Thiophanate-methyl

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 1997 - November 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Thiophanate-methyl

EC Number:

245-740-7

EC Name:

Thiophanate-methyl

Cas Number:

23564-05-8

Molecular formula:

C12H14N4O4S2

IUPAC Name:

methyl N-{[2-({[(methoxycarbonyl)amino]methanethioyl}amino)phenyl]carbamothioyl}carbamate

Test material form:

solid: particulate/powder

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc, Denver, Pennsylvania, USA
- Age at study initiation: 6 month
- Weight at study initiation: 3.27-4.51 kg
- Fasting period before study: no
- Housing: individually
- Diet: From the day of arrival until DG 6, each rabbit was given approximately 150 g of Certified Rabbit Chow® #5322 (Purina Mills, Inc.) daily in an individual stainless-steel "J-type" feeder attached to each cage. Beginning on DG 6, the first day of dosage, each rabbit was given approximately 180 g of Certified Rabbit Chow®#5322 daily.
- Water: ad libitum, local water that had been processed by passage through a reverse osmosis membrane
- Acclimation period: one day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations of the test substance were prepared weekly. The test substance was considered 100% pure for the purpose of dosage calculations.

VEHICLE
- Justification for use and choice of vehicle: Substance no-soluble in water.
- Amount of vehicle: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration and homogeneity analyses indicated no test substance contamination of the vehicle, all samples for the 0.5, 1.0, 2.0 and 4.0 mg/mL concentrations were within ±10% of target (5, 10, 20 and 40 mg/kg/day dosages, respectively) and the formulations were homogeneous. Stability analyses indicated the formulations were stable for a period of at least 7 days when stored refrigerated.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

gestation day 6 to 28

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a pilot study using two routes of oral exposure (stomach tube or diet) were selected to allow comparison of exposure. Six pregnant rabbits per test group were exposed to 0, 5, 10, 20, 40 and 80 mg/kg/day from GD 6 to 18. Based on data of this preliminary study, dosages of 80 mg/kg bw/day of thiophanate-methyl either in the diet or via gavage, appeared too high to be recommended for the developmental toxicity study in rabbits. The 10 mg/kg bw/day stomach tube dosage would be expected to be a no-observable-adverse-effect-level (NOAEL) for both maternal and embryo-foetal toxicity and the 40 mg/kg bw/day stomach tube dosage would be expected to produce maternal toxicity and minimal developmental toxicity. As comparable toxicity occurred via both the dietary and stomach tube routes, the gavage route was used in the study based on the ability to more accurately achieve the desired dosage.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Examination for clinical observations of effects of the test substance, abortions, premature deliveries and deaths

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on DGs 0 and 6 through 29

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
The thoracic, abdominal and pelvic viscera of each rabbit were examined for gross lesions. Uteri of rabbits that appeared nonpregnant were stained with 10% ammonium sulfide, to confirm the absence of implantation sites. Gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation (with the exception of parovarian cysts, which are common, spontaneous lesions in rabbits); all other tissues were discarded.

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

Fetal examinations:

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

Statistics:

Bartlett’s test, Fisher's exact test, Dunnet’s test, Kruskall-Wallis test; Dunn’s test

Historical control data:

Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The 40 mg/kg bw/day dosage group had significantly increased numbers of rabbits with abnormal faeces. Scant faeces and no faeces were observed for one or more does on DGs 7 to 24 and DGs 9 to 17, respectively. All other clinical observations were considered unrelated to the test substance because the incidences were not dosage-related.

Mortality:

no mortality observed

Description (incidence):

No deaths or abortions occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight gain for the entire dosage period was reduced in the 20 mg/kg/day dosage group (-12 %, p<0.05) and in the 40 mg/kg/day dosage group (-76%, p<0.01), as compared with the control group values. Within this period, the 20 mg/kg/day dosage group had significantly reduced weight gain on DGs 12 to 15 (-56%), and the 40 mg/kg/day dosage group had significant weight losses on DGs 6 to 12 (from -238% to -233%) and significantly reduced weight gain on DGs 12 to 15 (-67%). Reflecting these effects of the test substance, weight gains for the entire gestation period were significantly reduced in the 40 mg/kg/day dosage group (-68%). Maternal body weights were generally significantly reduced in the 40 mg/kg/day dosage group on DGs 10 to 29 (from -8% to -6%).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative feed consumption values were significantly reduced in the 20 and 40 mg/kg/day dosage groups for the entire dosage period (-12% and -12% at 20 mg/kg/day; -44% and -41 % at 40 mg/kg/day). Within this period, relative feed consumption values were significantly reduced in the 20 mg/kg/day dosage group on DGs 6 to 18 (from -20% to -16%), and absolute and relative feed consumption values were significantly reduced in the 40 mg/kg/day dosage group on DGs 6 to 24 (absolute, from -71 % to -15 %) or 21 (relative, from -70% to -24%).

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only necropsy observations were parovarian cysts occurring for one control group doe and four 10 mg/kg bw/day dosage does. The statistically significant increase in this gross lesion in the 10 mg/kg bw/day dosage group was not considered related to the test substance because 1) the incidence was not dosage-dependent and 2) this is a common observation in pregnant rabbits.

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:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

One control group litter consisted of two early resorptions, an event that is not unusual for a litter of only two conceptuses. Because such events may abnormally skew the data distributions, all values for this doe and litter were excluded from statistical analyses.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One 5 mg/kg bw/day dosage group doe had a litter consisting of eight live foetuses, two dead foetuses and one late resorption, a non-dosage dependent observation. No other dead foetuses occurred in this study.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Two premature deliveries occurred that were considered unrelated to effects of the test substance as they occurred in the 10 and 20 mg/kg bw/day dose groups and thus were not dosage-related.

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

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):

One 5 mg/kg bw/day dosage group fetus had an umbilical hernia with protrusion of the intestines. One 20 mg/kg bw/day dosage group fetus had a skin closure defect on the head and short 3rd and 4th digits on the right hindpaw with absence of the 3rd
and 4th phalanges (first observed at skeletal examination). No gross external variations occurred in the fetuses in this study.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The 40 mg/kg bw/day dosage group had a significant increase in the average for thoracic ribs (supernumerary ribs), with associated significant increases and reductions in the average for thoracic and lumbar vertebrae, respectively. This variation was considered an effect of the test substance because the values were statistically significant and exceeded the historical ranges of the testing facility. This variation in rib development was considered by the study authors to be a common finding at maternally toxic dosages and to be reversible.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One 40 mg/kg/day dosage group fetus had a diaphragmatic hernia as the only fetal alteration. The stomach and the left lateral lobe and a portion of the median lobe of the liver protruded through the hernia and into the left dorsal quadrant thoracic cavity.
One 40 mg/kg/day dosage group fetus had a pink 0.3 cm x 0.1 cm mass present on the cortex of the left kidney.
One control group fetus had fused apical and cardiac lung lobes and absence of the left diaphragmatic lung lobe. One control group fetus, one 5 mg/kg bw/kg/day dosage group fetus, two 10 mg/kg bw/day dosage group littermates, two 20 mg/kg bw/day dosage group littermates and two fetuses from different 40 mg/kg bw/day dosage litters had absence of the intermediate lung lobe, a common variation in lung development in this rabbit strain. No additional alterations occurred in these fetuses.
One 5 mg/kg bw/day dosage group fetus had a circumcorneal hemorrhage in one eye, a variation generally attributable to trauma during processing.
One 5 mg/kg bw/day dosage group fetus had no gallbladder as the only fetal alteration.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

Description (incidence and severity):

This variation in rib development was considered by the study authors to be a common finding at maternally toxic dosages and to be reversible.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL for maternal toxicity was 10 mg/kg bw/day based on reductions in maternal body weight gain and absolute and relative feed consumption. The developmental NOAEL was set at 20 mg/kg bw/day based on supernumerary thoracic ribs at 40 mg/kg bw/day.

Executive summary:

In a study according OECD TG 414 pregnant New Zealand White rabbits were used to investigate the teratogenic potential of the test item.

Dosages of 0, 5, 10, 20 or 40 mg/kg bw/day were investigated, administered by stomach tube to pregnant rabbits on GDs 6 through 28, with Caesarean-delivery of the foetuses on GD 29. All rabbits were observed for viability at least twice each day of the study. Additional examinations for clinical observations of effects of the test substance, abortions, premature deliveries and deaths were made before each daily intubation and approximately 60 minutes after intubation during dosage period. These observations were also made on the day the rabbits were sacrificed (DG29). Body weights were recorded on DGs 0 and 6 through 29. Feed consumption values were recorded daily after arrival at the Testing Facility. All rabbits were sacrificed on DG 29. The thoracic, abdominal and pelvic viscera of each rabbit were examined for gross lesions. The number of corpora lutea in each ovary was recorded. The uterus was excised and examined for pregnancy, number and distribution of implantations, early and late resorptions and live and dead foetuses. Each Caesarean-delivered foetus was weighed, examined for gross external alterations and individually identified with a tag noting study number, litter number and uterine distribution. After sacrifice, all live foetuses were examined internally to identify sex and visceral alterations; cavitated organs were evaluated by dissection. The brain was cross-sectioned and examined in situ. All foetuses were evaluated for skeleton alterations. Rabbits that prematurely delivered a litter were evaluated following the procedures described above. Delivered pups were weighed, examined for sex, and external and visceral alterations. Pups delivered on the day of scheduled Caesarean-sectioning were also examined for skeletal alterations.

No mortality was observed. The 40 mg/kg bw dosage group had significantly increased numbers of rabbits with abnormal faeces. Scant faeces and no faeces were observed for one or more does on DGs 7 to 24 and DGs 9 to 17, respectively. All other clinical observations were considered unrelated to the test substance because the incidences were not dosage-related.

Maternal body weight gain for the entire dosage period was reduced in the 20 mg/kg/day dosage group (-12 %, p<0.05) and in the 40 mg/kg/day dosage group (-76%, p<0.01), as compared with the control group values. Within this period, the 20 mg/kg/day dosage group had significantly reduced weight gain on DGs 12 to 15 (-56%), and the 40 mg/kg/day dosage group had significant weight losses on DGs 6 to 12 (from -238% to -233%) and significantly reduced weight gain on DGs 12 to 15 (-67%). Reflecting these effects of the test substance, weight gains for the entire gestation period were significantly reduced in the 40 mg/kg/day dosage group (-68%). Maternal body weights were generally significantly reduced in the 40 mg/kg/day dosage group on DGs 10 to 29 (from -8% to -6%).

Absolute and relative feed consumption values were significantly reduced in the 20 and 40 mg/kg/day dosage groups for the entire dosage period (-12% and -12% at 20 mg/kg/day; -44% and -41 % at 40 mg/kg/day). Within this period, relative feed consumption values were significantly reduced in the 20 mg/kg/day dosage group on DGs 6 to 18 (from -20% to -16%), and absolute and relative feed consumption values were significantly reduced in the 40 mg/kg/day dosage group on DGs 6 to 24 (absolute, from -71 % to -15 %) or 21 (relative, from -70% to -24%). At 40 mg/kg/day scant faeces or no faeces were also observed.

Concentration and homogeneity analyses indicated no test substance contamination of the vehicle, all samples for the 0.5, 1.0, 2.0 and 4.0 mg/ml concentrations were within ±1 0% of target (5, 10, 20 and 40 mg/kg/day dosages, respectively) and the formulations were homogeneous. Stability analyses indicated the formulations were stable for a period of at least 7 days when stored refrigerated.

No abortions occurred during the study. Two premature deliveries occurred that were considered unrelated to effects of the test substance as they occurred in the 10 and 20 mg/kg bw/day dose groups and thus were not dosage-related. One control group litter consisted of two early resorptions, an event that is not unusual for a litter of only two conceptuses. Because such events may abnormally skew the data distributions, all values for this doe and litter were excluded from statistical analyses. One 5 mg/kg/day dosage group doe had a litter consisting of eight live fetuses, two dead fetuses and one late resorption, as previously described a non-dosage dependent observation. No other dead fetuses occurred in this study.

The litter averages for corpora lutea, implantations, litter sizes, live and dead fetuses, early and late resorptions, percent resorbed conceptuses, percent male fetuses and fetal body weights, as well as the numbers of does with any resorptions or with viable fetuses did not differ significantly in the five dosage groups and were within the historical ranges of the testing facility. All placentae appeared normal.

No test substance-related fetal malformations were observed, however the 40 mg/kg/day dosage group had a significant increase in the average for thoracic ribs (supernumerary ribs), with associated significant increases and reductions in the average for thoracic and lumbar vertebrae, respectively. This variation was considered an effect of the test substance because the values were statistically significant and exceeded the historical ranges of the testing facility. This variation in rib development was considered by the study authors to be a common finding at maternally toxic dosages and to be reversible. The NOAEL for maternal toxicity was 10 mg/kg bw/day based on reductions in maternal body weight gain and absolute and relative feed consumption. The developmental NOAEL was set at 20 mg/kg bw/day based on supernumerary thoracic ribs at 40 mg/kg bw/day.