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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several in vitro studies of genotoxicity were performed with C(M)IT/MIT. Positive results were observed in three Ames assays and in three tests in mammalian cells (one chromosomal aberration test and two mouse lymphoma assays), with or without S9 activation. In contrast, C(M)IT/MIT was not mutagenic in primary culture of rat hepatocytes (UDS) and in a mouse cell transformation test.

A test was also performed with the major metabolite of C(M)IT/MIT, N-(methyl)malonamic acid (NMMA), which appeared not to be mutagenic when tested in a bacterial gene mutation assay test (Ames assay).

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine-gene

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 / 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

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

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'.

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 ***

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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.

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

tyrosine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: tyrosine kinase deficient

Metabolic activation:

with and without

Metabolic activation system:

arochlor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

Experiment 1, without S-9:
0.375 - 12 µg Acticide 14/ml
Experiment 1, with S-9:
0.1875 -6 µg Acticide14/ml
Experiment 2, without and with S-9:
0.5 - 6 µg Acticide14/ml

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: and Benzo[a]pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation; in suspension;

DURATION
- Preincubation period: 1ml freshly prepared S9 mix(+G6P,NADPH, KCl) were added to each cell culture (19ml). Cultures (with/without S9 mix) were incubated for 3 hours (shaking) at 37°C.
- Exposure duration: 3 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days (5-trifluorothymidine)


NUMBER OF REPLICATIONS: 2 / concentration/experiment


NUMBER OF CELLS EVALUATED:
Plating for survival (2*105 cells/ml, 10 days)
Expression period (1*106 cells/ml, TK- mutation expression, 2 days)
Plating for viability ( 1*104 cells/ml, 11 days)
Plating for 5-trifluororthymidine (TFT) resistance (104 cells/ml, 12 days)


DETERMINATION OF CYTOTOXICITY
-ability to grow as a colony after single cell plating

Evaluation criteria:

-mutation frequenc of negytive control within range of historical controls
-positive controls valid( in at least 1 concentration, diff. in mutation frequencies between control and positive control at least half of the historical range for controls)
-mutation frequency in at least one dose greater than negative control
-significant dose-relationship
-effect reproducible

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: >= 0.852 µg a.i./ml without and with S-9

Remarks on result:

other: other: Thymidine kinase (tk) locus in mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

table 1 : results of mutation experiment 1

without S-9			with S-9
treatment [µg/ml]	% relative survival	mutation frequency [per 10^6 cells]	treatment [µg/ml]	% relative survival	mutation frequency [per 10^6 cells]
0	100.0	93.08	0	100.0	115.46
0.375	82.6	103.52	0.1875	114.8
0.75	78.0	108.52	0.375	100.7	117.0
1.5	65.6	128.10	0.75	99.3	89.38
3	19.9	275.94*	1.5	98.5	143.23
6	0.5	1610.62*	3	37.5	185.53*
12	0.0		6	202	389.30*
positive control NQO			positive control BP
0.05	67.5	207.99	2	99.3	327.57
0.1	48.2	1123.89	3	71.5	736.77

table 2 : results of mutation experiment 2

without S-9			with S-9
treatment [µg/ml]	% relative survival	mutation frequency [per 10^6 cells]	treatment [µg/ml]	% relative survival	mutation frequency [per 10^6 cells]
0	100.0	145.17	0	100.0	173.52
0.5	103.6		0.5	91.8
1	81.9	263.86 *	1	95.8	247.18
2	72.9	403.43 *	2	80.8	367.92 *
3	35.3	837.73 *	3	39.7	539.59 *
4	18.7	742.91 *	4	25.7	418.33 *
5	5.6	739.25 *	5	21.7	322.29 *
6	0.5	1359.94 *	6	9.8	478.50 *
positive control NQO			positive control BP
0.05	74.0	806.12	2	81.4	589.90
0.1	48.2	1123.89	3	71.5	736.77

* significant at 5% level

Conclusions:

Interpretation of results (migrated information):
positive

When tested up to toxic concentrations, ACTICIDE 14 clearly induced mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9.
Under the conditions employed in this study, ACTICIDE 14 is mutagenic in this test system.

Executive summary:

The genotoxic 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 mammalian cell gene muatation assay according to OECD guideline 476. Cultured L5178Y mouse lymphoma cells that were deficient in thymidine kinase were treated with the test substance for 3 hours. Afterwards, test substance was removed from the system and cells were cultured for additional 2 days for expression of thymidine kinase, if mutation had taken place. After this period, cells were selected in 5 -trifluorothymidine-containing medium for 12 days. Cells without mutations died due to the 5 -trifluorothymidine-treatment, while those with mutations survived and formed colonies.

In the absence of S9, statistically significant increases were observed at the top 2 doses analysed in Experiment 1 (although due to extremely high toxicity little weight would be attached to the mutant frequency obtained at highest dose of 6 µg/ml). In Experiment 2, statistically significant increases in mutant frequency were obtained at all doses plated. A dose-relationship was also obtained in both experiments.

Similar results were obtained in the presence of S9. In Experiment 1, statistically significant increases in mutant frequency were obtained at the top 2 doses (extreme toxicity was observed at the highest dose of 6 gl/ml). In Experiment 2, statistically significant increases in mutant frequency were obtained at the top 5 doses. A dose-response was also obtained in both experiments.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

OECD Guideline 476 was not in effect at the time this study was conducted

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. SW 81/7211

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilution in water and dimethylsulfoxide (DMSO, 100 microliter/mL)
- Final dilution of a dissolved solid, stock liquid or gel: 0.25 to 30 nL/mL

OTHER SPECIFICS: Purity of test material was 15%

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9 homogenate mix.

Test concentrations with justification for top dose:

With metabolic activation: 1.22, 2.44, 4.88, 9.77, 10.0, 15.0, 19.5, 20.0, 25.0, 30.0 nL/mL
Without metabolic activation: 0.25, 0.50, 0.61, 1.0, 1.22, 1.5, 2.0 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water and Dimethylsulfoxide (DMSO) at 100 µL/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethylnitrosamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: n/a
- Exposure duration: 4 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): Not described

NUMBER OF REPLICATIONS: Not described

NUMBER OF CELLS EVALUATED: 3 x 10^6 cells

DETERMINATION OF CYTOTOXICITY
- Method: other: viable cell count

Evaluation criteria:

Mutant frequency

Statistics:

Not described

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

subtoxic concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: 0.0625 nL/mL to 10000 nL/mL

ADDITIONAL INFORMATION ON CYTOTOXICITY: Kathon™ 886 was excessively toxic at 2.44 nL/mL and lethal at higher concentrations without activation, and excessively toxic at 39.1 nL/mL and lethal at higher concentrations with activation.

Conclusions:

Kathon™ 886 (NAR) is considered active in the Mouse Lymphoma forward mutation assay, both in absence and presence of metabolic activation.

Executive summary:

OECD 476, In vitro mammalian cell gene mutation test. The treatment (without activation) that induced a higher toxicity induced a mutant frequency that was significantly above the background while the treatment that induced a lower toxicity was inactive. The test material was therefore considered positive without activation from 1 nL/mL at moderate to high toxicity. In the presence of S-9 mix, the test material was less toxic and could be assayed up to 30 nL/mL. However, it was considered active in mouse lymphoma cells from 2.44 to 30 nL/mL. Kathon™ 886 (NAR) is considered active in the Mouse Lymphoma forward mutation assay, both in absence and presence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

An additional S.typhimurium strain is required

Deviations:

yes

Remarks:

An additional S. typhimurium strain is required

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

An additional S.typhimurium strain is required

Deviations:

yes

Remarks:

An additional S.typhimurium strain is required

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. 10-2-81

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluted in water
- Final dilution of a dissolved solid, stock liquid or gel: 0.01 to 100 microgram/plate

OTHER SPECIFICS: Purity of test material was 16%

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

E. coli, other: B/r WP2/hcr

Metabolic activation:

with and without

Metabolic activation system:

S-9 prepared from male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

0.01, 0.05, 0.1, 0.5, 1, 5, 10 µg/plate without S9mix , and 0.1, 0.5, 1, 5, 10, 50 and 100 µg/plate in presence of S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water, DMSO was used for the positive controls (9-AA, 2-NF, 2-AA and B(a)P).

Negative solvent / vehicle controls:

yes

Remarks:

Water was used as the negative, solvent control

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: sodium,p-dimethylaminobenzenediazosulfonate; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Statistics:

Not described

Species / strain:

E. coli, other: B/r WP2hcr

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10µg/plate without S9mix, 100µg/plate with S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10µg/plate without S9mix, 100µg/plate with S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10µg/plate without S9mix, 100µg/plate with S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10µg/plate without S9mix, 100µg/plate with S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Other observations when applicable: At 10µg/plate without S9mix and 100µg/plate with S9mix, Kathon is cytotoxic for bacteria but not at the previous concentration.

Conclusions:

Kathon™ WT is mutagenic to S. typhimurium strain TA-100 at doses of 2.5 to 7.5mg/plate (without activation) and at doses of 50 to 75mg/plate (with activation). Kathon™ WT is not mutagenic to E. coli.

Executive summary:

OECD 471 and OECD 472 guidelines were not in effect at the time this study was conducted but were, in general, followed. Salmonella typhimurium and Escherichia coli reverse mutation assays were performed. A confirmatory assay was conducted in S. typhimurium strain TA-100 only. Assay was negative in E. coli strain WP2hcr, and S.Typhimurium TA98 and TA 1537, with and without metabolic activation. Assay was positive in S. typhimurium strain TA-100 with and without metabolic activation. Kathon™ WT is mutagenic to S. typhimurium strain TA-100 at doses of 2.5 to 7.5mg/plate (without activation) and at doses of 50 to 75mg/plate (with activation). Kathon™ WT is not mutagenic to E. coli. At 5 µg/plate the number of revertants was 8 times the water control in TA-100. At 50 µg/plate the number of revertants was 5 times the water control in TA-100. Negative in E.coli strain WP2 hcr as well as in S.typhimurium TA98 and TA 1537, with and without metabolic activation. At 10µg/plate without S9mix and 100µg/plate with S9mix, Kathon is cytotoxic for bacteria but not at the previous concentration.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

This was a screening test for S. typhimurium strains TA-98 and TA-1537 only

Deviations:

yes

Remarks:

This was a screening test for S. typhimurium strains TA-98 and TA-1537 only

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

yes

Remarks:

This was a screening test for S. typhimurium strains TA-98 and TA-1537 only

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. 2585

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Incorporation into agar plates
- Final dilution of a dissolved solid, stock liquid or gel: 1.6, 3, 5, 9, 16, 30 and 60 μg a.i./plate

OTHER SPECIFICS: Purity of test material was 14%

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

Microsomal enzyme preparation, liver extract from Aroclor 1254 pre-induced rats

Test concentrations with justification for top dose:

1.6, 3, 5, 9, 16, 30 and 60 μg a.i./plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: Kathon™ 886 pre-incubated with S9 for 2 h at room temperature
- Exposure duration: Not described

Evaluation criteria:

Not described

Statistics:

Not described

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

not specified

Remarks:

cytotoxic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic at all concentrations in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Kathon™ 886 up to 30 μg a.i./plate is mutagenic for S. typhimurium in the presence of metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

not specified

Remarks:

cytotoxic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic at all concentrations in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Kathon™ 886 up to 30 μg a.i./plate is mutagenic for S. typhimurium in the presence of metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic at all concentrations in the absence of metabolic activation. When Kathon™ 886 was pre-incubated with S-9, Kathon™ 886 was non-toxic in either strain up to 60 μg a.i./plate.

Conclusions:

Kathon™ 886 up to 30 μg a.i./plate is mutagenic for S. typhimurium in the presence of metabolic activation. However, where Kathon™ 886 is pre-incubated 2 h with S-9, no mutagenic activity was observed on bacteria.

Executive summary:

OECD Guideline 471, US EPA Guideline 84-2,in vitrogene mutation assay in bacteria in 2 strains of Salmonella typhimurium, TA-98 and TA-1537. A mutagenic response was detected in tester strains TA-98 and TA-1537 in the presence of metabolic activation up to 30 μg a.i./plate and were confirmed in an independent assay with 60 μg/plate exhibiting a toxic response. In the absence of metabolic activation, the test article was toxic at all concentrations. When Kathon™ 886 was pre-incubated with S-9, no mutagenic activity was detected up to 30 μg a.i./plate. At 60 μg a.i./plate, Kathon™ 886 was non-toxic in either strain and in TA-1537 produced a slight mutagenic response. Pre-incubation assay was not confirmed in an independent assay. Kathon™ 886 up to 30 μg a.i./plate is mutagenic for S. typhimurium in the presence of metabolic activation. However, where Kathon™ 886 is pre-incubated 2 h with S-9, no mutagenic activity was observed on bacteria.

Reference 6

Endpoint:

genetic toxicity in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Mouse embryo fibroblast cells in culture were exposed to Kathon™ 886 at concentrations from 0.05 to 0.8 nL of sample/mL.
- Short description of test conditions: Cells were exposed to the test compound for 24 h at 37 °C in Eagle’s Basal Medium supplemented with 10 % fetal calf serum
- Parameters analysed / observed: After 6 weeks of incubation, the plates were stained with 10 % Giemsa and scored for transformed foci.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. SW 81/7211

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilution in water
- Final dilution of a dissolved solid, stock liquid or gel: 0.0075 to 0.12 nL/mL a.i.

OTHER SPECIFICS: Purity of test material was 15%

Species / strain / cell type:

mammalian cell line, other: Mouse embryo C3H 10T1/2 fibroblast cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

None

Test concentrations with justification for top dose:

0.05, 0.1, 0.3, 0.5, 0.6 and 0.8 nL/mL of sample/mL (0.0075 to 0.12 nL/mL a.i.)

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: n/a
- Exposure duration: 24 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 20 plates per dose

NUMBER OF CELLS EVALUATED: 2000 cells were seeded

DETERMINATION OF CYTOTOXICITY
- Method: Not described

Evaluation criteria:

Type III transformed foci

Statistics:

Not described

Key result

Species / strain:

mammalian cell line, other: Mouse embryo C3H 10T1/2 fibroblast cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

not cytotoxic up to 0.8 nL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 25, 50 and 100 Kathon™ nL/mL (0.00015 to 15 nL/mL a.i.)

ADDITIONAL INFORMATION ON CYTOTOXICITY: Kathon™ 886 was toxic to cells at 2.5 nL/mL and higher

Conclusions:

Kathon™ 886 produced no foci transformation in the Mouse embryo fibroblast cells in primary culture in concentration up to 0.8 nL/mL (sublethal concentration).

Executive summary:

This was not a guideline study. Mouse embryo fibroblast cells in culture were exposed to Kathon™ 886 at concentrations from 0.05 to 0.8 nL of sample/mL. These treatment concentrations yielded 98 % to 33 % survival relative to control cells. Cells were exposed to the test compound for 24 h at 37 °C in Eagle’s Basal Medium supplemented with 10 % fetal calf serum. After 6 weeks of incubation, the plates were stained with 10 % Giemsa and scored for transformed foci. No type III transformed foci were seen in any of the 113 plates treated with the test compound.

Kathon™ 886 produced no adverse effect on the mammalian cell transformation test under the conditions of this study. Negative at 0.8 nL/mL; no Type III transformation foci in mouse embryo fibroplasts.

Kathon™ 886 produced no foci transformation in the Mouse embryo fibroblast cells in primary culture in concentration up to 0.8 nL/mL (sublethal concentration).

Reference 7

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan Guideline 59 Nohsan N°4200

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. 53083

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilution in water
- Final dilution of a dissolved solid, stock liquid or gel: 0.03475, 0.10425, 0.3475, 1.0425 and 3.475 microgram a.i./mL

OTHER SPECIFICS: Purity of test material was 13.9%

Species / strain / cell type:

primary culture, other: rat hepatocyte

Details on mammalian cell type (if applicable):

- Type and identity of media: Williams medium E

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Metabolic activation system:

n/a

Test concentrations with justification for top dose:

Range-finding: 0.0139 to 695 µg active substance/mL
Definitive: 0.03475, 0.10425, 0.3475, 1.0425 and 3.475 µg active substance/mL
Confirmatory: 0.0371, 0.1112, 0.371, 1.112 and 2.224 µg active substance/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

Positive control substance dissolved in ethanol at either 10 or 2.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: n/a
- Exposure duration: 18 hours

NUMBER OF REPLICATIONS: 3 plates/dose group

NUMBER OF CELLS EVALUATED: at least 9000 viable cells per plate; cells seeded per plate = 250,000

DETERMINATION OF CYTOTOXICITY
- Method: not described

Statistics:

Not described

Key result

Species / strain:

primary culture, other: rat hepatocyte

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2.22 µg/ml active substance

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: 0.0139 to 695 µg active substance/mL

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic at 2.22 µg/ml active substance ( 74.4% relative toxicity) and 3.475 µg active substance/mL (93.3 % relative toxicity). Survival index of the vehicle treatment is equal to 54%.

Conclusions:

Kathon™ 886 MW biocide did not induce DNA damage in rat hepatocytes primary cultures.

Executive summary:

OECD 482, US EPA 84-2, Japan Guideline 59 Nohsan N° 4200,in vitrogene mutation assay in primary rat hepatocytes with analytical confirmation of dosing solution stability and concentration. Kathon™ 886 MW biocide did not induce DNA damage in rat hepatocytes primary cultures.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

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.

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: In vitro cytogenetics assay using cultured human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

collected day before treatment; whole blood culture in RPMI medium with gentamycin and FCS
(lymphocyte-proliferation stimulated with PHA)

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9) prepared from Sprage Dawley rats, induced with Acrolor 1254

Test concentrations with justification for top dose:

The concentrations in the report are given for test article ACTICIDE 14.
Experiment 1, Trial 1, without and with S-9:
0.198 - 710 µg a.i./ml
Experiment 1, Trial 2 and Experiment 2, with S-9:
10.0 - 35.5 µg a.i./ml
Experiment 2, without S-9:
4.76 - 35.5 µg a.i./ml

Vehicle / solvent:

purified water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: and 4-nitroquinolone 1-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: without S9: 20 h; with S9: 3 hours, followed by a 17 h recovery phase (no recovery in expt. 2)
- Fixation time (start of exposure up to fixation or harvest of cells): overnight at 4°C in methanol/glacial acetic acid


SPINDLE INHIBITOR (cytogenetic assays): colchicin 90 min. prior to harvest
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS:
2

NUMBER OF CELLS EVALUATED:
100 metaphases (only cells with 44-46 chromosomes)


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Evaluation criteria:

statistical difference between treated/control, treated outside historical data range, effect confirmed in 2nd experiment

Statistics:

Fisher`s exact test

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: 53% mitotic inhibition at 103.6 µg/ml following treatment in absence of S-9. Treatment in presence of S-9 at 103.6 µg/ml gave apprapproximately 15% mitotic inhibition. At doses higher than this, severe mitotic inhibition was apparent

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: In vitro cytogenetics assay using cultured human peripheral blood lymphocytes

Remarks:

Migrated from field 'Test system'.

Experiment 1:

Table 1a: Results of chromosome analysis in human lymphocytes WITHOUT metabolic activation

Acticide 14 (μg/ml)		Control	43.75	67.31	103.6
		Mean +/- St. dev.
Chromatid aberrations	gaps (chr+ct)	0	0	6	6
	deletions	0	2	1	6
	interchanges	0	0	1	0
Chromosome aberrations	gaps (chr+ct)	0	0	0	0
	deletions	0	2	2	7
	interchanges	0	0	0	0
Mitotic index		5.6	3.9	4.7	2.7
Polyploidy		3	1	2	2
Endo reduplication		0	1	0	0

 

Table 1b: Results of chromosome analysis in human lymphocytes WITH metabolic activation

Acticide 14 (μg/ml)		Control	132.9	147.6	164
		Mean +/- St. dev.
Chromatid aberrations	gaps  (chr+ct)	4	0	2	3
	deletions	0	9	7	3
	interchanges	0	10	11	3
Chromosome aberrations	gaps (chr+ct)	4	0	2	3
	deletions	0	8	7	4
	interchanges	0	0	0	0
Mitotic index		6.5	2.8	3.9	2.1
Polyploidy		1	3	4	6
Endo reduplication		0	0	0	0

 

Experiment 2:

Table 2a: Results of chromosome analysis in human lymphocytes WITHOUT metabolic activation

Acticide 14 (μg/ml)		Control	65.54	81.92	102.4
		Mean +/- St. dev.
Chromatid aberrations	gaps (chr+ct)	1	1	4	5
	deletions	4	0	1	1
	interchanges	0	0	0	2
Chromosome aberrations	gaps (chr+ct)	1	1	4	5
	deletions	0	0	0	0
	interchanges	0	0	0	0
Mitotic index		5.7	3.5	3.6	1.8
Polyploidy		3	3	3	1
Endo reduplication		0	0	2	1

 

Table 2b: Results of chromosome analysis in human lymphocytes WITH metabolic activation

Acticide 14 (μg/ml)		Control	107.6	119.6	132.9
		Mean +/- St. dev.
Chromatid aberrations	gaps  (chr+ct)	3	5	4	9
	deletions	2	1	10	11
	interchanges	0	1	1	10
Chromosome aberrations	gaps (chr+ct)	3	5	4	9
	deletions	0	0	2	1
	interchanges	0	0	0	0
Mitotic index		4.8	3.5	3.7	1.9
Polyploidy		1	1	3	2
Endo reduplication		0	0	0	0

 

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation
positive with metabolic activation

ACTICIDE 14 induced chromosome aberrations in cultured human peripheral blood lymphocytes. The effect was seen most clearly following treatment in the presence of S-9.

Executive summary:

The 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 to induce chromosome aberrations was investigated in an in vitro genotoxicity study according to EPA OPP 84 -2 guideline. Human primary lymphocytes were incubated with the test substance in presence and absence of metabolic activation system (rat liver S9-mix). Cells were arrested in metaphase by addition of the spindel inhibitor colchicin, fixed and stained with Giemsa solution. Cells were microscopically evaluated for chromosome- and chromatide abnormalities.

Treatment with the test substance resulted in increased incidences of structural aberrations. While increased structural aberration n the absence of metabolic acitivation was observed in only one of two experiments, signbificant aberrations in the presence of metabolic activation were observed in both conducted experiments.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

C(M)IT/MIT was tested in one in vivo chromosomal aberration assay in mice (bone marrow) and one micronucleus test in mice (bone marrow). Negative results were observed in these in vivo studies. In the studies on tissue distribution of radiolabel in mouse presented in the dossier for MIT and C(M)IT (referenced A6.2.a/03 and A6.2.b/03, respectively in the doc IIIA), radioactivity has

been detected in bone marrow tissue following a single oral dose of the test material to adult male and female. This information provides support to the validity of the chromosome aberration test on bone marrow in mice and the micronuclei on bone marrow in mice, since it determines the extent of C(M)IT and MIT distribution to bone marrow of mice after oral exposure.

In the absence of genotoxicity, additional tests were carried out in tissue other than bone marrow.

Two UDS assays in rats confirmed the absence of genotoxicity of C(M)IT/MIT when tested in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guideline 59 Nohsan No. 4200

Deviations:

no

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. J70089

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilution in water prior to oral gavage

OTHER SPECIFICS: Purity of test material was 14.1%

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 21-32 g males, 20-30 g females
- Assigned to test groups randomly: Not described
- Fasting period before study: Not described
- Housing: Not described
- Diet (e.g. ad libitum): Not described
- Water (e.g. ad libitum): Not described
- Acclimation period: Not described

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: test substance in water administered by oral gavage

Duration of treatment / exposure:

n/a

Frequency of treatment:

Single oral gavage

Post exposure period:

6, 24 and 48 h after treatment

Dose / conc.:

30 mg/kg bw (total dose)

Remarks:

Based on a.i.

Dose / conc.:

15 mg/kg bw (total dose)

Remarks:

Based on a.i.

Dose / conc.:

3 mg/kg bw (total dose)

Remarks:

Based on a.i.

No. of animals per sex per dose:

7/sex/group per time point for water control and high dose, low and mid doses had 5/sex/group per time point (6, 24 and 48 h time points), and positive control (TEM) had 5/sex/group and were sacrificed at 24 h only

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine
- Route of administration: oral gavage
- Doses / concentrations: 1.0 mg/kg

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range finding test: 10, 20, 30, 40 and 50 mg a.i./kg. High mortality at 40 and 50 mg a.i./kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were given a single oral dose of the test article at concentrations of 3, 15 or 30 mg a.i./kg and and euthanized at 6, 24 or 48 h after treatment

DETAILS OF SLIDE PREPARATION: Bone marrow cells from both femurs were centrifuged, fixed, spread on glass slides, air-dried, stained with Giemsa stain, and mounted with cover glasses.

METHOD OF ANALYSIS: For each animal, at least 50 metaphases were read and scored using a microscope.

Evaluation criteria:

Chromosome aberrations

Statistics:

Not described

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

subtoxic concentrations

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 10, 20, 30, 40 and 50 mg a.i./kg
- Clinical signs of toxicity in test animals: High mortality at 40 and 50 mg a.i./kg.

Conclusions:

Kathon™ 886 MW biocide was negative up to 30 mg a.i./kg (didn’t cause chromosome aberrations in bone marrow cells) in this test system.

Executive summary:

OECD 475, US EPA 84-2 and Japan Guideline 59 Nohsan No. 4200. Adult CD-1 male and female mice were dosed with a single oral dose of the test article at concentrations of 3, 15 or 30 mg a.i./kg. Control animals received a single oral dose (vehicle) of distilled water or an i.p. injection of 1.0 mg/kg triethylenemelamine (TEM, positive control). Animals were euthanized at 6, 24 or 48 h after treatment. Positive control animals were euthanized only after 24 h of treatment. Bone marrow cells from both femurs were centrifuged, fixed, spread on glass slides, air-dried, stained with Giemsa stain, mounted with cover glasses. and read using a microscope.For each animal, at least 50 metaphases were scored. There were no significant increases in the number of aberrant cells in the treated groups relative to the control groups at any harvest time.

Reference 2

Endpoint:

in vivo insect germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)

Version / remarks:

OECD 477 was not in effect at the time this study was conducted

Deviations:

no

GLP compliance:

yes

Type of assay:

Drosophila SLRL assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. SW-8061

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilution in 5 % aqueous sucrose solution (pH 6.8) for oral exposures or 0.7% saline solution for intraperitoneal injection
- Final dilution of a dissolved solid, stock liquid or gel: Oral: 52 or 86 ppm a.i. (300 or 500 ppm of Kathon™ biocide); Intraperitoneal injection: 258 ppm a.i. (1,500 ppm of Kathon™ biocide)

OTHER SPECIFICS: Purity of test material was 17.2%

Species:

Drosophila melanogaster

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Males: Canton-S wild type stock from University of Wisconsin; Females: Basc stock from Brown University
- Age at study initiation: 1 day old for feeding study; 3 days old for injection study
- Weight at study initiation: Not described
- Assigned to test groups randomly: Not described
- Fasting period before study: Not described
- Housing: Glass vials
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): n/a
- Acclimation period: Not described

Route of administration:

other: oral in feed and intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Oral: sucrose; Intraperitoneal: sodium chloride
- Justification for choice of solvent/vehicle: Oral: feed; Intraperitoneal: Injection vehicle
- Concentration of test material in vehicle: Oral: 52 or 86 ppm a.i. (300 or 500 ppm of Kathon™ biocide); Intraperitoneal injection: 258 ppm a.i. (1,500 ppm of Kathon™ biocide).
- Amount of vehicle (if gavage or dermal): Oral: 5% sucrose ad libitum; Intraperitoneal: 0.3 µL of 0.7% sodium chloride

Duration of treatment / exposure:

Oral: Sucrose control and Kathon™ 886 flies: feeding for 3 days; Positive control: feeding for 24 h.
Intraperitoneal injection: 0.3 µL of test or control solution was injected once into the body at the base of a wing.

Frequency of treatment:

Oral: Continuous
Intraperitoneal injection: Single dose

Post exposure period:

Oral: males exposed by feeding were mated at the end of the third day of treatment
Intraperitoneal injection: males were mated 24 h after injection.

Dose / conc.:

52 ppm

Remarks:

Nominal a.i. in diet

Dose / conc.:

86 ppm

Remarks:

Nominal a.i. in diet

Dose / conc.:

258 ppm

Remarks:

Nominal a.i. injected

No. of animals per sex per dose:

15 males/vial, feeding study

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

Dimethylnitrosamine
- Route of administration: Oral and intraperitoneal
- Doses / concentrations: Oral 25 ppm and intraperitoneal 500 ppm

Evaluation criteria:

Induction of mutations in Drosophila melanogaster when administered to male flies orally or by intraperitoneal injection

Statistics:

Not described

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Poor food consumption; 43 % mortality in the 86 ppm a.i. oral feeding group

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Oral: 100 ppm to 10,000 ppm ; Intraperitoneal: 10 ppm to 5,000 ppm
- Clinical signs of toxicity in test animals: Oral: Significant mortality and poor food consumption at concentrations over 500 ppm Kathon™; Intraperitoneal: Not described
- Other: Maximum tolerable dose: Oral: 500 ppm Kathon™; Intraperitoneal: 1500 ppm Kathon™

Conclusions:

Kathon™ 886 biocide does not induce mutations in Drosophila melanogaster when administered to male flies orally or by intraperitoneal injection.

Executive summary:

Assessment of the mutagenic potential of Kathon™ 886 by evaluating the induction of sex-linked recessive mutations in the germ cells of Drosophila melanogaster by two routes of administration, adult feeding and adult injection. No increase in mutations at 86 ppm a.i. oral; 258 ppm a.i. injected. Kathon™ 886 biocide does not induce mutations in Drosophila melanogaster when administered to male flies orally or by intraperitoneal injection.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

revised draft guideline, March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UKEMS Test Guidelines 1993

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. 0517A001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No

OTHER SPECIFICS: Purity of test material was 14.76%

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, UK
- Age at study initiation: 37-52 days
- Weight at study initiation: 154-262 g
- Assigned to test groups randomly: Not described
- Fasting period before study: Not described
- Housing: Not described
- Diet (e.g. ad libitum): Not described
- Water (e.g. ad libitum): Not described
- Acclimation period: Not described

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance in water administered by oral gavage

Duration of treatment / exposure:

n/a

Frequency of treatment:

Single dose

Post exposure period:

2-4 h and 12-14 h after treatment

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

200 mg/kg bw (total dose)

No. of animals per sex per dose:

5/group per time point

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene for 12-14 h experiment; dimethylnitrosamine for 2-4 h experiment
- Route of administration: Oral gavage
- Doses / concentrations: 2-acetylaminofluorene: 75 mg/kg, dimethylnitrosamine: 10 mg/kg

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Rat LD50 = 457 mg/kg; in initial range-finder (200, 400, 500, 650 and 1000 mg/kg) there was mortality at 650 and 1000 mg/kg. At the 500 mg/kg dose level, sufficient clinical signs were observed for this dose to be considered the maximum dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 200 or 500 mg/kg Kathon™ 886F biocide, sampled 2-4 h and 12-14 h after treatment

DETAILS OF SLIDE PREPARATION: Hepatocytes were prepared from 3/5 animals in each dose group; 100 cells at 2-4 h, 50 cells at 12-14 h

METHOD OF ANALYSIS: Net grain count (number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm), determined in 2 of the 3 slides, each animal and each dose group.

Evaluation criteria:

Net grain count (number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm), determined in 2 of the 3 slides, each animal and each dose group.

Statistics:

Not described

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Lethargy, prostration, closing of eyes, mortality, hunching, piloerection, salivation.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 200, 400, 500, 650 and 1000 mg/kg
- Clinical signs of toxicity in test animals: Mortality at 650 and 1000 mg/kg. At the 500 mg/kg dose level, sufficient clinical signs were observed.
- Evidence of cytotoxicity in tissue analyzed: Not described
- Rationale for exposure: Not described
- Harvest times: Not described
- High dose with and without activation: 500 mg/kg

Conclusions:

Kathon™ 886F biocide had no genotoxic activity detectable in this system under the experimental conditions employed.

Executive summary:

OECD 485 (revised draft guideline, March 1996) and UKEMS Test Guidelines 1993, evaluation of the ability of Kathon™ 886F biocide to induce Unscheduled DNA Synthesis (UDS) in cultured primary rat hepatocytes from treated rats in anin vivo/in vitroprocedure.

Treatment with 200 or 500 mg/kg Kathon™ 886F biocide did not produce a group mean net grain count (NNG) value greater than –3.3 nor were there any cells found in repair at either dose.

Kathon™ 886F biocide had no genotoxic activity detectable in this system under the experimental conditions employed.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

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.

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at study initiation: 36-43 weeks
- Weight at study initiation: 25-32g (males) and 20-27g (females)
- allocated by randomy generated numbers
- Housing: not more than 3/cage, same sex
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 51-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dosing solutions prepared by dilution of the product win water within 1.5 hours before application;
0.625, 1.25. 2.0, 2.5 mg ACTICIDE 14 / ml;
20 ml/kg

Duration of treatment / exposure:

72 hours

Frequency of treatment:

once daily for 2 consecutive days

Post exposure period:

24/48 h after treatment, respectively

Remarks:

Doses / Concentrations:
12.5 mg Acticide 14/kg bw/day (=1.75 mg a.i./kg bw/day)
Basis:

Remarks:

Doses / Concentrations:
25 mg Acticide 14/kg bw/day (=3.5 mg a.i./kg bw/day)
Basis:

Remarks:

Doses / Concentrations:
40 mg Acticide 14/kg bw/day (=5.6 mg a.i./kg bw/day)
Basis:

Remarks:

Doses / Concentrations:
50 mg Acticide 14/kg bw/day (=7 mg a.i./kg bw/day)
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, CPA, single dose, 24 h

Tissues and cell types examined:

bone marrow (both femurs)

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: bone marrow flushed out of femur with 1 ml FBS, centrifuges, supernatant discarded, mixed with small amount of FBS, spotted on slide; slides air-dried, fixed in absolute methanol (5 min.), stained according to Gollapudi and Kamra (Giemsa)


METHOD OF ANALYSIS:
microscopic by a person not involved in study;
polychromatic/normochromatic erythrocytes ratio, Vernier coordinates of cells containing micronuclei

Evaluation criteria:

-acceptable, if control animals within historical range, at least 8 animals (m+f)/group available, positive control induced statistically significant increase in micronucleated cells
-positive, if statistically significant increase in micronucleated cells and increase outside historical control range

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs/mortality in 2 highest dose groups

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ACTICIDE 14 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 50 mg/kg/day (=7.2 mg a.i./kg/day), a dose at which limited mortality was observed.

No signs of clinical toxicity were apparent at 12.5 or 25 mg/kg/day. Signs of prostration, piloerection, tremors, coldness, sunken eyes and noisy breathing were seen at 40 mg/kg/day but only in a very small number of animals. Two males and three females were found dead (or were killed in extremis) at this dose level prior to the second administration. Prostration, hunched appearance, unsteady gait, piloerection, tremors, coldness and eye closure were observed at 50 mg/kg/day. Two males and one female were killed in extremis and one male and two females were found dead prior to the ferst sampling time.

Groups of mice treated with Acticide 14 exhibited PCE/NCE ratios which were similar to vehicle controls at both sampling times. Group mean frequencies of micronucleated PCE were also similar to those seen in vehicle control groups and were not significantly different by _x2 analysis.

Treatment (mg/kg bw/day)	kill time (hours)	group mean frequency of micronucleated PCE (per 1000)
vehicle	24	0.45
	48	0.55
12.5	24	0.45
	48	0.20
25	24	0.35
	48	0.35
40	24	0.56
	48	0.36
50	24	0.55
	48	0.55
positive control CPA, 80	24	23.18

Conclusions:

Interpretation of results (migrated information): negative
ACTICIDE® 14 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 50 mg/kg bw/day (=7 mg a.i./kg bw/day), a dose at which limited mortality was observed.

Executive summary:

The genotoxic 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 in vivo mouse bone marrow micronucleus test according to OECD guideline 474. Male and female CD-1 -mice were treatedw ith the test substance by oral gavage once daily for 2 days. Animals were killed 24 or 48 hours after the last treatment, and bone marrow cells were fixed on slides. Slides were stained and microscopically examined for micronucleated cells.

No signs of genotoxicity were identified. The highest evaluated doses were clearly in the toxic range (mortality and clinical signs).

Reference 5

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

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.

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Age at study initiation: 37-51 days
- Weight at study initiation: 202-286 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: not more than 3/cage in polypropylene cages with wire mesh lids and solid floors
- Diet / Water (e.g. ad libitum): ad lib.
- Acclimation period: at least 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 55-64
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 16-60 min. prior to administration (light-protected)

Duration of treatment / exposure:

12-14 hours (Experiment 1) and 2-4 hours (Experiment 2)

Frequency of treatment:

single

Post exposure period:

12-14 hours (Experiment 1) and 2-4 hours (Experiment 2)

Remarks:

Doses / Concentrations:
19 mg/kg bw (=2.66 mg a.i./kg bw/day)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
60 mg/kg bw (=8.4 mg a.i./kg bw/day)
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene(14 h experiment); N-dimethylnitrosamine (2h experiment);
- Route of administration: oral
- Doses / concentrations: 2-AAF: 75 mg/kg bw, DMN: 10 mg/kg bw

Tissues and cell types examined:

isolated primary hepatocytes

Details of tissue and slide preparation:

-primary hepatocytes prepared by two-step collagenase perfusion technique (Seglen et al.)
-after attachment (90 min.) cells were radiolabelled by addition of 10 µCi/ml [3H]-thymidine for 4 hours; labelled thymidine was removed by washing with "cold" thymidine-containing medium
-cells incubated overnight, fixed with methanol:acetic acid, mounted onto microscope slides
-autoradiography: radioactivity was visualised as cells were stained with Illford K2/Kodak developer/Illford Hypam fixer;
nuclei and cytoplasm were counterstained with Meyers haemalum/eosin Y
-grains were counted under the microscope, NG detemined (grains/nucleus)

Evaluation criteria:

-negative control NG of zero or less
-positive controls: 5 or more NG, 50% of the cells NG 5 or greater
-response is positive if 20% or more of the cells are responding and NG is zero or greater

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at higher dose levels

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

-tox range finding study: 12.5-100 mg/kg bw; LD50 = 89 mg/kg bw

table 1: 12 -14 hour-experiment

Dose (mg/kg bw)	net nuclear grain count (NG)	net grain count of cells in repair	% cells in repair
0	-0.7	6.7	1.0
19	-0.8	7.8	3.0
60	-0.8	5.9	1.4
positive control 2 -AAF	12.0	13.0	88.4

table 2: 2 -4 hour-experiment

Dose (mg/kg bw)	net nuclear grain count (NG)	net grain count of cells in repair	% cells in repair
0	-1.2	5.9	1.0
19	-0.9	6.7	1.8
60	-1.1	6.2	2.2
positive control 2 -AAF	11.8	13.7	83.0

Conclusions:

Interpretation of results (migrated information): negative
ACTICIDE 14 had no genotoxic activity detectable in the ex vivo UDS test system under the experimental conditions employed.

Executive summary:

The genotoxic 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 investigated in an ex-vivo unsceduled DNA-synthesis study according to EPA OPP 84 -2. Male Wistar rats were treated with the test substance by oral gavage, and 12 -14 and 2 -4 hours after tretament, primary hepatocytes were isolated by two-step collagenase technique. Heopatocytes were exposed to tritium-labelled thymidine, and thymidine was incorporated reflecting the activity of DNA repair.Heopatocytes were cultured overnight and incorporated radioactivity optically visualised. Microscopic evaluation revealed amount of responding cells and the grains/nucleus ratio (NG). The NG was compared to vehicle-treated and positive-control-treated cells.

When treated orally with ACTICIDE 14 at doses up to 60 mg/kg, male Wistar rats showed no induction of UDS in hepatocytes isolated after in vivo tretament approximately 12-14 or 2-4 hours after dosing.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

CMIT/MIT carries a mandatory classification in accordance with Annex VI Regulation EC 1272/2008 and is not classified for genotoxic effects.