Registration Dossier

Administrative data

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:
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.