Methyl isothiocyanate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study (OECD 471)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 2AA was used as positive control with S9.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl isothiocyanate

EC Number:

209-132-5

EC Name:

Methyl isothiocyanate

Cas Number:

556-61-6

Molecular formula:

C2H3NS

IUPAC Name:

isothiocyanatomethane

Details on test material:

Source and batch number : not precised.
Purity : 99.8%

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of the dose setting tests, there were six concentrations at a ratio of 2 for test (I) and test (II), with four or more concentrations believed not to inhibit the growth of bacteria, specifically 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μg/plate for TA100, TA1535 and TA1537; 9.77, 19.5, 39.1 and 78.1 μg/plate for WP2uvrA and 0.610, 1.22, 2.44, 4.88, 9.77 and 19.5 μg/plate for TA98, both with and without the S9 mix.

Vehicle / solvent:

Anhydrous ethanol (pharmaceutical product, lot number: LF5585, Wako Pure Chemical Industries, period of use: 5/11/2005 (set by their company), storage conditions: room temperature) was used as the vehicle for the test substance in the vehicle control substance.

Controls

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

anhydrous ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in glucose agar plate

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): no data

NUMBER OF REPLICATIONS: 3 replicates / 2 tests
NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY : Yes, "bacterial growth inhibition"

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Evaluation criteria:

The test results indicate that the number of revertant colonies on a plate treated with the test substance was double or more than that of the vehicle control, and were positive when increased according to the concentration.

Statistics:

The number of revertant colonies was used to calculate the mean values and standard deviations for each concentration. Significant differences were not noted.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 19.5µg/pl (TA98), 39.1 µg/pl (TA1535, TA1537), 78.1 µg/plate (TA100)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 156.3 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitate on plates treated with the test substance was not identified at any concentration at the start or completion of incubation, both with and without the S9 mix.
With and without the S9 mix, concentrations of TA100, TA1535 and TA1537 at 78.1 μg/plate or higher, concentrations of WP2uvrA at 312.5 μg/plate or higher and concentrations of TA98 at 19.5 μg/plate or higher were confirmed to inhibit growth of bacteria.
The number of revertant colonies was less than double that of the vehicle control for all of the bacteria strains, both with and without the S9 mix.
The number of revertant colonies in the vehicle control was the same level as the negative control for all of the bacteria strains.
The positive control substance demonstrated a clear increase in the number of revertant colonies. Additionally, the number of revertant colonies in the negative control and the positive control was within the range of background data for the test facility.
Reproducibility was confirmed in 2 tests.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table no.1 of results : Reverse mutation test of MITC in bacteria (test 1), without and with S9

	Test substance concentration (µg/plate)	Number of revertants (number of colonies/plate)
		Base-pair substitution			Frameshift type
		TA100	TA1335	WP2uvrA	TA98	TA1537
WITH S9	Negative control	93+/-11.3^1	9+/-2.3	40+/-7.0	20+/-3.5	10+/-0.6
	Vehicle control	97+/-12.2	11+/-1.0	43+/-1.2	31+/-5.9	10+/-3.2
	0.610	/	/	/	24+/-6.0	/
	1.22	/	/	/	24+/-4.9	/
	2.44	89+/-4.6	7+/-4.0	/	22+/-3.8	6+/-2.6
	4.88	99+/-7.2	7+/-3.2	/	22+/-3.2	11+/-4.5
	9.77	95+/-5.0	8+/-2.1	42+/-8.1	24+/-3.5	9+/-3.6
	19.5	100+/-7.8	5+/-2.3	45+/-2.1	25+/-9.5*	11+/-0.6
	39.1	110+/-2.5	12+/-1.5*	35+/-5.8	/	13+/-0.6*
	78.1	105+/-24.4*	9+/-2.6*	42+/-7.5	/	6+/-3.6*
	156.3	/	/	59+/-7.0*	/	/
	312.5	/	/	47+/-13.8*	/	/
	Positive control	AF-2	NaN3	AF-2	AF-2	9AA
	Number of colonies/plate	499+/-50.0	543+/-24.7	149+/-15.3	442+/-9.6	282+/-54.1
WITHOUT S9	Negative control	114+/-19.2	12+/-4.9	39+/-5.0	32+/-5.3	16+/-4.6
	Vehicle control	120+/-3.6	9+/-3.0	45+/-3.5	40+/-4.7	12+/-4.6
	0.610	/	/	/	31+/-4.7	/
	1.22	/	/	/	29+/-10.1	/
	2.44	108+/-7.2	8+/-3.6	/	37+/-2.9	15+/-4.6
	4.88	102+/-4.0	11+/-1.5	/	37+/-8.2	14+/-3.5
	9.77	101+/-9.5	8+/-1.2	48+/-3.8	33+/-7.0	12+/-1.0
	19.5	98+/-8.5	9+/-3.5	39+/-6.2	37+/-9.1	15+/-6.7
	39.1	111+/-6.0	11+/-3.5*	41+/-1.7	/	19+/-5.8*
	78.1	122+/-17.5*	8+/-5.0*	36+/-4.9	/	14+/-4.2*
	156.3	/	/	44+/-11.1	/	/
	312.5	/	/	51+/-3.6*	/	/
	Positive control	2AA	2AA	2AA	2AA	2AA
	Number of colonies/plate	1011+/-46.4	295+/-16.3	843+/-74.3	418+/-33.5	176+/-17.8
^1Mean of 3 replicates (mean +/-SD)

Table no.2 of results : Reverse mutation test of MITC in bacteria (test 2), without and with S9

	Test substance concentration (µg/plate)	Number of revertants (number of colonies/plate)
		Base-pair substitution			Frameshift type
		TA100	TA1335	WP2uvrA	TA98	TA1537
WITH S9	Negative control	128+/-5.1	8+/-3.5	36+/-1.2	17+/-3.6	14+/-4.5
	Vehicle control	133+/-19.1	7+/-2.5	26+/-6.1	20+/-6.6	15+/-3.1
	0.610	/	/	/	24+/-4.7	/
	1.22	/	/	/	23+/-3.2	/
	2.44	106+/-3.5	9+/-2.3	/	21+/-4.6	11+/-2.3
	4.88	118+/-13.6	9+/-2.1	/	27+/-4.5	12+/-5.0
	9.77	112+/-8.6	7+/-0.6	29+/-3.8	26+/-3.5	19+/-4.4
	19.5	107+/-16.6	9+/-2.1	30+/-2.3	30+/-5.0*	14+/-3.5
	39.1	120+/-20.4	7+/-3.1*	35+/-8.5	/	14+/-2.5*
	78.1	114+/-11.3*	12+/-2.3*	45+/-14.3	/	9+/-3.1*
	156.3	/	/	49+/-4.4*	/	/
	312.5	/	/	33+/-6.7*	/	/
	Positive control	AF-2	NaN3	AF-2	AF-2	9AA
	Number of colonies/plate	557+/-19.1	498+/-32.0	154+/-16.2	426+/-6	406+/-13.4
WITHOUT S9	Negative control	138+/-21.0	11+/-3.5	46+/-5.5	31+/-8.1	22+/-3.5
	Vehicle control	129+/-9.9	9+/-3.8	38+/-1.5	26+/-4.6	17+/-4.4
	0.610	/	/	/	31+/-3.5	/
	1.22	/	/	/	44+/-9.6	/
	2.44	111+/-12.5	8+/-2.0	/	39+/-5.7	19+/-2.1
	4.88	117+/-14.4	8+/-1.0	/	37+/-7.9	14+/-5.0
	9.77	118+/-11.1	8+/-3.6	34+/-1.7	34+/-2.5	22+/-3.2
	19.5	116+/-7.5	8+/-2.5	34+/-5.1	34+/-5.5*	15+/-2.5
	39.1	125+/-24.5	10+/-3.2*	50+/-3.6	/	19+/-4.6*
	78.1	128+/-4.0*	9+/-1.5*	39+/-2.6	/	18+/-6.7*
	156.3	/	/	47+/-8.1	/	/
	312.5	/	/	44+/-8.1*	/	/
	Positive control	2AA	2AA	2AA	2AA	2AA
	Number of colonies/plate	1034+/-75.5	294+/-6.5	970+/-113.2	407+/-4.2	171+/-16.2
^1Mean of 3 replicates (mean +/-SD)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

MITC did not induce genetic mutation under these test conditions.

Executive summary:

The presence of genetic mutation induction by methyl isothiocyanate was studied in reverse mutation tests performed using bacteria.

The bacterial strains used for the study included Salmonella typhimurium TA100, TA98, TA1535 and TA1537 as well as Escherichia coli WP2uvrA. The tests were conducted using the pre-incubation method, and performed both with and without the S9 mix. The test concentrations for test (I) and test (II) were set by performing dose setting tests. The test concentration included all strains 0.305~5000 μg/plate (ratio of 4, total of 8 concentrations), with and without the S9 mix.

Precipitate on plates was not identified on the plates, at any concentration, both with and without the S9 mix. With and without the S9 mix, concentrations of TA100, TA1535 and TA1537 at 78.1 μg/plate or higher,  concentrations of WP2uvrA at 312.5 μg/plate or higher and concentrations of TA98 at 19.5 μg/plate or higher were confirmed to inhibit growth of bacteria. The number of revertant colonies was less than double that of the vehicle control (anhydrous ethanol) for all of the bacteria strains, both with and without the S9 mix.