4-methylthiosemicarbazide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-21 to 2004-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methylthiosemicarbazide

EC Number:

229-563-2

EC Name:

4-methylthiosemicarbazide

Cas Number:

6610-29-3

Molecular formula:

C2H7N3S

IUPAC Name:

1-amino-3-methylthiourea

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

• Other name : MTSC
• batch number: 0404001019
• Sponsor's filing number: GRL 0035/04
• description:
- at receipt: white powder
- on the Study plan and on the test article description: white solid
• container: one glass flask
• date of receipt: 25 August 2004
• storage conditions: at room temperature, protected from light and humidity
• purity: 99.7%
• composition: see analytical certificate
• expiry date: 30 April 2006.

Method

Target gene:

Histidine operon.

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiments without S9 mix

Experiments with S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix was performed according to the direct plate
incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes, 37°C
- Exposure duration: 48 to 72 hours, 37°C in growth medium

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies and/or thinning of the bacterial lawn

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a
positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

Not applicable.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL.
Consequently, with a treatment volume of 50 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.
No noteworthy toxicity was noted towards the three strains used, with and without S9 mix.
The individual results of the preliminary toxicity test are provided in table 1.

TOXICITY AND MUTATIONS :
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level for the main test was 5000 μg/plate, according to the criteria specified in the international guidelines.
The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 μg/plate, for both mutagenicity experiments with and without S9 mix.
No toxicity was noted towards all the strains used, both with and without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.
The findings of the individual tests are provided in tables 2 (first experiment) and 3 (second experiment).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tables of results: Ames test with MTSC

 

1/ First experiment / Direct plate incorporation method

 

strain	compound	Dose level (µg/ plate)	S9-mix	Mean revertant colony counts	SD	Ration treated/ solvent	Individual revertant colony counts
TA 1535	DMSO		-	11	2		14	10	10
	Test item	312.5	-	9	3	0.8	8	13	7
		625	-	13	4	1.1	14	8	16
		1250	-	14	4	1.2	10	13	18
		2500	-	11	1	1.0	10	11	12
		5000	-	13	2	1.2	12	12	16
	NAN3	1	-	583	26	51.5	588	555	607
	DMSO		+	9	4		10	5	12
	Test item	312.5	+	12	1	1.4	13	11	13
		625	+	12	2	1.3	12	13	10
		1250	+	13	2	1.4	13	14	11
		2500	+	16	3	1.8	16	19	14
		5000	+	14	1	1.5	14	13	14
	2AM	2	+	219	22	24.4	201	214	243
TA 1537	DMSO		-	5	3		5	2	8
	Test item	312.5	-	7	2	1.5	10	6	6
		625	-	6	2	1.1	8	5	4
		1250	-	3	4	0.5	7	0	1
		2500	-	5	2	0.9	6	6	2
		5000	-	5	3	0.9	7	6	1
	9AA	50	-	657	332	131.3	999	335	636
	DMSO		+	6	3		2	8	8
	Test item	312.5	+	9	2	1.4	6	10	10
		625	+	9	2	1.6	7	11	10
		1250	+	10	2	1.7	12	8	11
		2500	+	9	3	1.4	11	10	5
		5000	+	7	3	1.2	7	5	10
	2AM	2	+	62	11	10.4	74	60	53
TA 98	DMSO		-	22	5		24	17	36
	Test item	312.5	-	15	7	0.7	8	22	14
		625	-	17	9	0.8	18	8	25
		1250	-	20	6	0.9	26	14	20
		2500	-	33	3	1.5	30	32	36
		5000	-	27	8	1.2	20	36	26
	2NF	0.5	-	274	17	12.3	260	269	292
	DMSO		+	27	4		24	32	25
	Test item	312.5	+	27	6	1.0	34	24	24
		625	+	27	4	1.0	22	30	29
		1250	+	26	2	1.0	28	24	26
		2500	+	22	8	0.8	18	17	31
		5000	+	17	7	0.6	11	24	16
	2AM	2	+	775	383	28.7	525	583	1216
TA 100	DMSO		-	167	25		176	176	139
	Test item	312.5	-	147	16	0.9	166	138	137
		625	-	139	25	0.8	155	110	152
		1250	-	130	13	0.8	145	119	126
		2500	-	112	9	0.7	104	110	121
		5000	-	103	7	0.6	104	109	96
	NAN3	1	-	828	54	5.0	881	773	928
	DMSO		+	129	14		120	122	146
	Test item	312.5	+	132	1	1.0	132	133	132
		625	+	131	9	1.0	126	126	141
		1250	+	122	6	0.9	127	125	115
		2500	+	90	17	0.7	81	80	110
		5000	+	119	39	0.9	92	163	101
	2AM	2	+	1312	983	10.1	773	716	2447
TA 102	DMSO		-	316	7		319	322	308
	Test item	312.5	-	321	21	1.0	298	338	328
		625	-	342	19	1.1	340	362	325
		1250	-	350	10	1.1	343	346	361
		2500	-	298	16	0.9	283	296	314
		5000	-	281	27	0.9	311	272	260
	MMC	0.5	-	2370	62	7.5	2298	2405	2406
	DMSO		+	342	12		333	355	337
	Test item	312.5	+	383	69	1.1	454	378	316
		625	+	393	27	1.2	365	419	396
		1250	+	429	7	1.3	423	437	428
		2500	+	377	55	1.1	398	419	315
		5000	+	342	32	1.0	362	359	305
	2AM	10	+	1809	206	5.3	2025	1788	1614

SD: standard deviation

- : absence S9

+: presence S9

 

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under these experimental conditions, the test item MTSC did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Executive summary:

The potential of 4 -methylthiosemicarbazide to induce reverse mutation was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

A preliminary toxicity test was performed to define the dose-levels of MTSC to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The test item 4-METHYLTHIOSEMICARBAZIDE was dissolved in dimethylsulfoxide (DMSO). The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level for the main test was 5000 μg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 μg/plate, for both mutagenicity experiments with and without S9 mix. No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. Under these experimental conditions, the test item MTSC did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.