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Toxicological information

Genetic toxicity: in vitro

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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
Study period:
20 April - 7 May 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
The test article Kordek 573T (Lot No. B-1103) containing 97.5% of the active ingredient 2-methyl-4-isothiazolin-3-one, was used.

Note: All concentrations were adjusted for active ingredient.

Method

Target gene:
histidine requiring bacteria
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Strains of Salmonella typhirnurium used for this study included: TA98, TA100, TA1535, TA1537 and TA102 obtained from Dr. B. Ames, University of California, Berkeley. Strains were characterized for nutritional requirements, crystal violet sensitivity, and ampicillin resistance prior to initiation of the study.
Metabolic activation:
with and without
Metabolic activation system:
The S-9 used for metabolic activation was obtained from the livers of rats treated with Aroclor 1254 (obtained from Molecular Toxicology, Inc. [Moltox]), Lot No. 835).
Test concentrations with justification for top dose:
Based on the range finding data, the test article was initially evaluated at 20, 50, 200, 500 and 1000 ug/plate with metabolic activation and 5, 20, 50, 200, and 500 ug/plate without metabolic activation.

A confirmatory assay was conducted at concentrations of 60, 90, 160, 300 and 600 pglplate in all strains with metabolic activation and in TA1537 and TA102 without metabolic activation, and at concentrations of 30, 60, 90, 160 and 300 ug/plate in strains TA98, TA100 and TA1.535 without metabolic activation
Vehicle / solvent:
The solvent for the test article was distilled water. The solvent for the positive control articles (with the exception of sodium azide, mitomycin-c and 9-aminoacridine) was dimethyl sulfoxide (DMSO) (Sigma Chemical Co., Lot No. 117H3602). The solvent for sodium azide and mitomycin-c was distilled water. The solvent for 9-aminoacridine was 95% ethanol (Pharmco USP, Lot No. 970805 1).
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-anthramine with S9 and 2-nitrofluorene (2NF) for strain TA98; sodium azide (SA) for strains TA 100 and TA1535, 9-aminoacridine (9AA) for strain TA1537, and mitomycin-c (MMC) for strain TA102.
Details on test system and experimental conditions:
The test article was evaluated for mutagenic activity at concentrations ranging from 5 to 1000 ug/plate, with and without metabolic activation, in Salmonella strains TA98, TA100, TA1535, TA1537, and TA102. Control plates were run to: check for sterility, determine the background reversion rate, and measure the response of each tester strain to a positive control compound.

For the activated portion of the assay the following were added, in order, to sterile test tubes: 2 ml of top agar, 0.1 ml of the bacteria inoculum, 0.1 ml of the appropriate concentration of test compound, and 0.5 ml of phosphate buffer mix (with S-9 and NADP). For the non-activated portion of the assay, the above procedure was followed, except that the 0.5 ml of phosphate buffer mix (without S-9 or NADP) was added to the tubes directly after addition of the top agar. Each test article concentration was tested in triplicate, in minimal plates (minimal-glucose agar medium). The controls were tested in six replicates in minimal plates. The contents of the tubes were mixed and poured onto petri dishes containing approximately 20 ml of the appropriate agar. Plates were allowed to set for several minutes then placed in covered plastic boxes and incubated at 37 (+/-1) degrees Celsius for at least 48 hours prior to colony counting. The results were confirmed in an independent assay.
Evaluation criteria:
Following the incubation period, sterility plates were checked for contamination. Following the sterility check, the number of colonies on each plate was determined. The mean and standard deviation for each concentration was calculated. Background growth was checked for each experimental point to observe any toxic response.

A mutagenicity assay is considered valid if the following conditions are met. First, the spontaneous reversion rate, with and without metabolic activation, must be reasonably consistent with the expected range for the strain being used; i.e., 20-60 colonies per plate for TA98, 100-250 for TA100, 13-50 for TA1535,7-20 for TA1537, and 240-360 for TA102. Second, the positive control materials must elicit a positive response. And third, strains must maintain their genotype, i.e., nutritional requirements, crystal violet sensitivity and ampicillin resistance.

A test article is considered positive (mutagenic) if it elicits in independent assays a number of revertants per plate at least 2 times that observed in the solvent control (background). A response that does not meet this criterion but elicits a potential biologically significant response (e.g., a dose related increase in revertants per plate over 3 concentrations) is considered an equivocal response and requires further evaluation.

A test article is considered negative (non-mutagenic) if the criteria for a positive assay were not met and the test article was tested up to either 5,000 ug/plate, the limit of solubility, or the limit of toxicity. Toxicity is defined as the elimination of a uniform background lawn.
Statistics:
Statistical methods beyond the calculation of the mean and standard deviation are not considered necessary for the interpretation of this study

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In all strains at 1000 ug/plate with metabolic activation and in strains TA98, TA100 and TA1535 at 500 ug/plate without metabolic activation (Table 1) and toxicity was observed in TA100 at 600 ug/plate with metabolic activation (Table 2).
Vehicle controls validity:
other: yes, except for TA98 which was retested (Table 1)
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Because the test substance has bactericidal properties, a range finding study was conducted to select dose levels for the definitive assay. Initial range finding data showed toxicity in all strains, with and without metabolic activation, at 1000 ug/plate but not at 100 ug/plate. A subsequent range finding investigation employing strains TA98, TA100, TA1535, and TA1537 showed toxicity at 500 ug/plate without activation but not with activation. Based on the range finding data, the test article was initially evaluated at 20, 50, 200, 500 and 1000 ug/plate with metabolic activation and 5, 20, 50, 200, and 500 ug/plate without metabolic activation. All concentrations were adjusted for active ingredient.

In the definitive assay, the solvent control values for TA98 were not reasonably consistent with the historical data base and the expected range of spontaneous revertants for this strain. TA98 was therefore not scored, but was repeated in an independent assay. Toxicity was observed in the definitive assay in all strains at 1000 ug/plate with metabolic activation and in strains TA98, TA100 and TA1535 at 500 ug/plate without metabolic activation (see Table 1 for mean summary data).

A confirmatory assay was conducted at concentrations of 60, 90, 160, 300 and 600 ug/plate in all strains with metabolic activation and in TA1537 and TA102 without metabolic activation, and at concentrations of 30, 60, 90, 160 and 300 ug/plate in strains TA98, TA100 and TA1535 without metabolic activation (see Table2 for mean summary data). In the confirmatory assay toxicity was observed in TA100 at 600 ug/plate with metabolic activation.

At these test article concentrations, a mutagenic response was not detected in any of the Salmonella tester strains examined (TA98, TA100, TA1535, TA1537 and TA102).

Samples of the dosing solutions were submitted for independent chemical analysis of the test article concentration for the definitive assay. The results indicate that the concentration of the Kordek 573T dosing solutions agreed with the expected target concentrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 - Definitive Assay

MEAN SUMMARY DATA

 Test                Mean Revertants/Plate
 Material (ug/plate)  S9  TA98  TA100  TA1535  TA1537  TA102
 Solvent Controls            
 Water  +

 --- (---)

31 (3)

 205 (18)  14 (2)  14 (2)  324 (24)
 Water  -

 --- (---)

30 (5)

 221 (29)  13 (4)  12 (2)  257 (28)
 Positive Controls            
 2 -anthramine (2)  +

 --- (---)

832* (33)

 1046* (73)  172* (28)  35* (2)  N.D.
 2 -anthramine (12)  + N.D.  N.D.  N.D.  N.D.  1274* (121)
   2 -nitrofluorene (3)  -

   --- (---)

775* (134)

N.D.  N.D.  N.D.  N.D.
 sodium azide (2)  -  N.D.    740* (46)   785* (42)  N.D.  N.D.
   9 -aminoacridine (100)  -  N.D.  N.D.  N.D.   464* (148)  N.D.
 mitomycin-c (2)  -  N.D.  N.D.  N.D.  N.D.  867* (28)
 Kordek 573T (1000)  +  ---(---)---(---)F  ---(---)F  ---(---)F  ---(---)F  ---(---)F
 (500)  +  ---(---)28 (6)  223 (7)  12 (4)  14 (1)  257 (34)
 (200)  +  ---(---)35 (5)  230 (32)  10 (1)  11 (1)  330 (11)
 (50)  +  ---(---)37 (3)  235 (11)  15 (3)  15 (3)  291 (25)
 (20)  +  ---(---)33 (8)  246 (5)  18 (5)  16 (5)  299 (38)
 (500)  -  ---(---)---(---)F  ---(---)F  ---(---)F  7 (3)  239 (21)
 (200)  -  ---(---)33 (4)  200 (61)  12 (3)  10 (1)  280 (26)
 (50)  -  ---(---)30 (6)  214 (6)  14 (2)  10 (5)  306 (21)
 (20)  -  ---(---)26 (2)  238 (9)  15 (2)  15 (7)  291 (12)
 (5)  -  ---(---)28 (5)  179 (6)  17 (2)  12 (3)  305 (7)

* Positive Response: Greater than or equal to 2 x Solvent (TA98, TA100, TA1535, TA1537 and TA102)

F: Toxicity of Strain Observations

Excluded from Calculations: F

--- TA98 Value out of Range (Controls not consistent with historical data base and expected values for this strain)

Data Reported as: Mean (Standard Deviation)

N.D. = Not Determined.

Table II - Confirmatory Assay

MEAN SUMMARY DATA

 Test                Mean Revertants/Plate
 Material (ug/plate)  S9  TA98  TA100  TA1535  TA1537  TA102
 Solvent Controls            
 Water  +  25 (4)  263 (22)  17 (5) 13 (2)   350 (11)
 Water  -  28 (6)  226 (8)  18 (6)  15 (3)  311 (24)
 Positive Controls            
 2 -anthramine (2)  +  301* (53)  601* (26)  148* (20)  31* (5)  
 2 -anthramine (12)  +          1330* (97)
 2 -nitrofluorene (3)  -  693* (126)        
 sodium azide (2)  -    752* (27)  918* (35)    
 9 -aminoacridine (100)  -        432* (150)  
mitomycin-c (2) -          1048* (43)
 Kordek 573T (600)  +  29 (2)  ---(---)F  10 (1)  12 (2)  266 (26)
 (300)  +  29 (2)  247 (17)  13 (1)  15 (2)  334 (35)
 (160)  +  39 (6)  252 (11)  11 (1)  13 (1)  348 (14)
 (90)  +  29 (5)  251 (33)  14 (4)  17 (3)  320 (52)
 (60)  +  33 (4)  248 (36)  11 (6)  22 (2)  366 (28)
 (600)  -  N.D.  N.D.  N.D.  5 (1)  210 (16)
 (300)  -  31 (4)  257 (10)  8 (3)  18 (4)  313 (15)
 (160)  -  32 (6)  276 (11)  11 (4)  12 (2)  287 (21)
 (90)  -  27 (2)  284 (7)  14 (5)  18 (3)  330 (26)
 (60)  -  32 (6)  241 (10)  12 (3)  18 (2)  345 (34)
 (30)  -  23 (2)  255 (13)  15 (2)  N.D.  N.D.

* Positive Response: Greater than or equal to 2 x Solvent (TA98, TA100, TA1535, TA1537 and TA102)

F: Toxicity of Strain

Observations Excluded from Calculations: F

Data Reported as: Hean (Standard Deviation)

N.D. = Not Determined.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, Kordek 573T was not mutagenic in the Salmonella gene mutation assay.
Executive summary:

Kordek 573T, (sample number TD 99-019), lot number B-1103, 97.5% active ingredient) was evaluated for mutagenic activity in the Salmonella typhimurium gene mutation assay (Ames test). Tester strains were TA98, TA100, TA1535, TA1537, and TA102 in the presence and absence of a metabolic activation system (S-9 liver fraction from Aroclor 1254 treated rats). Distilled water was used as the solvent for the test article and as the solvent control. In the presence of metabolic activation, 2- anthramine was used as the positive control for all strains. In the absence of metabolic activation, the positive controls used were: 2- nitrofluorene (TA98), sodium azide (TA100 and TA1535), 9- aminoacridine (TA1537), and mitomycin-c (TA102). The test article concentrations were selected from range finding data to establish toxic levels in the tester strains. The test article was evaluated at concentrations ranging from 5 to 1000 μg/plate (all concentrations were adjusted for active ingredient) and the number of revertants was determined. Results were confirmed in an independent assay.

The test article did not induce an increase in revertants when compared to solvent controls. This was true for all tester strains both with and without metabolic activation. Toxicity was observed in the definitive assay in all strains at 1000 μg/plate with metabolic activation and in strains TA98, TAl00 and TA1535 at 500 μg/plate without metabolic activation. In the confirmatory assay toxicity was observed in TA100 at 600 μg/plate with metabolic activation.

Under the conditions of this study, Kordek 573T was not mutagenic in the Salmonella gene mutation assay.