Captan

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 March and 12 April 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Captan

EC Number:

205-087-0

EC Name:

Captan

Cas Number:

133-06-2

Molecular formula:

C9H8Cl3NO2S

IUPAC Name:

2-[(trichloromethyl)sulfanyl]-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Physical state: white powder
- Analytical purity: 91.2 % (w/w)
- Batch number: WRC 11240-37-1
- Storage conditions: in the dark, room temperature

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna UK, Huntingdon, Cambridgehire, UK.
- Weight at study initiation: 3.2-4.2 kg on the day of insemination (Day 1 of gestitaion)
- Housing: Individually in cages.
- Diet (e.g. ad libitum): CRB pellets supplied by Labsure Animal Diets, Lavender Mill, Manea, Cambridgehire UK.
- Water (e.g. ad libitum): Via an automatic watering system.
- Acclimation period: Yes, seven days prior to insemination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14-19 °C
- Humidity (%): 40-70%
- Air changes (per hr): 25-30 air changes per hour.
- Photoperiod (hrs dark / hrs light):12 hours light / 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Corn oil.
- Storage temperature of food: At room temperature.

IN-LIFE PHASE
- 5 March and 12 April 1990.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of each preparation owas analysed prior to the start of dising to verify the achieved concentrations of captan. The chemical stability of captan in corn oil was determined by re-analysis of the 10mg/kg formulation over a period of 45 days. The homogeneity of captan in corn oil was determined for the 10 and 100 mg/ml formulations.

Details on mating procedure:

Artificial Insemination Proven male New Zealand White rabbits previously obtained from Interfauna UK were used for this study. A total of 15 males was used for collection of semen. Semen was collected from each male using an artificial vagina. The volume of the sample obtained was measured and then diluted with sufficient physiological saline to inseminate one replicate. Approximately 1 ml of the diluted semen was inseminated into each female using a plastic catheter (18FG Nelaton supplied by Portex Limited, Hythe, Kent, UK). Each replicate of females was inseminated with semen from one male. After Insemination, each female was given an intravenous Injection of 25IU PROFASI 500 to promote ovulation. Eighty females were artificially inseminated for the study. The females were within a weight range of 3.2 -4.2 kg on the day of insemination (Day 1 of gestation).

Duration of treatment / exposure:

dosed once daily from days 7-19

Frequency of treatment:

dosed once daily

Duration of test:

30 days

No. of animals per sex per dose:

The study consisted of one control group and three treatment groups. Each contained twenty female rabbits:
Grroup 1: 20 female, Control: 0 mg/kg bw/day
Group 2: 20 female, 10 mg/kg bw/day
Group 3: 20 female, 30 mg/kg bw/day
Group 4: 20 female, 100 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked at lest once each day throughout the study.
- Details of changes in behaviour or clinical condition including no abnormalities detected were recorded daily during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: The bodyweight of each animal was recorded on Days 1 and 4, 7-19 (inclusive) and on Days 22, 26 and 30 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: The amount of food consumed by each animal was measured by giving a weighed quantity of food on Days 1, 4, 7, 10, 13, 16, 19, 22 and 26 and calculating the amount consumed from the residue on Days 4, 7, 10, 13, 16, 19, 22, 26 ad 30.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 30.
- Organs examined: gravid uterus,

Ovaries and uterine content:

On day 30 females were sacrificed, examined macroscopically and the uteri examined for number of corpora lutea, live foetuses and intra uterine deaths. Foetuses were weighed, sexed and examined for external, visceral and skeletal abnormalities.
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: Individual foetal weights

Fetal examinations:

- External examinations: Yes, all foetuses
- Soft tissue examinations: Yes, all foetuses
- Skeletal examinations: Yes, all foetuses
- Head examinations: Yes, all foetuses

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The incidence of few/no faeces on tray was increased in all captan treated groups compared with controls.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related reduction in maternal body weight gain in 30 and 100 mg/kg bw/day dams, which was most marked during days 7 to 10. Compensatory body weight gain was evident during the post-dosing period in the 100 mg/kg bw/day dams.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A dose-related reduction in food consumption was also seen in the 30 and 100 mg/kg bw/day females, followed by a compensatory increase in food consumption by 100 mg/kg bw/day dams, particularly on days 26 to 30. The low dose group was unaffected.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related reduction in maternal body weight gain in 30 and 100 mg/kg bw/day dams, which was most marked during days 7 to 10. Compensatory body weight gain was evident during the post-dosing period in the 100 mg/kg bw/day dams.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

One 100 mg/kg bw/day female was killed intercurrently following signs of abortion.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The incidence of pre-implantation loss in 100 mg/kg bw/day dams was not significantly affected. Post-implantation loss in 100 mg/kg bw/day dams was significantly increased compared to the control and reflected the increased incidences of intra-uterine deaths (early and late).

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Mean total implantation loss was not affected by captan treatment and as a result there was no adverse effect on the mean number of live foetuses compared to the controls.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean foetal weight in the 100 mg/kg bw/day dose group was significantly lower than the control, with a consequent reduction (not statistically significant) in mean gravid uterine weight.

Reduction in number of live offspring:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Mean foetal weight in the 100 mg/kg bw/day dose group was significantly lower than the control, with a consequent reduction (not statistically significant) in mean gravid uterine weight.

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of foetuses with major defects of the head in both the 30 and 100 mg/kg bw/day groups

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: yes.
Embryotoxic effect (100 mg/kg/day): increased post-implantation loss;
foetotoxic effect (100 mg/kg/day): reduced foetal weight.

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Levels of captan in dosing solutions: Achieved concentrations of captan in dosing preparations were ± 4% of nominal levels. Homogeneity and stability of captan in solution were satisfactory.

General observations: Treated dams had an increased incidence of few or no faeces compared to the control. Increased incidences of diarrhoea in all treated groups and mucus in the faeces in 100 mg/kg bw/day dams were observed. One 100 mg/kg bw/day female was killed intercurrently following signs of abortion. There was a dose-related reduction in maternal body weight gain in 30 and 100 mg/kg bw/day dams, which was most marked during days 7 to 10. Compensatory body weight gain was evident during the post-dosing period in the 100 mg/kg bw/day dams (Table 7.8.2-2). A dose-related reduction in food consumption was also seen in the 30 and 100 mg/kg bw/day females, followed by a compensatory increase in food consumption by 100 mg/kg bw/day dams, particularly on days 26 to 30. The low dose group was unaffected.

 

Table 7.8.2-2 Developmental toxicity study in the rabbit: body weight of dams

Period (days)	Body weight gain (g) at dose (mg/kg bw/day)
	0	10	30	100
1 – 7	145.2	137.5	172.9	159.6
7 – 19	238.2	205.5	57.2*	-159.3**
7 – 10	15.6	40.2	-68.0*	-142.5**
10 – 13	85.9	52.5	68.8	11.2**
13 – 16	145.9	122.8	59.3*	23.0**
16 – 19	-9.3	-9.8	-2.9	-51.0
19 – 30	249.4	288.8	300.3	502.3**
19 – 22	31.1	53.1	51.4	150.4**
22 – 26	125.6	141.9	146.7	176.9
26 – 30	91.7	93.8	102.2	175.00**
1 – 30	632.7	631.8	530.4	502.6

Significantly different from the control, * (p < 0.05), ** (p < 0.01)

 

Pregnancy and litter data: There was no treatment-related effect on pregnancy rate. The incidence of pre-implantation loss in 100 mg/kg bw/day dams was not significantly affected. Post-implantation loss in 100 mg/kg bw/day dams was significantly increased compared to the control and reflected the increased incidences of intra-uterine deaths (early and late). Mean total implantation loss was not affected by captan treatment and as a result there was no adverse effect on the mean number of live foetuses compared to the controls. Mean foetal weight in the 100 mg/kg bw/day dose group was significantly lower than the control, with a consequent reduction (not statistically significant) in mean gravid uterine weight. A summary of litter data is given in Table 7.8.2-3.

 

Table 7.8.2-3. Developmental toxicity study in the rabbit: litter data

 

Litter data parameter	Dose (mg/kg bw/day)
	0	10	30	100
Corpora lutea (mean)	10.54	10.23	9.80	10.42
Pre-implantation loss (transformed mean)	0.490	0.428	0.369	0.291
Post-implantation loss (transformed mean)	0.311	0.337	0.325	0.485*
Implantations (mean)	8.46	8.23	8.50	0.50
Intra-uterine deaths (mean % early)	3.1	4.7	0.0	10.7
Intra-uterine deaths (mean % late)	3.5	4.2	9.5	11.9
Live foetuses (mean)	7.85	7.38	7.50	7.17
Mean gravid uterine weight (g)	514.8	481.2	472.7	440.3
Mean litter weight (g)	339.2	319.9	310.3	271.4
Mean foetal weight (g)	45.64	44.90	43.30	37.99**

Significantly different from the control, * (p < 0.05), ** (p < 0.01)

 

Foetal assessment (major defects): The incidence of foetuses with major defects was significantly increased in the 100 mg/kg bw/day dose group compared to the control. Eight foetuses from five litters had at least one major abnormality compared to one in the control. Four of the eight foetuses affected in the 100 mg/kg bw/day dose group were from one litter. There was no compound-related effect on the incidence of defects involving the cardiac blood vessels but there was an increased incidence of foetuses with major defects of the head in both the 30 and 100 mg/kg bw/day groups. In the latter group, two of the three affected foetuses were litter mates. Of the remaining types of major defects all but one occurred in the 100 mg/kg bw/day group. The possibility of the major defects being treatment-related cannot be dismissed though the small numbers involved and type of specific minor defects and the fact that several foetuses were litter mates, provides no strong evidence for a compound relationship.

Foetal assessment (minor defects): The incidence of minor external/visceral defects was increased in the 30 and 100 mg/kg bw/day dose groups compared to the control and was mainly due to an increase in the incidence of cysts on the liver. The overall incidence of minor skeletal defects was unaffected by treatment, although the incidence of unossified 6^thand 7^thlumbar transverse processes and asymmetric alignment of the pelvic girdle was increased in the 100 mg/kg bw/day dose group.

Foetal assessment (variants): There were no external or visceral variants. Each foetus had at least one skeletal variant. Treatment-related effects were seen in the incidence of the following variants in the 100 mg/kg bw/day dose group; odontoid partially ossified (increase); normal length extra 13th ribs (increase); 27 pre-sacral vertebrae (increase): asymmetric development of 1st and 2nd sacral vertebrae (increase); 4th, 5th, 6th and 7th lumbar transverse processes partially ossified; 2nd lumbar transverse processes partially ossified; 3^rdlumbar transverse processes fully ossified. The effects on ossification of the lumbar transverse processes reflect reduced ossification which may be associated with reduced foetal weight. The majority of these variants were also seen in the 30 mg/kg bw/day dose group.

The overall incidence of foetuses with extra 13th rib was increased in 30 and 100 mg/kg bw/day dose groups compared to the control (60.8, 52.1, 77.3 and 77.6% affected in the control, 10, 30 and 100 mg/kg bw/day dose groups).

Foetal assessment (manus and pes assessment): An increase in manus scores, reflecting the decrease in ossification was observed in the 100 mg/kg bw/day dose group (mean score 2.56, p <0.01) compared to the control (mean score 2.24). The mean pes score was 2.0 for all dose groups. A summary of the incidence of defects and variants is given in Table 7.8.2-4.

 

Table 7.8.2-4 Developmental toxicity study in the rabbit: summary of foetal defects and variants

 

Incidence of foetal defects and variants	Dose (mg/kg bw/day)
	0	10	30	100
Number of litters examined	13	13	10	12
External and visceral:
Number of foetuses examined	102	96	75	86
Number of foetuses with major defects	1	0	2	6*
Number of foetuses with minor defects only	1	3	6*	8**
Skeletal defects:
Number of foetuses examined	102	96	75	85
Number of foetuses with major defects	0	0	1	4*
Number of foetuses with minor defects only	37	41	25	36
Variants:
Number of foetuses examined	102	96	75	85
Number of foetuses showing variants	102	96	75	85

Significantly different from the control, * (p < 0.05), ** (p < 0.01)

 

Gross pathology dams: There was no evidence of treatment-related effects on the incidence of macroscopic findings

 

Applicant's summary and conclusion

Conclusions:

The administration of captan at dose levels of 30 and 100 mg/kg bw/day was associated with maternal toxicity and retarded foetal development. Although increases in major defects at these dose levels provided no consistent evidence of a treatment-relationship due to the diverse nature of the abnormalities seen, an association with the administration of captan cannot be dismissed. The overall NOEL (maternal and foetal) was 10 mg/kg bw/day.

Executive summary:

The purpose of this study was to investigate the effects of captan on the embryonic and foetal development of the rabbit when administered by gavage during organogenesis.

The rabbit is one of the species generally recommended for assessment of teratogenicity and the New Zealand White strain was used because of its known susceptibility to a number of teratogens and because of the substantial background data within this laboratory relating to studies of this type. The oral route was chosen for administration of captan as this represents a possible route of human exposure.

Groups of 20 female New Zealand White rabbits were dosed by gavage with 0, 10, 30 or 100 mg captan/kg/day in corn oil from Days 7-19 (inclusive) of gestation which thus included the period of major organogenesis. A control group of animals received corn oil alone.

The day of insemination was designated Day 1 of gestation. On Day 30 of gestation the females were killed and their uteri examined for live foetuses and intra-uterine deaths. The foetuses were weighed, examined for external and visceral abnormalities, sexed, eviscerated and stained for skeletal examination.

Administration of captan at dose levels of 30 and 100 mg/kg/day was associated with maternal toxicity and developmental toxicity.

An increase in major defects at these dose levels provided no consistent evidence of a compound relationship due to the diverse nature of the abnormalties seen. The overall no-effect level was 10 mg captan/kg/day.