Chlorothalonil

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Chlorothalonil

EC Number:

217-588-1

EC Name:

Chlorothalonil

Cas Number:

1897-45-6

Molecular formula:

C8Cl4N2

IUPAC Name:

tetrachlorobenzene-1,3-dicarbonitrile

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: rats of approximately 150 g (males)
- Diet: ad libitum
- Water: ad libitum
- Acclimation: All animals will be held for a minimal period of time to assure that all the animals used for the preliminary or main test are in good health.
- Housing: Currently acceptable practices of good animal husbandry was followed at all times

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % (w/w) Methocel E15 Premium (hydroxy-propyl methylcellulose) in ion-exchanged water.
- Amount of vehicle: 2 mL

Details on exposure:

All the suspensions or solutions were prepared extemporeanously in a solution at 0.5 % methocel. Test substance was crushed in a mortar in small quantities of aqueous solution of methacel. The volume was then adjusted with the same vehicle. The animals were not be fasted prior to the administration of the test material, vehicle or the positive control.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Two doses with a 24 hours interval in between

Post exposure period:

6 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group II, suspension at 0.4 mg/mL

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group III, suspension at 2 mg/mL

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group IV, suspension at 10 mg/mL

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group V, suspension at 50 mg/mL

Doses / concentrations 5

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

Group VI, suspension at 250 mg/mL

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

Substance: Methy methasulfonate (MMS)
Route of administration: oral gavage
Doses: 65 mg/kg, suspended at 2.5 μL/mL (Group VII)

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCEs) in bone morrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study was conducted to determine the maximum dose of test substance which could be administered by incubation.

METHOD OF ANALYSIS: The principle of the method consists to search the presence of a rest of chromosome in the young erythrocits of the marrow. Clastogenic agents can cause breakdown of a chromosome at the mitosis and the fragment which is not, at the telophase, included in the cell, appears in the cytoplasm as a small ball (from where the name of micronucleus). Micronuclei have been observed on animals treated with agents considered as mutagenic, at the level of different cells of the marrow, namely the myelocyts and the erythroblasts. However, the most are observed in the polychromatophilic erythrocits, cells particularly interesting for this study, because a few hours after the end of their last mitosis, these cells expel their nucleus. This chromosomal rest is easily recognisable. The young erythrocits, less than 24 hours old, have besides, the advantage to possess tinctorial properties different from the adult erythrocits.
The product is administered in two shots at 24 hours interval and the animals are sacrificed 6 hours after the second administration. The femur is taken and cut at its two ends in order to loosen the medular tubular. The marrow is recovered in serum of veal’s embryon. After centrifugation, the bottom is homogenized and one drop is laid out on a lamina. The smear is dried and coloured by the May Grünwald-Giemsa. The counting of the cells carrying micronuclei is realized on 2000 polychromatophilic erythrocits. The readings are done by two observers which read each on a different lamina 1000 polychromatophilic erythrocits. The average of these two reading is then calculated.

Statistics:

The statistical analysis consisted by a comparison of two measures done with the Student’s method, valid for small samples. The value of t calculated has been compared to the value of t theoric given in a distribution table of t.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

One animal died approximately 4 hours after the second administration.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In the preliminary study four rats were orally dosed with 5000 mg/kg test substance. The rats were dosed twice with a 25 hour interval between doses. One rat died before the second dosing, the other three animals survived at least six hours after the second dose. The maximum dose selected for the micronucleus test was set at 5000 mg/kg.

MICRONUCLEUS TEST
- Dose levels for the micronucleus test in the rat were 0, 8, 40, 200, 1000 and 5000 mg/kg. Ten males rats per group were dosed twice with a 24 hour interval between doses. One high dose rat died four hours after the second administration of test material and was not included in the micronuclei evaluation. The mean per cent polychromatic erythrocytes with micronuclei did not differ statistically from the vehicle control. The mean per cent polychromatic erythrocytes with micronuclei from rats treated with MMS was statistically higher than controls.

Any other information on results incl. tables

Table 1: Polychomatophilic erythrocits average (P.E.) carying micrnuclei in rats

Products	Doses mg/kg (PO)	Number of animals	% of P.E. with micronuclei m -/+ 2 Sm
Control methocel		10	0.16 ± 0.08
Test substance	2 x 8	10	0.21 ± 0.06
	2 x 40	10	0.22 ± 0.04
	2 x 200	10	0.17 ± 0.07
	2 x 1000	10	0.23 ± 0.04
	2 x 5000	9	0.23 ± 0.06
MMS	2 x 65	10	3.22 ± 1.10

Applicant's summary and conclusion

Conclusions:

Orally administered test substance to male rats at 2 x 8, 2 x 40, 2 x 200, 2x 1000 and 2 x 5000 mg/kg has not determined at the level of the marrow any increasing of the average of polychromatophilic erythrocits carrying micronuclei (Schmid’s micronuclei technic). By the same way methylmethanesulfonate utilised as positive control at the dose of 2 x 65 mg/kg appeared as a clastogenic agent. The absence of clastogenic property of the test substance indicates then, according to the interpretation admitted by the technic applied, an absence of mutagenic effect.

Executive summary:

An OECD 474 -like micronucleus test in compliance with GLP was conducted in rats to determine if the substance, orally administered, caused formation of micronuclei in polychromatic erythrocytes. Only male animals were used for the tests. Animals were given two doses of substance by gavage with a 24 hour interval between doses. Polychromatic erythrocytes in the bone marrow were evaluated for the presence of micronuclei. Oral administration of test substance to rats did not cause an increased incidence of micronuclei in polychromatophilic erythrocytes in the bone marrow of these animals.

A possible Mutagenic potentiality has been searched for by the technic of Micronucleus in Rat. Suspension of test substance in an aqueous solution at 0.5 % of methocel has been orally administered in two shots at 24 hours interval to groups of 9 or 10 males rats, 8, 40, 200, 1000 or 5000 mg/kg/day. The animals were sacrificed by cervical dislocation 6 hours after the second shot. Hematogenic marrow smears taken from the femur, were prepared. The polychromatophilic erythrocits count which carry micronuclei was done on 2000 elements per animal, by two observers which read each 1000 elements.

The administration of test substance has not induced any significant increasing of the polychromatophilic erythrocits average carrying micronuclei; an increasing should have indicated a clastogenic effect of the product. On the other hand, methyl-methanesulfonate, utilised as positive control and orally administered at 65 mg/kg/day, increases the polychromatophilic erythrocits average with micronuclei. In conclusion, the absence of the clastogenic effect of test substance expresses, according to the interpretation of the technic utilized, the absence of Mutagenic effect.