2-methyl-2H-isothiazol-3-one hydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was concluded that MIT.HCl did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations limited by toxicity in the absence and in the presence of a rat liver metabolic activation system (S9), utilizing the plate incorporation methodology (absence and presence of S9) or pre-incubation methodology (presence of S9).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2017 to 1 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Identification: MIT.HCl (Methyl isothiazolinone hydrochloric acid)
- CAS no.: 26172-54-3
- Source and lot/batch No.of test material: Sigma-Aldrich / 10013913
- Expiration date of the lot/batch: 30 Nov 2018
- Purity test date: 23 Nov 2016
- Purity: >99.9%
- Apperance: White to light beige powder
- Moelcular weight: 151.61 g/mol
- Storage condition of test material: 2 to 8°C, protected from light

Target gene:

see below

Species / strain / cell type:

S. typhimurium 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:

β-Naphthoflavone/Phenobarbital

Test concentrations with justification for top dose:

Experimental 1 (plate incorporation): +/- S9: All strains: 0, 5, 16, 50, 160, 500, 1600, 5000 ug/plate
Experiment 2 (plate incorporation): - S9: All strains: 0, 9.375, 18.75, 37.5, 75, 150, 300, 600 ug/plate
Experimental 2 (plate incorporation): + S9: TA98, TA1535, TA102: 0, 18.75, 37.5, 150, 300, 600, 1200 ug/plate
Experimental 2 (plate incorporation): + S9: TA100, TA1537, TA102: 0,9.375, 18.75, 37.5, 150, 300, 600 ug/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

-S9: TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-S9: TA100, TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

-S9: TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

-S9: TA102

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9: TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

+S9: TA100, TA1535, TA1537 TA102

Details on test system and experimental conditions:

The plate incorporation methodology was used for Experiment 1 (-/+ S9) and Experiemtn 2 (-S9). In addition the pre-incubation methodology was in Experiment 2 (+S9). Triplicate plates were used for the main tests.

Evaluation criteria:

A concentration related increase in revertant numbers was =>1.5-fold (in strain TA102), =>2-fold (in strains TA98 or TA100) or =>3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.

The test article was considered positive in this assay if the above criterion was met.

The test article was considered negative in this assay if the above criterion was not met

Statistics:

Statistics not warranted

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

A. Mutation experiment 1:

Mutation Experiment 1 treatments of all tester strains were performed utilizing the plate incorporation methodology in the absence and in the presence of S‑9, using final concentrations ofMIT.HClat 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity ranging from a slight thinning of the background bacterial lawn with a marked reduction in revertant numbers to a complete killing of the test bacteria, was observed at 500, 1600 and 5000 µg/plate in all strains in the absence and presence of S-9.

 

Table 7.6.1/01-1: Bacterial (reverse) gene mutation – Main mutation experiment 1

Conc. (ug/plate)	TA98		TA100		TA1535			TA1537		TA102
	-S9	+S9	-S9	+S9	-S9		+S9	-S9	+S9	-S9	+S9
0	28.7	36.0	111.0	109.3	8.3		20.7	7.0	8.0	259.3	258.7
5	23.7	35.3	93.3	111.7	15.0		15.0	10.0	10.7	258.7	270.3
16	27.0	34.0	100.3	105.3	14.3		14.7	10.7	11.5	298.0	265.7
50	30.0	36.3	111.0	113.0	15.0		17.0	10.3	8.7	261.7	271.7
160	27.3	33.7	91.7	96.7	13.7		9.7	10.7	8.3	246.0	250.0
500	T	16.7S	T	T	T		4.7V	T	T	T	37.0V
1600	T	T	T	T	T		T	T	T	T	T
5000	T	T	T	T	T		T	T	T	T	T
+ve	561.7	193.3	553.0	946.7	501.0		137.0	84.3	147.3	610.0	1150.7
S: Slight thinning of background bacterial lawn T: Toxic, no revertant colonies V: Very thin background bacterial lawn -S9 (absence of metabolic activation) TA98: 2-nitrofluorene TA100, TA1535: Na azide TA1537: 9-aminoacridine TA102: mitomycin C						+S9 (presence of metabolic activation) TA98: Benzo[a]pyrene TA100, TA1535, TA1537, TA102: 2-aminoanthracene

B.Mutation experiment 2:

Mutation Experiment 2 treatments of all the tester strains were performed in the absence (utilising the plate incorporation methodology) and in the presence of S-9. The maximum test concentration was reduced based on strain specific toxicity observed in Mutation Experiment 1 and narrowed concentration intervals were employed in order to examine more closely those concentrations of MIT.HCl approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. The concentration ranges used in Mutation Experiment 2 were as follows:

·        9.375, 18.75, 37.5, 75, 150, 300 and 600 µg/plate for all strains in the absence of S-9

·        9.375, 18.75, 37.5, 75, 150, 300 and 600 µg/plate for strains TA100 and TA1537 in the presence of S-9

·        18.75, 37.5, 75, 150, 300, 600 and 1200 µg/plate for strains TA98, TA1535 and TA102 in the presence of S-9.

In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system.

 

Following treatments in the absence of S-9, evidence of toxicity in the form of a complete killing of the test bacteria, was observed at 600 µg/plate in all strains. A slight thinning of the background bacterial lawn was also noted in all strains except TA1537 at 300 µg/plate and at 150 µg/plate in strain TA100.

 

Following treatments in the presence of S-9, evidence of toxicity ranging from a slight thinning of the background bacterial lawn to a complete killing of the test bacteria, was observed at 150 µg/plate and above in strain TA1537, at 300 µg/plate and above in strains TA98, TA100 and TA1535 and at 600 µg/plate and above in strain TA102.

 

Table 7.6.1/01-2: Bacterial (reverse) gene mutation – Main mutation experiment 2

Conc. (ug/plate)	TA98		TA100		TA1535			TA1537		TA102
	-S9	+S9	-S9	+S9	-S9		+S9	-S9	+S9	-S9	+S9
0	25.7	39.7	121.3	149.3	27.0		23.3	7.7	10.7	309.0	367.7
9.375	22.0	-	113.3	125.7	18.0		-	9.0	15.0	319.0	-
18.75	16.7	39.7	127.7	139.0	17.0		28.3	11.0	16.7	328.7	331.0
37.5	19.3	28.0	120.3	144.3	18.3		23.7	9.0	12.7	314.0	348.3
75	19.7	42.7	127.7	132.3	14.7		19.7	8.7	13.3	298.7	338.7
150	21.0	39.0	130.3S	148.0	13.0		20.0	10.7	11.0S	297.0	277.7
300	14.7S	32.7S	174.0S	217.3S	8.7S		13.3S	9.7	T	201.0S	261.3
600	T	T	T	T	T		T	T	T	T	T
1200	-	T	-	-	-		T	-	-	-	T
+ve	462.7	270.0	534.3	1167.7	523.7		135.0	287.7	103.0	773.7	2132.7
S: Slight thinning of background bacterial lawn T: Toxic, no revertant colonies -S9 (absence of metabolic activation) TA98: 2-nitrofluorene TA100, TA1535: Na azide TA1537: 9-aminoacridine TA102: mitomycin C						+S9 (presence of metabolic activation) TA98: Benzo[a]pyrene TA100, TA1535, TA1537, TA102: 2-aminoanthracene

The positive controls induced an acceptable increase in revertant colony numbers, thereby demonstrating the sensitivity and specificity of the test system.

 

Following MIT.HCltreatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed =>1.5-fold (in strain TA102), =>2-fold (in strains TA98or TA100) or =>3-fold (strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any MIT.HCl mutagenic activity in this assay system

Conclusions:

It was concluded that MIT.HCl did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations limited by toxicity in the absence and in the presence of a rat liver metabolic activation system (S9), utilizing the plate incorporation methodology (absence and presence of S9) or pre-incubation methodology (presence of S9).

Executive summary:

MIT.HCl was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535,TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by β -naphthoflavone / Phenobarbital induced rat liver post-mitochondrial fraction (S9), in two independent experiments.

 

All MIT.HCl treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).

 

In Mutation Experiment 1, treatments of all tester strains were performed in the absence and in the presence of S9, using final concentrations of MIT.HCl at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed at 500, 1600 and 5000 µg/plate in all strains in the absence and presence of S9.

 

In Mutation Experiment 2, treatments of all the tester strains were performed in the absence and in the presence of S9. The maximum test concentration was reduced based on strain specific toxicity observed in Mutation Experiment 1 and narrowed concentration intervals were employed in order to examine more closely those concentrations of MIT.HCl approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. The concentration ranges used in Mutation Experiment 2 were as follows:

·        9.375, 18.75, 37.5, 75, 150, 300 and 600 µg/plate for all strains in the absence of S9

·        9.375, 18.75, 37.5, 75, 150, 300 and 600 µg/plate for strains TA100 and TA1537 in the presence of S9

·        18.75, 37.5, 75, 150, 300, 600 and 1200 µg/plate for strains TA98, TA1535 and TA102 in the presence of S9

·         

In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system.

 

Following treatments in the absence of S9, evidence of toxicity in the form of a complete killing of the test bacteria, was observed at 600 µg/plate in all strains. A slight thinning of the background bacterial lawn was also noted in all strains except TA1537, at 300 µg/plate and at 150 µg/plate in strain TA100.

 

Following treatments in the presence of S9, evidence of toxicity ranging from a slight thinning of the background bacterial lawn to a complete killing of the test bacteria, was observed at 150 µg/plate and above in strain TA1537, at 300 µg/plate and above in strains TA98, TA100 and TA1535 and at 600 µg/plate and above in strain TA102.

 

The test article was completely soluble in the assay system at all concentrations treated.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies counts fell within acceptable ranges for vehicle control treatments and were elevated by positive control treatments.

 

Following MIT.HCl treatments of all the test strains in the absence and presence of S9, no biologically relevant increases in revertant numbers were observed. This study was considered therefore to have provided no evidence of anyMIT.HClmutagenic activity in this assay system.

 

It was concluded that MIT.HCl did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations limited by toxicity in the absence and in the presence of a rat liver metabolic activation system (S9), utilizing the plate incorporation methodology (absence and presence of S9) or pre-incubation methodology (presence of S9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

n/a

Additional information

n/a

Justification for classification or non-classification

Comparison with the CLP criteria

There was no indication that MIT.HCl has a mutagenic effect in the in vitro bacterial (reverse) gene mutation assay.