 360/300 mg/kg/day had anasarca.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant increase in the incidence of minor skeletal variations at 90 and 360/300 mg/kg/day including increases in the incidence of rudimentary ribs and sternabrae not ossified. In the absence of other foetal effects, these skeletal variations are attributed to the maternal effects observed rather than a specific foetal effect.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

One foetus at 90 mg/kg/day a cleft/hare lip. (Total incidence of cleft/hare lip at 90 mg/kg/day = 2 including the one seen on external observation). At 360/300 mg/kg/day, one foetus had cleft palate and one cleft palate, hydromelia and protruding tongue.
There was a statistically significant increase in the incidence of visceral variations at 360/300 mg/kg/day for renal papilla(e) short, renal papilla(e) absent, and dilated ureters. These variations are thought to represent a slight delay in the normal development of the urinary system and were secondary to the maternal toxicity observed in the high-dose groups and were not representative of any direct foetotoxicity.

Although the incidence of cleft palate is not statistically significant in this study, the occurrence of this malformation in 3 foetuses from 3 litters (one intermediate and two high-dose) is remarkable because cleft palate is an uncommon malformation in CD rats. While cleft palate has not been seen in control CD rats at this facility, data from other laboratories show that cleft palate does occur in control animals at incidence of 0-0.35%. In this study, the incidence of cleft palate in the intermediate dose group was 0.33%, while in the high-dose group the incidence was approximately 0.70%. The cleft palate and foetal variations were thought to be secondary to the maternal toxicity.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Remarks on result:

other: increased incidence of cleft palate at 360/300 mg/kg bw/day, increased incidences of visceral variation at 360/300 mg/kg bw/day and skeletal variations at 90 and 360/300 mg/kg bw/day

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

external: paw

visceral/soft tissue: urinary

Description (incidence and severity):

One female at 90 mg/kg/day had a cleft lip and palate, micromelia and a club foot and a female at 360/300 mg/kg/day had anasarca. At 360/300 mg/kg/day, one foetus had cleft palate and one cleft palate, hydromelia and protruding tongue. There was a statistically significant increase in the incidence of visceral variations at 360/300 mg/kg/day for renal papilla(e) short, renal papilla(e) absent, and dilated ureters.
These effects are a secondary non-specific consequence of maternal toxicity effects.

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Table 1. Intergroup comparison of body weight gain (g) – selected times

	Dose level (mg/kg/day)
Days of gstation	0 (control)	30	90	360/300
0-6	29.05	32.00	32.41	28.09
6-8	7.14	3.76	3.14*	2.68*
12-16	23.09	22.19	23.91	23.27
6-16	46.82	44.38	45.62	40.18*
16-20	49.27	51.33	59.68	59.91
0-20	125.14	127.71	133.05	128.18
0-20#	57.23	52.24	56.11	50.95
* Statistically significant difference from control group mean, p<0.05 # body weight gain excluding gravid uterus weight Table 2. Intergroup comparison of food consumption (g) – selected times   Dose level (mg/kg/day) Days of gestation 0 (control) 30 90 360/300 6-7 21.00 21.90 18.73 18.82 7-8 21.00 21.43 20.32 16.82 8-9 22.18 22.24 19.68* 18.58* 9-10 21.77 23.19 20.82 16.68* 10-11 22.77 22.38 19.82* 20.36 19-20 25.00 25.62 26.45 26.27 * Statistically significant difference from control group mean, p<0.05 ** Statistically significant difference from control group mean, p<0.01 Table 3. Caesarean section observations for all pregnant females Observation Dose level (mg/kg/day) 0 (control) 30 90 360/300 # Animals Assigned (Mated) 24 24 24 24 # Animals Pregnant 23 21 22 22    #Intercurrent deaths 1 0 0 0 % Pregnant 95.8 87.5 91.7 95.7 Corpora Lutea/Dam 16.9 16.7 17.3 16.5 Implantations/Dam 13.5 14.2 14.3 14.0 Total # viable foetuses 270 284 302 285 Total # Litters (viable) 22 21 22 22 Live Foetuses/Dam 12.3 13.5 13.7 13.0 Mean number early resorptions 1.1 0.7 0.5 1.0 Mean number late resorptions 0 0 0 0.1 Mean number resorptions (total) 1.1 0.7 0.6 1.1 Mean number pre-natal death 0.08 0.04 0.04 0.08 % dead foetuses 0.3 0 0 0 Mean Foetal Weight (g) 3.5 3.5 3.5 3.5                Males (g) 3.6 3.7 3.6 3.6                Females (g) 3.3 3.4 3.4 3.4 Sex Ratio (% Males per litter) 51.9 49.3 48.3 46.0 Table 6. Visceral variations  Visceral variations Dose level (mg/kg/day) 0 (control) 30 90 360/300 No. foetuses with renal papilla(e) short 32/141 27/148 40/156 57/148** No. foetuses with renal papilla(e) absent 4/141 4/148 8/156 16/148** No. foetuses with dilated ureter(s) short 38/141 21/148 38/156 63/148** **Statistically different from control at P<0.01 Table 7. Skeletal variations  Skeletal variations Dose level (mg/kg/day) 0 (control) 30 90 360/300 No. foetuses with rudimentary ribs 0/129 1/136 4/146 53/137 No. litters with foetuses with rudimentary ribs 0/22 1/21 4/22* 16/22** No. foetuses with sternebrae not ossified 49/129 54/136 83/146* 99/137** **Statistically different from control at P<0.05 *Statistically different from control at P<0.01

Applicant's summary and conclusion

Conclusions:

In a developmental study performed in compliance with GLP and following an EPA OPP 83-3 guideline, the no observed adverse effect level (NOAEL) for maternal toxicity and developmental toxicity is 30 mg/kg/day. The NOAEL for teratogenicity is 30 mg/kg/day.

Executive summary:

In a developmental study performed in compliance with GLP and following an EPA OPP 83-3 guideline, the test substance was evaluated for its embryotoxic, foetotoxic and/or teratogenic potential in rats. the test substance technical was administered to three groups of 24 pregnant Crl:COBS CD (SD)BR VAF/PLUS rats at doses of 30, 90 or 360/300 mg/kg once daily by gavage during gestational days 6-15. A fourth (control) group received an equivalent volume (10 mL/kg/day) of vehicle only (3% cornstarch in 0.5% Tween 80). On gestational day 20, all dams were sacrificed and a laparohysterectomy was performed followed by an examination of the reproductive tract and its contents.

Severe signs of maternal toxicity were observed at the 360 mg/kg/day dose level. Consequently, the dosage was lowered to 300 mg/kg/day and the severity of the side-effects decreased. However, other signs of treatment-related effects were observed in dams of both the intermediate and high-dose groups and included decreased food consumption and decreased body weight gain.

Administration of the test substance did not have a statistically significant effect on any of the reproductive parameters examined, the incidence of foetal gross, visceral or skeletal malformations, foetal body weights or foetal sex ratios. Cleft palate was observed in three foetuses from three dams, one in an intermediate and two in high-dose group litters. However, historical data from both in-house and other laboratories show that cleft palate does occur sporadically in both untreated rats and in rats treated with non-teratogenic compounds and may be associated with maternal toxicity. The cleft palate was thought to be secondary to the maternal toxicity. Signs of foetotoxicity were observed in both the intermediate and high-dose groups as an increased incidence of minor skeletal and visceral variations. These foetotoxic effects are thought to be secondary to the maternal toxicity rather than due to any direct foetal effects. There was no evidence of any treatment-related embryotoxicity observed in this study. The no-effect level was 30 mg/kg/day in this study for both the foetus and the dam. Evidence of maternal and foetotoxicity was noted at 90 mg/kg/day (lowest effect level). The highest dose, 360 mg/kg/day, was not well tolerated by the dams; lowering the dose to 300 mg/kg/day produced what is considered as the maximum tolerated dose in the study.

The no observed effect level (NOAEL) for maternal toxicity and developmental toxicity is 30 mg/kg/day. The NOAEL for teratogenicity is 30 mg/kg/day.