reaction mass of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2H -isothiazol-3-one

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: Guideline-conform study under GLP without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

histidine-gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

-bacterial suspension and test substance mixed withg molten agar
-checked for strain characteristis within last 2 hours of treatment (Maron et al., DeSerres et al.)

Additional strain / cell type characteristics:

other: histidine-requiring

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

-bacterial suspension and test substance mixed withg molten agar
-checked for strain characteristis within last 2 hours of treatment (Maron et al., DeSerres et al.)

Additional strain / cell type characteristics:

other: histidine-requiring

Metabolic activation:

with and without

Metabolic activation system:

Acrolor 1254 induced rat liver post-mitochondrial fraction (S-9) of the male Sprague Dawley strain.

Test concentrations with justification for top dose:

Mutation experiment 1:
Without S9: 1.25, 2.5, 5, 10, 15 µg/plate
With S9 (10%): 6.25, 12.5, 25, 50, 100 µg/plate
Mutation experiment 2:
Without S9: 1.25, 2.5, 5, 10, 15µg/plate
With S9 (10%): 10, 20, 40, 60, 80 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: nitrofluoroene, aminoacridine, gluataldehyde, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure /selection duration: 3 days

NUMBER OF REPLICATIONS: 3/concentration, two experiments

CELL COUNTING
-electronically: SeeScanplc

DETERMINATION OF CYTOTOXICITY
- examination of the background lawn

VERIFICATION OF RESULTS
-revertant colonies transferred to fresh plates; all colonies grew

Evaluation criteria:

valid if
-mean of negative control within historical control data
-positive controls clearly increase number of revertants, starin dependent
-no more than 5% of the pülates lost due to contamination/unforseen events
positive, if:
-statistically significant (Dunnett`s, p < 0.01) increase in no. of revertants
-positive effects reproducible

Statistics:

Dunnett`s test

Results and discussion

Test results

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: Toxicity range-finder experiment with TA100: |3.55 - 7.10 µg a.i./plate without S-9 mix|>= 17.75 µg a.i./plate with S-9 mix

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
in TA100:
toxic at >25 Acticide14/plate without S-9, toxic at >125 µg Acticide14/plate with S-9

COMPARISON WITH HISTORICAL CONTROL DATA:

controls: within historical controls

Remarks on result:

other: other: Salmonella typhimurium (strains TA98, TA100, TA1535, TA1537 and TA102)

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

table 1: Strain Mean Revertants (Experiment 2)

with S-9						without S-9
Treatement [µg/plate] Treatment [µg/plate]	TA 98	TA 100	TA 1535	TA 1537	TA 102	Treatment [µg/plate]	TA 98	TA 100	TA 1535	TA 1537	TA 102
0	37.4	105.8	6.6	9.8	380.6	0	28.6	85.6	5.8	7.6	322.0
10	38.0	193.3 ***	8.0	12.3	503.0	1.25	35.3	183.3 ***	3.0	11.7	435.3 ***
20	40.7	258.3 ***	5.3	13.3	500.7	2.5	34.3	277.7 ***	5.7	14.0 *	527.7 ***
40	46.0	301.7 ***	5.7	12.3	497.0	5	37.7 *	495.3 ***	4.7	19.7 ***	693.3 ***
60	43.3	323.3 ***	8.0	13.7	598.0	10	46.3 ***	1016 ***	8.0	22.3 ***	890.0 ***
80	41.3	460.7 ***	6.3	13.7	673.7	15	69.3 ***	1411 ***	13.3 ***	32.0 ***	1016.0 ***

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

ACTICIDE 14 was mutagenic in the Ames-test. Presence of metablic activation system reduced mutagenicity.

Executive summary:

The mutagenic potential of a 14% aqueous solution of 3 parts 5 -chloro-2 -methyl-2H-isothiazol-3 -one and 1 part 2 -methyl-2H-isothiazol-3 -one (cited as ACTICIDE 14 in the report) was evaluated in a bacterial mutation assay according to guideline EPA OPP 84 -2. Five histidine-requiring test strains of Salmonella typhimurium were treated with the test substance for 3 days in the presence or absence of a metabolical activation system under histidine-exclusion. The number of colonies observed represented the number of suviving bacteria and thus the number of back-mutations to the histidine-gene.

Acticide 14 clearly demonstrated an ability to induce mutation in 5 strains aof Salmonella typhimurium, when tested up to concentrations close to, or within, the toxic range. The mutagenic activity observed was greatly reduced (but nevertheless still detectable) when testing was carried out in the presence of a rat liver metabolic activation system.