Thiophanate-methyl

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 1998 - 12 January 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

February 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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:

mouse

Strain:

other: B6D2F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: 40 days
- Weight at study initiation: 20 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: each group was kept, with the sexes separated, in plastic disposable cages
- Diet: ad libitum, RM1(E)SQC standard laboratory pelleted rodent diet (Special Diet Services
- Water: ad libitum, tap water
- Acclimation period: 5 to 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 48 - 58
- Air changes (per hr): controlled environment
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Methylcellulose
- Justification for choice of solvent/vehicle: MC was selected since it provides homogeneously suspended condition.
- Amount of vehicle: 20 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test substance were freshly prepared on the morning of the test (using identical methods for each phase of the test) and were diluted to the concentrations shown overleaf in aqueous 1% methylcellulose, obtained from Courtaulds, batch number T70654. All animals in all groups were dosed with the standard volume of 20 mL/kg bodyweight. All groups were treated orally by intragastric gavage.

Duration of treatment / exposure:

24 h or 48 h

Frequency of treatment:

once

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5 (per group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Carbendazim (Methyl 1H-benzimidazol-2-ylcarbamate)
- Justification for choice of positive control(s): The test substance is metabolised to carbendazim in vivo, a known spindle poison which is positive in the micronucleus test following oral administration.
- Route of administration: oral, gavage
- Doses / concentrations: prepared as a suspension in aqueous 1 % methylcellulose (details as above) at a concentration of 50 mg/mL just prior to administration.

Examinations

Tissues and cell types examined:

bone marrow of femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
From the results obtained in the preliminary toxicity study, dose levels of 500, 1000 and 2000 mg/kg bodyweight were chosen for the micronucleus test.

TREATMENT AND SAMPLING TIMES:
Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Five males and five females from each group were sacrificed 24 and 48 hours after dosing, with the exception that animals in the positive control group were sampled at the 24 hour time point only. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone.

DETAILS OF SLIDE PREPARATION:
The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner. Eight smears were made from each animal to allow conventional staining (using Giemsa) and optional centromere-specific staining for determining the mechanism of formation of micronuclei. The prepared smears were fixed in methanol (> 10 minutes) then allowed to air-dry before storing in a dust-free environment. Smears for conventional analysis were stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of QUIT'S improved R66 Giemsa (BDH) with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined.

OTHER: Centromeric staining
One unstained reserve slide from each animal in the positive control (carbendazim) and high level treatment group sampled at the 24 hour point and a total of ten slides from 9 animals treated with a chromosome-breaking agent (Mitomycin C) were stained and analysed. Slides from animals treated orally with Mitomycin C at 12 mg/kg and sampled 24 hours later were surplus reserve slides produced during the course of a separate parallel micronucleus test employing CD1 outbred mice, therefore no additional animal experimentation was required to produce the slides. Slides were encoded then stained with the mouse major satellite pan-centromeric probe, which was supplied conjugated to a fluorescent dye (FITC) to allow detection.
Only the hybridised region of each smear was examined using a fluorescence microscope. Micronucleated immature erythrocytes were identified by propidium iodide staining using a green excitation filter and a red barrier filter. Identified micronuclei were checked for the presence of an FITC-stained centromere using a blue excitation filter with a green barrier filter under a 100x oil immersion objective. Centromere-positive micronuclei contained a relatively large (compared with non-specific background staining) yellow-green slightly diffuse signal which was rounded or elongated in shape and lying in the same position and plane as the micronucleus.

THE PROPORTION OF IMMATURE ERYTHROCYTES
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01). This decrease would normally be evident at the 48 hour sampling time; a decrease at the 24 hour sampling time is not necessarily expected because of the relatively long transition time of erythroid cells [late normoblast -> immature erythrocyte (approximately 6 hours); mature erythrocyte (approximately 30 hours)]

Evaluation criteria:

The presence of a high proportion of centromere-positive micronuclei is indicative of chromosome-lagging and, therefore spindle damage. The presence of a low proportion of centromere-positive micronuclei is indicative of chromosome-breaking activity for the test substance. The presence of a high proportion of small micronuclei is indicative of a chromosome-breaking agent, while the presence of a high proportion of large micronuclei implies the presence of whole chromosomes and is indicative of aneugenic activity associated with spindle poisons.

Statistics:

The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (Agresti et al. 1990); for individual inter-group comparisons (Je the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects (see next section) on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000, 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No mortalities or clinical signs of reaction were obtained at any time after administration of the standard limit dose (2000 mg/kg) of the test substance during this preliminary study.
- Harvest times: 1, 3, 6, 12 and 24 hours after treatment (plasma levels), 48 h (toxicity)
- Other: The substance appears to have been rapidly absorbed and systemically distributed before being cleared to reach very low levels between 12 to 24 hours after treatment; clearance was slowest in the high level group. Substantial amounts of the metabolite (carbendazim) were also seen in all groups treated with the test substance. In general the levels of carbendazim were lower but parallel to the detected levels of parent.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: No mortalities and no clinical signs of reaction were seen in any group at any time point in the micronucleus test.

- Induction of micronuclei: Animals treated with test item showed small but highly statistically significant dose-related increases in the number of micronucleated immature erythrocytes at both sampling times [P<0.001] with several mean values lying outside the laboratory historical control range while results for some individual animals lay at the extreme end of the historical control range. Carbendazim caused large, highly significant increases [P<0.001] in the frequency of micronucleated immature erythrocytes.

Centromeric staining
Carbendazim induced the expected high proportion (68%) of micronuclei containing centromeres, while Mitomycin C caused a low proportion of centromere-positive micronuclei (24%). The substance appeared to cause an intermediate proportion of centromere-positive micronuclei (34%).

Size analysis
Carbendazim induced a high proportion of large micronuclei while Mitomycin C induced mainly small micronuclei (mean sizes 40.1 and 25.4 units respectively). The substance appeared to produce micronuclei of intermediate size distribution (mean size 31.9 units)

- Proportion of immature erythrocytes:
A statistically significant dose-related decrease in the proportion of immature erythrocytes was obtained for animals treated with Thiophanate-methyl and sampled at the 24 hour time point [P<0.01]. Since this decrease was only very slight and was not seen at the later sampling time, it is possible that the apparent decrease was the result of chance variation rather than being evidence of treatment-related toxicity. Carbendazim caused a small but statistically significant decrease in the proportion [P<0.001]. It should be noted that cytotoxic compounds such as carbendazim only cause a small decrease in this proportion at the 24 hour sampling time because of the lag caused by erythrocyte maturation, although a much larger decrease would be expected if a later sampling time had been used.

- Appropriateness of dose levels and route: The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.

Applicant's summary and conclusion

Conclusions:

The test substance has shown clear evidence of weak genotoxic activity in the mouse micronucleus test. Qualitative analysis of micronuclei to determine whether the substance operates primarily by causing spindle dysfunction in a similar manner to carbendazim (and would, therefore, be expected to exhibit a threshold dose for activity) were inconclusive. This was because of the large difference in effective dose levels of the two compounds in this study.

Executive summary:

A study according OECD TG 474 was conducted. Male and female B6D2F1 mice were treated by single oral gavage with the test item at dose levels of 500, 1000 or 2000 mg/kg bw. A concurrent negative control group received the vehicle only, while a positive control (PC) group was treated with carbendazim at 1000 mg/kg bw. Bone marrow smears were obtained each from 5 mice/sex/dose group at 24 and at 48 hours after dosing with the exception that mice in the PC group were sampled at the 24 hour time-point only.

A preliminary toxicity test had previously shown that a dose of 2000 mg/kg, the standard limit dose for the micronucleus test, was tolerated; this level was therefore selected as an appropriate maximum for use in this study. Serum analysis of mice treated with the substance in the course of this preliminary study showed that the test item was absorbed and systemically distributed following oral administration, so that significant bone marrow exposure could reasonably be expected. In addition, serum analysis showed that a proportion was metabolized to carbendazim (a known spindle-poison with aneugenic activity).

One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

Mice treated with the test substance showed a small dose-related and highly statistically significant increase in the frequency of micronucleated immature erythrocytes at both sampling times. Increases obtained at the low and intermediate dose levels were very small, with group mean values falling just outside the laboratory historical control range for mice.

A statistically significant dose-related decrease in the proportion of immature erythrocytes was obtained for animals treated with the substance and sampled at the 24 hour time point; since this decrease was only very slight and was not seen at the later sampling time, it is possible that the apparent decrease was the result of chance variation rather than being treatment-related. The positive control compound, carbendazim, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes together with a significant decrease in the proportion of immature erythrocytes.

Centromere-specific staining and size analysis of micronuclei in bone marrow smears prepared from these animals subsequently confirmed that carbendazim produced micronuclei predominantly from lagging chromosomes with the remainder being formed from chromosome fragments. In comparison, a smaller proportion of the micronuclei induced by the substance was apparently formed from lagging chromosomes. However, this difference in proportion could relate to the much lower rate of induction of micronuclei by the test item rather than indicating any fundamental difference in mechanism of activity between the two compounds.

The test substance has been shown to induce low numbers of micronucleated cells in the bone marrow of mice. Qualitative analysis of micronuclei to determine whether the substance operates primarily by causing spindle dysfunction in a similar manner to carbendazim (and would, therefore, be expected to exhibit a threshold dose for activity) was inconclusive. This was because of the large difference in effective dose levels of the two compounds with the test substance inducing much lower numbers of micronuclei than carbendazim at comparable dose levels.