Thiram

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan - 26 Feb 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

Missing strain for AT reversion site (S. thyphimurium TA 102 or E.coli WP2)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Thiram

EC Number:

205-286-2

EC Name:

Thiram

Cas Number:

137-26-8

Molecular formula:

C6H12N2S4

IUPAC Name:

thiram

Method

Target gene:

His operon

Species / strain

Species / strain / cell type:

bacteria, other: TA 98, 100, 1535, 1538 and 1537

Additional strain / cell type characteristics:

other: His auxotrophy, defective DNA repair activity (delta uvrB) and defective LPS barrier (rfa) Ampicillin resistance (TA 98 and TA 100 only)

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats which received a single injection of 500 mg/kg bw Aroclor 1254 in olive oil 5 days before sacrifice.

The protein concentration of the S9 preparation was usually between 20 and 45 mg/mL.

The S9 fraction was mixed with cofactors to final concentrations of: 8 mM MgCI2, 33 mM KCI, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate buffer (pH 7.4).

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 5.6 % v/v in the S9 mix. Each batch of S9 was routinely tested for its capability to activate the known mutagen 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

The standard plate incorporation assay was performed using 1, 10, 33.3, 100, 333.3, 666.6 and 1000 µg/plate of the test item with S9 mix using Salmonella strains TA 98, TA 100, TA 1535, TA 1538 and TA 1537.

Without S9 mix Salmonella strains TA 100, TA 1535,TA 1538 and TA 1537 were used in the presence of 1, 3.3, 10, 33.3, 66.6 and 100 µg test substance/plate.

Strain TA 98 was tested with 10, 33.3, 100, 333.3, 666.6 and 1000 µg test substance/plate, without S9 mix.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine, 4-NOPD, TA 1537, TA 1538, TA 98, 50 μg/plate in DMSO; - S9 mix, 2-aminoanthracene, 2-AA, TA 1535, TA 1537, TA 1538, TA 98, TA 100, 10 µg/plate in DMSO, + S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2 (with and without metabolic activation, plus a pre-test for strains TA 98 and TA 100)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

A pre-experiment for testing the toxicity of the test article was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutagenicity with each 3 plates.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 100) or thrice (strains TA 1535, TA 1537, TA 1538, TA 98) the colony count of the corresponding solvent control is observed. Also, a significant dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential.

Acceptability:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data

Statistics:

No statisitcal analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects occurred with and without metabolic activation at 1000 µg/plate (exp.II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects occurred with and without metabolic activation at 1000 µg/plate (exp.II).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects occurred at 333.3 and 1000µg/plate (exp.I) with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

dose-dependent and significant increase in revertant colony numbers with and without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

normal background growth up to 1000 µg/plate with S9 mix and up to 100 µg/plate in the absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

dose-dependent and significant increase in revertant colony numbers with and without metabolic activation.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

normal background growth up to 1000 µg/plate with S9 mix and up to 100 µg/plate in the absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 1 – 5000 μg/plate and only in strains TA 98 and 100. Genotoxicity was not observed up to the highest concentration tested. At 5000 µg/mL, no spontanous revertants occurred. Data can be found in Attachment 1 in the attached background material.

STUDY RESULTS
The test material produced a dose-dependent and significant increase in revertant colony numbers observed in strains TA1535 and TA100 in the two experiments performed. Signs of toxicity with and without metabolic activation. With and without S9 mix, toxic effects occurred in strains TA98 at 333.3 and 1000µg/plate (exp.I) and in the strains TA1537 and TA1538 at 1000 µg/plate (exp.II). With metabolic activation toxic effects occurred in strains TA1537 (exp.I) and TA100 (exp.I and II) at 1000 µg/plate. Strains TA1535, TA1537, TA1538 and TA100 showed normal background growth up to 1000 µg/plate with S9 mix and up to 100 µg/plate in the absence of metabolic activation.

For results of both experiment I and experiment II as well as the pre-experiment, please refer to the attached background material
(Attachment 2) under "Attached background material"



HISTORICAL CONTROL DATA
- Positive historical control data: no
- Negative (solvent/vehicle) historical control data: yes, can be found in Attachment 3 in the attached background material.

Any other information on results incl. tables

Table 1: Revertant colonies mean from three plates with and without metabolic activation

Treatment	Conc. [µg/plate]	S9 mix	TA 1535 Exp I/ ExpII	TA 1537 Exp I/ ExpII	TA 1538 Exp I/ ExpII	TA 98 Exp I/ ExpII	TA 100 Exp I/ ExpII
Negative control		-	22 ; 9	10 ; 9	18 ; 12	19 ; 19	95 ; 77
Solvent control^A		-	18 ;16	6 ; 7	12 ; 12	18 ; 20	89 ; 75
test substance	1.0	-	27 ; 18	9 ; 15	10 ; 14	- ; -	107 ; 104
	3.3	-	17; 22	11 ; 8	15 ; 15	- ; -	132 ; 109
	10.0	-	33; 23	16 ; 17	11 ; 17	25 ; 32	205 ; 147
	33.3	-	54 ;41	12 ; 17	12 ; 15	33 ; 31	277 ; 220
	66.6	-	56; 53	12 ; 16	16 ; 14	- ; -	271 ; 274
	100.0	-	44; 51	12 ; 12	14 ; 21	34 ; 31	368 ; 256
	333.3	-				8 ; 33
	666.6	-				7 ; 16
	1000.0	-				1 ; 12
Positive control^B		-	133 ; 1100	305 ; 220	2594 ; 2421	2031 ; 170	1299 ; 1069
Negative control		+	11 ; 11	10 ; 13	16 ; 13	33 ; 31	104 ; 87
Solvent control^A		+	11 ; 12	11 ; 13	18 ; 13	32 ; 29	89 ; 92
test substance	1.0	+	20 ; 14	9 ; 12	17 ; 13	31 ; 30	95 ; 98
	10.0	+	23 ; 27	11 ; 13	20 ; 15	38 ; 28	145 ; 187
	33.3	+	52 ; 44	14 ; 18	12 ; 16	35 ; 40	220 ; 269
	100.0	+	70 ; 54	19 ; 11	16 ; 19	40 ;42	298 ; 293
	333.3	+	45 ; 34	11  ; 11	10 ; 17	33 ; 27	247 ; 304
	666.6	+	53 ; 39	12 ; 11	15 ; 9	47 ;45	247 ; 266
	1000.0	+	37 ; 26	0 ; 4	11 ; 3	31 ; 43	0 ; 0
Positive control^B		+	507 ; 109	414 ; 89	2243 ; 152	2604 ; 1672	2564 ; 837
^ADMSO,^B2-aminoanthracene,SD - Standard deviation

Applicant's summary and conclusion

Conclusions:

The study was performed according to OECD guideline 471 and compliant with GLP. Under the conditions of this study, the test substance induced point mutations by base pair changes in the genome of the strains TA1535 and TA100 with and without metabolic activation.