Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following 3 studies were identified as key studies -

AMES - 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 mitomycinc (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.

Chromsomal aberration test -

The objective of this in vitro assay was to evaluate the ability of Kordek 573T (Sample identification: TD 99-087, Lot No. B1103, active ingredient 97.5%) to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation. Based on the results of the study, Kordek 573T (Sample identification: TD 99-087, Lot No. B1103, active ingredient 97.5%) may be interpreted as being positive for inducing chromosome aberrations in CHO cells with and without metabolic activation. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index). The highly nonlinear dose response for the induction of chromosomal

aberrations and the associated correlation of the chromosomal aberrations with high level of cytotoxicity suggest that the chromosomal damage was induced by an indirect process associated with cytotoxicity. As noted by Hilliard et. al. (1998), chromosomal damage in a CHO assay that is limited to concentrations that are highly cytotoxic (suggested to be in the 30-40% or more range) may be interpreted as a "false positive."

Gene mutation study in mammalian cells (HGPRT) -

The test article, Kordek® 573T (Sample identification: TD 99-087, Lot No. B1103, active ingredient 97.5%) did not cause a significant increase in the mutant frequency at the HGPRT locus among the test article-treated cultures in the presence and absence of exogenous metabolic activation. There was no dose-dependent response in the test article treated cultures. The mutant frequencies of the test article solvent and the solvent for the positive controls were within

SITEK's historical negative control values. The positive controls caused a significant increase in the mutant frequencies. All criteria for a valid assay were met. Thus, Kordek® 573T (Sample No. TD99-087, Lot No. B1103, 97.5 % active ingredient) was considered to be non-mutagenic in CHO cells when tested under the conditions of this study.

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
histidine requiring bacteria
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).
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
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'.

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.

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.

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
Study period:
30 September - 15 November 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
EPA OPP 84-2
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chromosome aberration: structural chromosome damage expressed as breakage, or breakage followed by reunion, of both sister chromatids at an identical site.

Chromatid aberration: structural chromosome damage expressed as breakage of single chromatids or breakage followed by reunion between chromatids. This is the most common type of structural aberration
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The Chinese hamster ovary cells (CHO-WBL) used in this assay were from a permanent cell line and were originally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. The cells have since been recloned to maintain karyotypic stability. This cell line has an average cycle time of 12 to 14 hours with a modal chromosome number of 21.
Metabolic activation:
with and without
Metabolic activation system:
Rats were treated once with 500 mg/kg of Aroclor 1254 and S9 prepared approximately 5 days later (Molecular Toxicology, Inc., Lot #929).
Test concentrations with justification for top dose:
In the initial chromosomal aberrations assay, concentrations of 0.0785, 0.157, 0.313, 0.625, 1.25, 2.50, 5.00, 9.53, 12.7, 16.9, 22.5, 30.0, and 40.0 μg/mL were tested with and without metabolic activation.

In a confirmatory chromosomal aberrations assay, concentrations of 0.157, 0.3 13, 0.625, 1.25, 2.50, 3.75, 5.00, 7.50, 10.0, 12.5, 15.0, and 20.0 μg/mL without metabolic activation and 1.25,2.50,5.00,7.50, 10.0, 12.5, 15.0, 17.5, and 20.0 μg/mL with metabolic activation were tested.
Vehicle / solvent:
Sterile Deionized Water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC) for the nonactivation series and cyclophosphamide (CP) in the metabolic activation series.
Details on test system and experimental conditions:
Aberrations Assay Without Metabolic Activation
Cultures were initiated by seeding approximately 1.2 x 10(6) cells per 75 cm2 flask into 10 mL of complete McCoy's 5a medium. One day after culture initiation, the cells were incubated at ~37 C with the test article at predetermined concentrations for 3.0 hours for the initial assay or 17.8 hours in the confirmatory assay. The cultures were then washed with buffered saline. In the initial assay, the cells were refed with complete McCoy's 5a medium and incubated for the rest of the culture period up to the time of harvest with 0.1 ug/mL ColcemidB present during the last ~2.0 hours of incubation. In the confirmatory assay, cells were refed with complete McCoy's 5a medium with 0.1 ug/mL ColcemidB and harvested 2.0 hours later.

Aberrations Assay With Metabolic Activation
Cultures were initiated by seeding 1.2 x 10(6) cells per 75 cm2 flask into 10 mL of complete McCoy's 5a medium. One day after culture initiation, the cultures were incubated at 37°C for 3.0 hours in the presence of the test article and the S9 reaction mixture in McCoy's 5a medium without FBS. The cells were then washed twice with buffered saline and the cells were refed with complete McCoy's 5a medium. The cells were incubated for the rest of the culture period up to the time of harvest with 0.1 ug/mL Colcemidm present during the last ~2.0 hours of incubation. The cultures were then harvested.

Harvest Procedure
Prior to the harvest of the cultures, visual observations of toxicity were made. These observations included an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic (large rounded cells) or dead cells floating in the medium. The cultures were then trypsinized to collect mitotic and interphase cells. An aliquot was removed to count cells. The rest of the cultures were treated with 0.075 M KCl hypotonic solution. This treatment helps to swell the cells and thus disperse the chromosomes. The cultures were then fixed with an absolute methanol: glacial acetic acid (3: 1, v:v) fixative before air-dried slides were prepared.

Slide Preparation and Staining
Slides were prepared by dropping the harvested cultures on clean slides and airdried. The slides were stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were then air-dried and mounted permanently.

Aberrations Analysis and Assay Evaluation
Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 +/- 2 (range 19-23) were analyzed.
One hundred cells, if possible, from each replicate culture from at least four concentrations of the test article and from the negative, vehicle, and one dose of the positive control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962, 1976). At least 25 cells were analyzed from those cultures that had greater than 25% of cells with one or more aberrations. Mitotic index was evaluated from the negative control, vehicle control, and a range of concentrations by analyzing the number of mitotic cells in 1000 cells and the ratio expressed as a percentage of mitotic cells. Percent polyploidy and endoreduplication were also analyzed by evaluating 100 metaphases, if available, and tabulated.

References:
Evans, H. J. (1 962) Chromosomal aberrations produced by ionizing radiation, International Review of Cytology, 13: 22 1-32 1.

Evans, H.J. (1976) Cytological Methods for Detecting Chemical Mutagens. In: Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed.), Plenum Press, New York and London, pp. 1-29
Evaluation criteria:
An assay was considered acceptable for evaluation of test results only if all of the following criteria were satisfied. The metabolic activation and nonactivation sections of the aberrations assay were independent units and would be repeated independently, as needed, to satisfy the acceptance criteria.

Acceptable Controls
The negative (untreated) and the vehicle control cultures must contain less than approximately 5% cells with aberrations The positive control result must be significantly higher (p
Acceptable High Dose
If the aberration results are negative and there is no significant reduction (approximately >/= 50%) in mitotic index, and/or cell counts, the assay must include the highest applicable dose (a target dose of 10 mM or 5 mg/mL, whichever is lower) or a dose exceeding the solubility limit in culture medium. Testing was conducted at insoluble concentrations when a well-dispersed suspension in culture medium was obtained that did not settle rapidly.

Acceptable Number of Doses
The assay must include at least three analyzable concentrations.

Acceptable Dose-Related Response
If significant increases are observed but not in consecutive concentrations, there should be a clear evidence for a dose-response.

Assay Evaluation Criteria
The following factors are taken into account in evaluation of the test article data:
Percentage of cells with aberrations.
Percentage of cells with more than one aberration.
Evidence for increasing amounts of damage with increasing dose, i.e., a dose related increase in aberrations.
Statistics:
The experimental unit is the cell, and therefore the percentage of cells with structural aberrations was the basis for evaluation. Statistical analysis employed a Cochran-Arrnitage test for linear trend and Fisher's Exact Test (Thakur et al., 1985) to compare the percentage of cells with aberrations (and, if applicable, the percentage of cells with more than one aberration) in treated cells to the results obtained for the vehicle controls.

Statistical analysis was also performed for cells exhibiting polyploidy andlor endoreduplication in order to indicate significant (p < 0.01) increases in these events as indicators of possible induction of numerical aberrations; however, the test article was evaluated only for structural aberrations and not for numerical aberrations by this protocol.

Evaluation of a Positive Response
A test article was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p
Evaluation of a Negative Response
A test article was considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cells with chromosomal aberrations at any of the concentrations.

Equivocal Evaluation
Although most assays gave clearly positive or negative results, in rare cases the data set would preclude making a definitive judgment about the activity of the test article. Results might remain equivocal or questionable regardless of the number of times the assay is repeated.

Continued below:
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
other: False positive due to cytotoxicity
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Chromosomal Aberrations Assay Without Metabolic Activation
Initial Assay
Dead cell monolayers, >90% reduction in the cell monolayer confluence, floating dead cells, debris, and a severe reduction in the number of visible mitotic cells were observed in the cultures treated with 40.0 ug/mL. Very unhealthy cell monolayers, ~ 55% reduction in the cell monolayer confluence, floating dead cells, debris, and a severe reduction in the number of visible mitotic cells were observed in the cultures treated with 30.0 ug/mL. Very unhealthy cell monolayers, ~ 15% reduction in the cell monolayer confluence, floating dead cells, debris, and a severe reduction in the number of visible mitotic cells were observed in the cultures treated with 22.5 ug/mL. Unhealthy cell monolayers, ~ 15% reduction in the cell monolayer confluence, floating dead cells, debris, and a severe reduction in the number of visible mitotic cells were observed in the cultures treated with 16.9 ug/mL. Slightly unhealthy cell monolayers and a slight reduction in the number of visible mitotic cells were observed in the cultures treated with 12.7 ug/mL. Slight reductions in the number of visible mitotic cells were observed in the cultures treated with 5.00 and 9.53 ug/mL. Slight reductions of 8% and 23% were observed in the mitotic indices of the cultures treated with 9.53 and 12. ug/mL, respectively, as compared with the vehicle control cultures (Table 1). Reductions of 2%, 1%, 39%, 42%, 57%, 78%, 82%, 93%, and 98% were observed in the cell counts of the cultures treated with 1.25, 2.50, 5.00, 9.53, 12.7, 16.9, 22.5, 30.0, and 40.0 ug/mL, respectively, as compared with the vehicle control cultures (Table 1). Chromosomal aberrations were analyzed from the cultures treated with 1.25,2.50, 5.00, 9.53, and 12.7 ug/mL (Table 2). A significant increase in cells with chromosomal aberrations was observed in the cultures treated with 9.53 and 12.7 ug/mL. No significant increase in polyploidy or endoreduplication was observed in the cultures analyzed, except for a weakly significant response in endoreduplication in the cultures treated with 9.53 ug/mL. However, the increase observed, 5.3% is within the historical control data of 0-10% and 0-9.5% in the negative and vehicle control cultures, respectively, and therefore of no biological relevance. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index). The highly nonlinear dose response for the induction of chromosomal aberrations and the associated correlation of the chromosomal aberrations with high level of cytotoxicity suggest that the chromosomal damage was induced by an indirect process associated with cytotoxicity. As noted by Hilliard et. al. (1998), chromosomal damage in a CHO assay that is limited to concentrations that are highly cytotoxic (suggested to be in the 30-40% or more range) may be interpreted as a "false positive."

Based on the results from the initial assay, the confirmatory nonactivation aberrations assay was conducted testing concentrations of 0.157, 0.313, 0.625, 1.25, 2.50, 3.75, 5.00, 7.50, 10.0, 12.5, 15.0, and 20.0 ug/mL. Treatment period was for 17.8 hours and the cultures were harvested 20.0 hours from the initiation of treatment.

Confirmatory Assay
Dead cell monolayers, ~ 85% reduction in the cell monolayer confluence, debris, and no visible mitotic cells were observed in the cultures treated with 20.0 ug/mL. Unhealthy cell monolayers, floating dead cells, debris, and no visible mitotic cells were observed in the cultures treated with 12.5 and 15.0 ug/mL. Slightly unhealthy cell monolayers, floating dead cells, debris, and a reduction in the number of visible mitotic cells were observed in the cultures treated with 7.50 and 10.0 ug/mL. Slight reductions in the number of visible mitotic cells were observed in the cultures treated with 3.75 and 5.00 ug/mL. Reductions of 36%, 32%, 61%, and 85% were observed in the mitotic indices of the cultures treated with 5.00, 7.50, 10.0, and 12.5 ug/mL, respectively, as compared with the vehicle control cultures (Table 3). Reductions of 29%, 39%, 42%, 49%, 50%, 78%, and 98% were observed in the cell counts of the cultures treated with 3.75, 5.00, 7.50, 10.0, 12.5, 15.0, and 20.0 ug/mL, respectively, as compared with the vehicle control cultures (Table 3). Chromosomal aberrations were analyzed from the cultures treated with 1.25, 2.50, 3.75, and 7.50 ug/mL (Table 4). A significant increase in cells with chromosomal aberrations was observed in the cultures treated with 3.75 and 7.50 ug/mL. No significant increase in polyploidy or endoreduplication was observed in the cultures analyzed. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index). The highly nonlinear dose response for the induction of chromosomal aberrations and the associated correlation of the chromosomal aberrations with high level of cytotoxicity suggest that the chromosomal damage was induced by an indirect process associated with cytotoxicity. As noted by Hilliard et. al. (1998), chromosomal damage in a CHO assay that is limited to concentrations that are highly cytotoxic (suggested to be in the 30-40% or more range) may be interpreted as a "false positive."

The sensitivity of the cell cultures for induction of chromosomal aberrations is shown by the increased frequency of aberrations in the cells exposed to mitomycin C, the positive control agent. The test article is considered positive for inducing chromosomal aberrations under nonactivation conditions. The test article is considered negative for inducing polyploidy and endoreduplication under nonactivation conditions.

Chromosomal Aberrations Assay With Metabolic Activation
Initial Trial
Very unhealthy cell monolayers, >90% reduction in the cell monolayer confluence, floating dead cells, debris, and a severe reduction in the number of visible mitotic cells were observed in the cultures treated with 40.0 ug/mL. Unhealthy cell monolayers, ~30% reduction in the cell monolayer confluence, floating dead cells, debris, and a severe reduction in the number of visible mitotic cells were observed in the cultures treated with 30.0 ug/mL. Unhealthy cell monolayers, ~ 15% reduction in the cell monolayer confluence, floating dead cells, debris, and a severe reduction in the number of visible mitotic cells were observed in the cultures treated with 22.5 ug/mL. Unhealthy cell monolayers, floating dead cells, debris, and a reduction in the number of visible mitotic cells were observed in the cultures treated with 16.9 ug/mL. Slightly unhealthy cell monolayers, debris, and a slight reduction in the number of visible mitotic cells were observed in the cultures treated with 12.7 ug/mL. Slight reductions in the number of visible mitotic cells were observed in the cultures treated with 5.00 and 9.53 ug/mL. Reductions of 10%, 10%, 27%, 56% and 70% were observed in the mitotic indices of the cultures treated with 1.25, 5.00,9.53, 12.7, and 16.9 ug/mL, respectively, as compared with the vehicle control cultures (Table 5). Reductions of 15%, 24%, 28%, 27%, 52%, 59%, 77%, and 90% were observed in the cell counts of the cultures treated with 2.50, 5.00, 9.53, 12.7, 16.9, 22.5, 30.0, and 40.0 ug/mL, respectively, as compared with the vehicle control cultures (Table 5). Chromosomal aberrations were analyzed from the cultures treated with 2.50, 5.00, 9.53, 12.7, and 16.9 ug/mL (Table 6). A significant increase in cells with chromosomal aberrations was observed in the cultures treated with 12.7 and 16.9 ug/mL. No significant increase in polyploidy or endoreduplication was observed in the cultures analyzed. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index). The highly nonlinear dose response for the induction of chromosomal aberrations and the associated correlation of the chromosomal aberrations with high level of cytotoxicity suggest that the chromosomal damage was induced by an indirect process associated with cytotoxicity. As noted by Hilliard et. al. (1 998), chromosomal damage in a CHO assay that is limited to concentrations that are highly cytotoxic (suggested to be in the 30-40% or more range) may be interpreted as a "false positive."

Based on the results from the initial assay, the confirmatory aberrations assay with metabolic activation was conducted testing concentrations of 1.25,2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 17.5, and 20.0 ug/mL. Treatment period was for 3.0 hours and the cultures were harvested 20.0 hours from the initiation of treatment.

Confirmatory Assay
Unhealthy cell monolayers, floating dead cells, debris, and a severe reduction in the number of no visible mitotic cells were observed in the cultures treated with 17.5 and 20.0 ug/mL. Slightly unhealthy cell monolayers, floating dead cells, debris, and a reduction in the number of visible mitotic cells were observed in the cultures treated with 15.0 ug/mL. Slightly unhealthy cell monolayers, floating dead cells, debris, and a slight redwction in the number of visible mitotic cells were observed in the cultures treated with 12.5 ug/mL. Slight reductions in the number of visible mitotic cells were observed in the cultures treated with 7.50 and 10.0 ug/mL. Reductions of 21%, 25%, 48%, 44%, 39%, 48%, and 77% were observed in the mitotic indices of the cultures treated with 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, and 17.5 ug/mL, respectively, as compared with the vehicle control cultures (Table 7). Reductions of 14%, 9%, 23%, 42%, 52%, and 76% were observed in the cell counts of the cultures treated with 2.50, 10.0, 12.5, 15.0, 17.5, and 20.0 ug/mL, respectively, as compared with the vehicle control cultures (Table 7). Chromosomal aberrations were analyzed from the cultures treated with 1.25, 2.50, 5.00, and 7.50 ug/mL (Table 8). A significant increase in cells with chromosomal aberrations was observed in the cwltures treated with 7.50 ug/mL. No significant increase in polyploidy or endoreduplication was observed in the cultures analyzed. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index). The highly nonlinear dose response for the induction of chromosomal aberrations and the associated correlation of the chromosomal aberrations with high level of cytotoxicity suggest that the chromosomal damage was induced by an indirect process associated with cytotoxicity. As noted by Hilliard et. al. (1998), chromosomal damage in a CHO assay that is limited to concentrations that are highly cytotoxic (suggested to be in the 30-40% or more range) may be interpreted as a "false positive."

The successful activation by the metabolic system is illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent. The test article is considered positive for inducing chromosomal aberrations under activation conditions. The test article is considered negative for inducing polyploidy and endoreduplication under activation conditions.

Reference:
Hilliard, C.A., Armstrong, M.J., Bradt, C.I., Hill, R.B., Greenwood, S.K., and Galloway, Sheila M. (1998) Chromosome aberrations in vitro related to cytotoxicty of nonmutagenic chemicals and metabolic poisons, Environmental and Molecular Mutagenesis, 31: 3 16-326.
Remarks on result:
other: False positive due to cytotoxicity

Solubility and Dose Determination

The highest concentration used in this assay was limited by toxicity. Kordek 573T (Sample identification: TD 99-087) was dissolved/suspended in sterile deionized water (Lot # 33, prepared at Covance-Vienna) at 50.0 mg/mL after heating to 40-44C in a water-bath for ~45 minutes. This was a translucent, light yellow suspension. This suspension and its dilutions were dosed using a 10% (100 uL/mL) dosing volume and the vehicle control cultures were treated with 100 uL/mL of sterile deionized water. The initial chromosomal aberrations assay was conducted with a top stock concentration of 50.0 mg/mL and concentrations of 33.9, 48.4, 69.1, 98.7, 141, 202, 288, 412, 588, 840, 1200, 1720, 2450, 3500, and 5000 ug/mL with and without metabolic activation were tested with a treatment period of 3.0 hours and a harvest of 20.1 hours from the initiation of treatment. Due to the excessive toxicity observed while conducting visual observations in the cultures tested, the initial chromosomal aberrations assay was repeated testing concentrations of 0.0785, 0.157, 0.313, 0.625, 1.25, 2.50, 5.00, 9.53, 12.7, 16.9, 22.5, 30.0, and 40.0 ug/mL with and without metabolic activation with a treatment period of 3.0 hours and a harvest of 20.0 hours from the initiation of treatment. All dosing for this repeat assay and the confirmatory assay was achieved using a 1% (10.0 uL/mL) dosing volume and the vehicle control cultures were treated with 10.0 uL/mL of sterile deionized water. Analytical verification of the dosing solutions were performed by the Sponsor.

Osmolality

Osmolality of the test article in the culture medium was evaluated using a Digimatic Osmometer, Model 3D2, manufactured by Advanced Instruments, Inc. The instrument is a freezing point depression osmometer. The osmometer was calibrated using 100, 290, and 900 mOsm/kg reference controls. Sample and control materials were measured in duplicate, at room temperature, using 250 ul in disposable cuvettes. Osmolality of the test material was evaluated in culture medium at 5000 ug/mL. Results are presented in Table 1. The reading for the reference solution was 289 mOsm/kg water and was within the normal range. McCoy's 5a culture medium was 299 mOsm/kg water and McCoy's 5a culture medium with sterile deionized water at 100 uL/mL was 270 mOsm/kg water. The reading for the test concentration of Kordek 573T (TD99-087) at 5000 ug/mL, 316 mOsm/kg, was similar to McCoy's 5a culture medium.

Analysis of Dosing Solutions

The dosing solutions from both the definitive and confirmatory assays were analyzed by the Rohm and Haas Toxicology Analytical Group. The samples were shipped and stored at ambient temperatures until analysis. The results of the analysis showed that the average active ingredient of the dosing solutions ranged from 92.1-115 percent of the target concentrations.

TABLE 1

ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY

Metabolic Activation: -S9 .......................................................................................................3.0 hour treatment, 20.0 hour harvest

 Confluencea %  % Mitotic Index  Average %  % Mitotic  Total Cell Count (x 106)     Average Cell  % Cell % Relative 
   Treatment   Solvent Control   A culture    B culture   Mitotic Index  Index Reduction A culture  B culture Count (x 106 Count Reduction  Cell Counts 
 Negative Control McCoy's 5a  100  8.3  9.0  8.7  ---  8.21  6.12  7.17  ---  ---
 Vehicle Control water 10.0 uL/mL  100  8.0  7.3  7.7  0  6.52  6.34  6.43  0  100
 Test Article 0.0785 ug/mL  100  ---  ---  ---  ---  7.20  8.24  7.72  0  120
 0.157 ug/mL  100  ---  ---  ---  ---  8.10  6.23  7.17  0  112
 0.313 ug/mL  100  ---  ---  ---  ---  7.49  7.45  7.47  0  116
 0.625 ug/mL  100  10.7  10.7  10.7  0  8.50  6.91  7.71  0  120
 1.25 ug/mL  100  11.1  11.8  11.5  0  6.44  6.16  6.30  2  98
 2.50 ug/mL  100  13.4  10.1  11.8  0  6.23  6.55  6.39  1  99
 5.00 ug/mL  100  12.1  9.1  10.6  0  3.64  4.18  3.91  39 61
 9.53 ug/mL  100  8.0  6.1  7.1  8  3.92  3.49  3.71  42  58
 12.7 ug/mL  100  5.4  6.3  5.9  23  2.66  2.81  2.74  57  43
 16.9 ug/mL  86  ---  ---  ---  ---  1.22  1.62  1.42  78  22
 22.5 ug/mL  86  ---  ---  ---  ---  1.22  1.12  1.17  82  18
 30.0 ug/mL  43  ---  ---  ---  ---  0.61  0.22  0.42  93  7
 40.0 ug/mL  <7  ---  ---  ---  ---  0.14  0.14  0.14  98  2

aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

McCoy's 5a = culture medium

Water = Sterile deionized water

TABLE 2

CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS

Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment

Metabolic Activation: -S9

 Test Material  Cells Scored  # of Aberrations/Cell  % Cells with Aberrations  % Cells with >1 Aberrations  % PP  %E
 Controls            
Negative McCoy's 5a  200  0.02  1.5  0.0  2.0  2.0
  Solvent Water 10 uL/mL  200  0.01  1.0  0.0  0.5  0.0
 Positive            
 MMC 1.50 ug/mL  50  1.94  72.0*  56.0*  3.5  1.5
 Test Article 1.25 ug/mL  200  0.01  0.5  0.0  1.0  1.0
 2.50 ug/mL  200  0.01  0.5  0.0  1.5  1.5
 5.00 ug/mL  200  0.05  3.5  0.5  3.0  1.5
 9.53 ug/mLa  200  0.29  14.5*  8.0*  2.8  5.3*
 12.7 ug/mL  50  0.54  30.0*  12.0*  3.0  2.5

a 200 cells were analyzed for polyploidy and endoreduplication.

McCoy's 5a = culture medium

Water = Sterile deionized water

MMC = Mitomycin

* Significantly greater than the vehicle controls, p<0.01.

TABLE 3

ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY

Metabolic Activation: -S9...............................................................................17.8 hour treatment, 20.0 hour harvest

 Confluencea%  % Mitotic Index  Average %  % Mitotic  Total Cell Count (x 106)     Average Cell  % Cell % Relative 
   Treatment   Solvent Control   A culture    B culture   Mitotic Index  Index Reduction A culture  B culture Count (x 106 Count Reduction  Cell Counts 
 Negative Control McCoy's 5a  100  7.6  7.7  7.7  ---  5.51  4.75  5.13  ---  ---
 Vehicle Control Water 10.0 uL/mL  100  7.6  7.1  7.4  0  4.32  3.96  4.14  0  100
 Test Article 0.157 ug/mL  100  ---  ---  ---  ---  7.13  5.15  6.14  0  148
 0.313 ug/mL  100  ---  ---  ---  ---  5.69  5.80  5.75  0  139
 0.625 ug/mL  100  11.8  9.2  10.5  0  4.61  5.22  4.92  0  119
 1.25 ug/mL  100  13.0  14.0  13.5  0  4.07  4.50  4.29  0  104
 2.50 ug/mL  100  7.0  8.3  7.7  0  4.72  4.07  4.40  0  106
 3.75 ug/mL  100   7.2  8.4  7.8  0  3.82  2.02  2.92  29  71
 5.00 ug/mL  100  4.4  4.9  4.7  36  2.45  2.56  2.51  39  61
 7.50 ug/mL  100  4.5  5.4  5.0  32  2.09  2.74  2.42  42  58
 10.0 ug/mL  100  3.5  2.3  2.9  61  2.27  1.98  2.13  49  51
 12.5 ug/mL  100  0.3  1.8  1.1  85  2.05  2.05  2.05  50  50
 15.0 ug/mL  100  ---  ---  ---  ---  0.68  1.12  0.90  78  22
 20.0 ug/mL  14  ---  ---  ---  ---  0.18  0.00  0.09  98  2

aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

McCoy's 5a = culture medium

Water = Sterile deionized water

TABLE 4

CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS

Cells Fixed 20.0 Hours After Initiation of Treatment, 17.8 Hour Treatment

Metabolic Activation: -S9

Test Material  Cells Scored  # of Aberrations/Cell  % Cells with Aberrations  % Cells with >1 Aberrations  % PP  %E
 Controls            
 Negative McCoy's 5a  200  0.01  0.5  0.0  1.5  0.0
 Vehicle Water 10.0 uL/mLa  200  0.03  2.5  0.0  1.8  0.3
 Positive            
 MMC 0.400 ug/mL  50  0.82  44.0*  18.0*  3.0  0.5
 Test Article 1.25 ug/mLa  200  0.00  0.0  0.0  1.3  0.8
 2.50 ug/mLa  200  0.08  3.5  2.0  1.8  0.0
 3.75 ug/mL  50  0.66  28.0*  20.0*  4.0  0.5
 7.50 ug/mL  50  0.72  34.0*  18.0*  2.5  1.0

a 200 cells were analyzed for polyploidy and endoreduplication.

McCoy's 5a = culture medium

Water = Sterile deionized water

MMC = Mitomycin C

* Significantly greater than the vehicle controls, p<0.01

TABLE 5

ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY

Metabolic Activation: +S9................................................................................................3.0 hour treatment, 20.0 hour harvest

 Confluencea%  % Mitotic Index  Average %  % Mitotic  Total Cell Count (x 106)     Average Cell  % Cell % Relative 
   Treatment   Solvent Control   A culture    B culture   Mitotic Index  Index Reduction A culture  B culture Count (x 106 Count Reduction  Cell Counts 
 Negative Control McCoy's 5a  100  16.0  12.9  14.5  ---  4.68  3.49  4.09  ---  ---
 Vehicle Control Water 10.0 uL/mL  100  15.1  14.2  14.7  0  3.74  3.78  3.76  0  100
 Test Article 0.0785 ug/mL  100  ---  ---  ---  ---  4.32  3.82  4.07  0  108
 0.157 ug/mL  100  ---  ---  ---  ---  4.36  4.25  4.31  0  115
 0.313 ug/mL  100  ---  ---  ---  ---  3.89  3.96  3.93  0  105
 0.625 ug/mL  100  ---  ---  ---  ---  4.21  4.18  4.20  0  112
 1.25 ug/mL  100  12.0  14.5  13.3  10  4.03  4.72  4.38  0  116
 2.50 ug/mL  100  15.9  16.6  16.3  0  3.20  3.17  3.19  15  85
 5.00 ug/mL  100  12.5  13.9  13.2  10  2.59  3.13  2.86  24  76
 9.53 ug/mL  100  11.4  10.1  10.8  27  3.02  2.41  2.72  28  72
 12.7 ug/mL  100  6.8  6.1  6.5  56  2.56  2.95  2.76  27  73
 16.9 ug/mL  100  4.2  4.6  4.4  70  1.80  1.84  1.82  52  48
 22.5 ug/mL  86  ---  ---  ---  ---  1.55  1.55  1.55  59  41
 30.0 ug/mL  71  ---  ---  ---  ---  0.72  1.04  0.88  77  23
 40.0 ug/mL  <7  ---  ---  ---  ---  0.29  0.43  0.36  90  10

a This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

McCoy's 5a = culture medium

Water = Sterile deionized water

TABLE 6

CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS

Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment

Metabolic Activation: +S9

Test Material  Cells Scored  # of Aberrations/Cell  % Cells with Aberrations  % Cells with >1 Aberrations  % PP  %E
 Controls            
 Negative McCoy's 5a  200  0.00  0.0  0.0  1.0  0.0
 Solvent Water 10.0 uL/mL  200  0.02  1.0  0.5 1.0  1.0
 Positive            
 CP 5.00 ug/mL  50  0.42  32.0*  8.0*  2.5  1.5
 Test Article 2.50 ug/mL  200  0.01  0.5  0.0  2.0  1.0
 5.00 ug/mL  200  0.01  0.5  0.0  1.5  2.0
 9.53 ug/mL  200  0.05  3.0  1.0  2.0  3.5
 12.7 ug/mL  200  0.18  11.0*  6.0*  3.0  2.5
 16.9 ug/mL  125  0.14  12.0*  2.4  3.0  3.0

McCoy's 5a = culture medium

Water = Sterile deionized water

CP = Cyclophosphamide

* Significantly greater than the vehicle controls, p<0.01.

TABLE 7

ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY

Metabolic Activation: +S9.................................................................................................3.0 hour treatment, 20.0 hour harvest

 Confluencea%  % Mitotic Index  Average %  % Mitotic  Total Cell Count (x 106)     Average Cell  % Cell % Relative 
   Treatment   Solvent Control   A culture    B culture   Mitotic Index  Index Reduction A culture  B culture Count (x 106 Count Reduction  Cell Counts 
 Negative Control McCoy's 5a  100  7.5  7.2  7.4  ---  4.82  5.58  5.20  ---  ---
 Vehicle Control Water 10.0 uL/mL  100  9.5  11.7  10.6  0  4.25  4.18  4.22  0  100
 Test Article 1.25 ug/mL  100  11.0  11.2  11.1  0  4.72  4.00  4.36  0  103
 2.50 ug/mL  100  8.7  8.0  8.4  21  3.96  3.28  3.62  14  86
 5.00 ug/mL  100  7.0  9.0  8.0  25  5.04  9.00  7.02  0  166
 7.50 ug/mL  100  4.4  6.5  5.5  48  3.89  4.72  4.31  0  102
 10.0 ug/mL  100  6.3  5.5  5.9  44  2.99  4.68  3.84  9  91
 12.5 ug/mL  100  6.7  6.2  6.5  39  3.38  3.13  3.26  23  77
 15.0 ug/mL  100  5.2  5.8  5.5  48  2.23  2.63  2.43  42  58
 17.5 ug/mL  100  2.9  1.8  2.4  77  2.20  1.87  2.04  52  48
 20.0 ug/mL  100  ---  ---  ---  ---  1.12  0.94  1.03  76  24

aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

McCoy's 5a = culture medium

Water = Sterile deionized water

TABLE 8

CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS

Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment

Metabolic Activation: +S9

Test Material  Cells Scored  # of Aberrations/Cell  % Cells with Aberrations  % Cells with >1 Aberrations  % PP  %E
 Controls            
 Negative McCoy's 5a  200  0.02  1.0  0.5  1.5  1.0
 Vehicle Water 10.0 uL/mL  200  0.02  1.5  0.0  1.5  1.0
 Positive            
 CP 5.00 ug/mL  50  0.56  40.0*  8.0*  6.0  0.5
 Test Article 1.25 ug/mL  200  0.00  0.0  0.0  1.0  0.5
 2.50 ug/mL  200  0.02  1.5  0.5  1.5  1.5
 5.00 ug/mL  200  0.03  1.5  0.5  2.5  1.0
 7.50 ug/mL  200  0.19  9.0*  5.0*  2.5  1.0

McCoy's 5a = culture medium

Water = Sterile deionized water

CP = Cyclophosphamide

* Significantly greater than the vehicle

Conclusions:
Interpretation of results (migrated information):
ambiguous only positive at cytotoxic concentrations

1.25, 2.50, 3.75, 5.00, 7.50, 10.0, 12.5, 15.0, and 20.0 μg/mL without metabolic activation and 1.25,2.50,5.00,7.50, 10.0, 12.5, 15.0, 17.5, and 20.0 μg/mL with metabolic activation were tested. Cultures treated with concentrations of 1.25,2.50,3.75, and 7.50 μg/mL without metabolic activation and 1.25,2.50,5.00, and 7.50 μg/mL with metabolic activation were analyzed for chromosomal aberrations. A significant increase in cells with chromosomal aberrations was observed in the cultures treated with 3.75 and 7.50 μg/mL without metabolic activation and 7.50 μg/mL with metabolic activation. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index). Kordek 573T (Sample identification: TD 99-087, Lot No. B1103, active ingredient 97.5%) may be interpreted as being positive for inducing chromosome aberrations in CHO cells with and without metabolic activation. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index). The highly nonlinear dose response for the induction of chromosomal aberrations and the associated correlation of the chromosomal aberrations with high level of cytotoxicity suggest that the chromosomal damage was induced by an indirect process associated with cytotoxicity. As noted by Hilliard et. al. (1998), chromosomal damage in a CHO assay that is limited to concentrations that are highly cytotoxic (suggested to be in the 30-40% or more range) may be interpreted as a "false positive."
Executive summary:

The objective of this in vitro assay was to evaluate the ability of Kordek 573T (Sample identification: TD 99-087, Lot No. B1103, active ingredient 97.5%) to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation.

The highest concentration analyzed in this assay was based on cytotoxicity induced by Kordek 573T (TD identification: 99-087). The test article was solubilized/suspended in sterile deionized water. All dosing was achieved using a dosing volume of 1% (10.0 μL/mL) and the vehicle control cultures were treated with 10.0 μL/mL of sterile deionized water. Cytotoxicity assessments were based on reductions in mitotic index and cell counts.

In the initial chromosomal aberrations assay, the treatment period was for 3.0 hours with and without metabolic activation and cultures were harvested 20.0 hours from the initiation of treatment. Concentrations of 0.0785, 0.157, 0.313, 0.625, 1.25, 2.50, 5.00, 9.53, 12.7, 16.9, 22.5, 30.0, and 40.0 μg/mL were tested with and without metabolic activation. Cultures treated with

concentrations of 1.25, 2.50, 5.00, 9.53, and 12.7 μg/mL without metabolic activation and 2.50, 5.00, 9.53, 12.7, and 16.9 μg/mL with metabolic activation was analyzed for chromosomal aberrations. A significant increase in cells with chromosomal aberrations was observed in the cultures treated with 9.53 and 12.7 μg/mL without metabolic activation and 12.7 and 16.9 μg/mL with metabolic activation. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced greater than 40% cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index).

In a confirmatory chromosomal aberrations assay, the treatment period was for 17.8 hours without metabolic activation and 3.0 hours with metabolic activation, and cultures were harvested ~20.0 hours from the initiation of treatment. Concentrations of 0.157, 0.3 13, 0.625, 1.25, 2.50, 3.75, 5.00, 7.50, 10.0, 12.5, 15.0, and 20.0 μg/mL without metabolic activation and 1.25, 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 17.5, and 20.0 μg/mL with metabolic activation were tested. Cultures treated with concentrations of 1.25, 2.50, 3.75, and 7.50 μg/mL without metabolic activation and 1.25, 2.50, 5.00, and 7.50 μg/mL with metabolic activation were analyzed for chromosomal aberrations. A significant increase in cells with chromosomal aberrations was observed in the cultures treated with 3.75 and 7.50 μg/mL without metabolic activation and 7.50 μg/mL with metabolic activation. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index).

Kordek 573T (Sample identification: TD 99-087, Lot No. B1103, active ingredient 97.5%) may be interpreted as being positive for inducing chromosome aberrations in CHO cells with and without metabolic activation. The increase in chromosomal aberrations was only observed at Kordek 573T concentrations that induced significant cytotoxicity (expressed either as a reduction in cell count or as reduction in mitotic index). The highly nonlinear dose response for the induction of chromosomal aberrations and the associated correlation of the chromosomal aberrations with high level of cytotoxicity suggest that the chromosomal damage was induced by an indirect process associated with cytotoxicity. As noted by Hilliard et. al. (1998), chromosomal damage in a CHO assay that is limited to concentrations that are highly cytotoxic (suggested to be in the 30-40% or more range) may be interpreted as a "false positive."

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
Study period:
2 November 1999 - 13 April 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPP 84-2
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO cells were used as the indicator cells in this study. The hypodiploid CHO cell line (CHO-K1) was originally derived from the ovary of a Chinese hamster (4). A clone of this cell line, CHO-K1-BH4, was isolated by Dr. A. W. Hsie and has been shown to be sensitive in detecting chemical mutagens from a wide range of chemical classes (1,2,3).

The CHO-Kl-BH4 cell line used in this study was originally obtained from Dr. Patrick O'Neill, University of Vermont, on November 6, 1984. The cells were subcultured and cryopreserved in a large number of ampules for HGPRT mutation studies. The cell identity of this cell line was established by karyotypic analysis and documented at SITEK Research Laboratories. These cells exhibit a high cloning efficiency and a short doubling time of 12-14 hours.

References:
1. Hsie, A. W., D. A. Casciano, D. B. Couch, D. F. Krahn, J. P. O'Neill and B. L. Whitfield. The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals. Mutation Res., 86: 193-214, 1981.
2. O'Neill, J. P., and A. W. Hsie. Phenotypic expression time of mutagen-induced 6-thioguanine resistance in cells (CHOMGPRT system). Mutation Res., 59: 109- 1 18, 1979.
3. O'Neill, J. P., P. A. Brimer, R. Machanoff, G. P. Hirsch and A. W. Hsie. A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl
transferase locus in cells (CHOMGPRT system): Development and definition of the system. Mutation Res.,a:91-101, 1977
Metabolic activation:
with and without
Metabolic activation system:
The S-9 fractions were prepared by SITEK Research Laboratories in 0.15M KC1 from induced, male, Sprague-Dawley rats
Test concentrations with justification for top dose:
Range finding test - O.1,0.5, 1.0, 5.0, 10, 50, 100, 500, 1000 and 5000 ug/mL with and without activation.

Definitive assay - 0.5, 1.0, 5.0, 10, 15 and 25 ug/mL with and without activation:

Confirmatory Assay - 5.0, 10, 15, 25 and 40 ug/mL with and without activation
Vehicle / solvent:

Sterile, deionized, distilled water was used to dissolve and dilute the test article.

Dimethyl sulfoxide (DMSO) was used to dissolve and dilute the positive control EMS. Acetone was used to dissolve and dilute the positive control DMBA
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulfonate (EMS) was used without S9 and 7,12-Dimethylbenz(a)anthracene (DMBA) was used with S9
Details on test system and experimental conditions:
DETERMINATION OF OSMOLALITY AND pH OF THE DOSING SOLUTIONS
Stock solutions of the test article, Kordek 573T, were prepared in sterile, deionized, distilled water. The senun-free culture medium with and without S-9 mixture containing water and 5000 ug/mL of the test article were tested for osmolality in a vapor pressure osmometer (5 100 B Vapor Pressure Osmometer frorn Wescor, NC) (4). The osmolality for 5000 ug/mL was tested because it was the highest test article concentration tested in the Range Finding Test.

The culture medium was observed for any precipitate formation or change in color (pH) due to the addition of the test article.

PREPARATION OF TEST CULTURES
The CHO stock cultures were grown in complete Hx-free medium. Stock cultures growing in T-75 cm2 tissue culture flasks and showing approximately 80-90% confluency were harvested and used to prepare the test cultures. The medium from the T-75 cm2 flasks was discarded, and the cells were washed with Ca++-free and Mg++-free Hanks' Balanced Salt Solution (HBSS). The cells were then dissociated by adding 2.0 mL of 0.05 % trypsin to each flask. The cells were rinsed with trypsin, and the excess trypsin was then removed with a Pasteur pipet. The flasks were incubated at 37 + 1°C until the cells dissociated.

Five mL of complete Hx-free medium was then added to each of the stock culture flasks, and the cell suspension was aspirated to obtain a single cell suspension. The cells from a number of stock culture flasks were pooled, centrifuged and resuspended in Hx-free medium. An aliquot of the cell suspension was diluted to the appropriate concentration prior to counting in a cell counter. Based on the cell counts, a separate cell suspension with 3x10(5) cells/ml was prepared to seed the test flasks. An appropriate number of T-75 cm2 plastic culture flasks were seeded with 10 mL of cell suspension to obtain test cultures with 3x10(6) cells/flask. The flasks were incubated at 37 +/- 1°C in an atmosphere of approximately 5% CO, and 95 % air for 18-20 hours.

PREPARATION OF METABOLIC ACTIVATION SYSTEM
Immediately prior to treatment, the S-9 fraction was mixed with the cofactor pool to obtain the S-9 cofactor mixture which was kept in an ice bath until used. The S-9 cofactor mixture consisted of 50mM sodium phosphate (pH 7.5), 4mM NADP, 5mM glucose-6-phosphate, 30mM KCl, 10mM MgCl,, l0mM CaCl, and 100 uL/mL S-9 fraction.

The protein content of the S-9 cofactor mixture was 3.68 mg/mL for the S-9 batch No. 101299. Prior to its use, the S-9 cofactor mixture was diluted 1:5 (by volume) with Hx-free Ham's F-12 nutrient medium supplemented with 2mM L-glutaxnine (serum-free culture medium) and used for refeeding the flasks for the activated portion of the experiment.

PREPARATION OF THE TEST ARTICLE DOSING SOLUTIONS
The test article was melted in a 44°C waterbath prior to weighing, as per Sponsor's instruction. The test article was weighed, dissolved and diluted in sterile, deionized, distilled water to prepare the stock solutions. The test article solutions were prepared just before treatment. All of the test article and control treatments were done under UV-filtered lights to avoid photoinactivation or photoactivation of chemicals. The stability of the test article under experimental conditions was not determined by SITEK Research Laboratories.

Approximately 5.0 mL of each test article concentration (and 10 mL of the solvent) from the definitive and confiitory Assays were saved at room temperature and shipped to the Sponsor's Toxicology Department for concentration verification.

RANGE FINDING TEST (A-1)
Test cultures used in the Range Finding Test were seeded approximately 18-20 hours prior to use. Single cultures were used at each test article and solvent control level. Kordek 573T was tested at dose levels of 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000 and 5000 ug/mL with and without activation.

The medium was removed and 10 mL of serum-free medium was added to each of the culture flasks in the non-activated system, and 10 mL, of serum-free culture medium containing S-9 mixture was added to each of the culture flasks in the activated system. The cells were treated by adding 100 uL of stock solutions to the cultures to achieve the correct final concentration. The final dosing volume was 1 % (10 uL/mL culture media). The solvent control flasks received 100 uL of water in each flask. The cells were exposed to the test article for 4 hours in an incubator set at 37 f 1°C with approximately 5 % CO, and 95% air. After the exposure period, the cells were washed with Ca++- and Mg++-free HBSS, trypsinized and seeded for cytotoxicity determination.

The test article did not show any signs of precipitate in the treatment media. However, the pH .of the treatment media became .slightly acidic, which was noticed due. to a slight change m the color of the phenol red lndicator in the media, at the top concentration of 5000 ug/mL. Since the change in pH was not very significant, it was not necessary to adjust it back to normal.

To determine cytotoxicity, the cells from each flask were trypsinized and seeded in triplicate at a density of 200 cells/60 mm dish. Hx-free medium, supplemented with 10% HIFBS, 2mM L-glutamine, 50 units/ml penicillin and 50 ug/mL streptomycin, was used in seeding the cells. The cells were allowed to grow for a period of 8 days without any disturbance to minimize the formation of satellite colonies. The colonies were then washed with phosphate buffered saline (PBS), fixed with methanol, stained with Giemsa stain, and counted. A cluster of more than 50 cells growing within a confined area was considered a colony. The average number of colonies per plate was calculated, and the Relative Cloning Efficiency (RCE) was determined by the following formula:

RCE =[( Average No. of Colonies in Test Plates)/(Average No. of Colonies in Solvent Plates)] x 100

These calculations were performed using the Lotus 123 (ver . 3.4) program 25 10B. WK3.

CHO/HGPRT DEFINITIVE AND CONFIRMATORY MUTATION ASSAYS (B-1 and B-4)
In this study, the CHO/HGPRT Mutation Assay was performed in two independent trials: a definitive and a confirmatory assay. The definitive assay (B-1) was performed at following test article concentrations:

With and Without Activation: 0.5, 1 .O, 5.0, 10, 15 and 25 ug/mL

The test cultures for the Mutation Assays were prepared as described in "Preparation of Test Cultures." Duplicate cultures, seeded with 3x10 cells/flask, were used at each dose level.

The medium was removed and 10 mL of serum-free medium was added to each of the culture flasks in the non-activated system and 10 mL of serum-free culture medium containing S-9 mixture was added to each of the culture flasks in the activated system. The cells were treated by adding 100 uL of stock solutions to the cultures. The cells were exposed to the test article for 4 hours in an incubator set at 37 +/- 1°C with approximately 5 % CO, and 95% air. After the exposure period, the cells were washed with Ca++- and Mg++-free HBSS and seeded for parallel toxicity and mutant expression. The parallel toxicity plates were seeded with 200 cellslplate in three plates for each replicate. The plates were incubated for 8 days in a humidified 5 % CO, incubator set at 37 +/- 1°C.

For the expression of 6-thioguanine (TG)-resistant mutants (HGPRT locus mutants), the cells from each of the duplicate culture flasks were subcultured in Hx-free Ham's F-12 nutrient medium supplemented with 5 % dialyzed HIFBS, 2mM L-glutamine, 50 units/ml penicillin and 50 ug/mL streptomycin (cloning medium) at a density of 2x10(6) cells/150 cm2 flask. The cells were subcultured as described above at 2- to 3 day intervals for a period of 9 days, prior to selecting the mutant phenotypes. The cells were grown as attached cultures in T-150 cm2 tissue culture flasks. The cell counts were performed using an automatic cell counter, and the counts were entered into Lotus 123 program 25 10CNTS. WK3.

After the expression period, the cells from each of the treated replicates were harvested and seeded in twelve 100 mm tissue culture plates at a density of 2x10(5) cells/plate in 10 mL of cloning medium containing 10uM TG. To determine the cloning efficiency of the cells at the time of selection, 200 cells/60 mm dish were plated in triplicate in the cloning medium. All of the clonable test doses and appropriate positive and solvent controls were cloned for mutant selection. The cultures were then incubated for 7-8 days without disturbing the plates to minimize the formation of satellite colonies.

After the incubation period, the colonies were then washed with PBS, fixed, stained, and counted for cloning efficiency and mutant selection. The calculations were performed using Lotus 123 program 25 10B.WK3. The average number of clones from the triplicate plates were calculated and the Percent Clonable Cells (Cloning Efficiency) for eath treatment condition was determined. The number of TG-resistant mutants for 2.4x10(6) cells seeded was calculated by totaling the number of mutants from the twelve replicate plates. Based on the Cloning Efficiency, the number of TG-resistant mutants per 1x10(6) surviving cells was calculated for each dose.

In this study, a confiitory Mutation Assay (B-4) was performed to confirm the results obtained in the definitive Mutation Assay (B-1). The earlier confirmatory Mutation Assays, B-2 and B-3, were lost due to lack of colonies in the parallel toxicity plates. The experimental procedures for the confirmatory assay were essentially the same as in the first Mutation Assay, except the test article concentrations were altered based on the parallel toxicity results in the definitive assay.

Concentrations tested in the Confirmatory Assay (B-4) were:

With and Without Activation: 5.0, 10, 15, 25 and 40 ug/mL

The concentration of 40 ug/mL without activation could not be cloned due to toxicity.
Evaluation criteria:
The results of the CHO/HGPRT Locus Muption Assay were evaluated on the basis of the number of TG-resistant mutants per 1x10(6) surviving cells. Mutant data from test concentrations which showed more than 90% reduction in RCE were not included in evaluating the results. The significance of the test results was determined by one of the following methods:

1. The test results were considered sipficant if a dose showed more than a two-fold increase in the number of mutants per 1x10(6) surviving cells over that of the concurrent and historical solvent controls.
2. If no dose group yielded an average mutant frequency greater than 15 mutants per 1x10(6) surviving cells, the test article was considered negative.
3. Alternatively, the test results could have been analyzed by statistical methods to determine if there was a significant increase in the mutation frequency if necessary. A statistical analysis was not performed on the mutation frequency data, therefore, statistical analysis was not used in the interpretation or reporting of the results for this study. In evaluating the test results, biological significance, such as reproducibility and dose response, was also considered.

Positive Response
The test article is considered to have caused a positive response if:
1. The test article showed a positive dose response and on: test dose showed a statistically significant increase in the number of mutants per 1x10(6) surviving cells or a twofold increase in the number of mutants per 1x10(6) surviving cells. In the event that the test article caused a significant increase in the number of mutants per 1x10(6) surviving cells due to an unusually low number of mutants in the concurrent solvent control, the data from the test article-treated cultures were compared to the historical solvent control data.

Continued below
Statistics:
A statistical analysis was not performed on the mutation frequency data, therefore, statistical analysis was not used in the interpretation or reporting of the results for this study.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Table 1
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE FINDING TEST (A-1)
The results of the Range Finding Test are summarized and presented in Table 1. In the non-activated system, the concentrations of 0.1 to 10 ug/mL had Relative Cloning Efficiencies (RCEs) ranging from 90% to 39%. The concentrations of 500, 1000 and 5000 ug/mL were not seeded for colony formation due to toxicity, and the concentrations of 50 and 100 ug/mL had no surviving colonies. In the activated system, the test article concentrations of 0.1 to 10 ug/mL had RCEs ranging from 101 % to 50%. The concentrations of 1000 and 5000 ug/mL were not seeded for colony formation due to toxicity, and the concentrations of 50, 100 and 500 ug/mL had no surviving colonies in this system.

DEFINITIVE MUTATION ASSAY (B-1)
The definitive assay was performed at the following test article concentrations:
With and Without Activation: 0.5, 1.0, 5.0, 10, 15 and 25ug/mL.

The results of the parallel toxicity test from the definitive assay (B-1) are presented in Table 2.

The RCEs among the test article-treated cultures ranged from 29% to 79% and 42% to 80%, for the test article concentrations of 0.5 to 25 pg/mL with and without exogenous metabolic activation.

The assay results are presented in Tables 3 (non-activated system) and 4 (activated system.

In the non-activated system, the number of mutants per 1x10(6) surviving cells in the concurrent solvent controls, water and DMSO, ranged from 4 to 36. Among the test article treated cultures at concentrations of 0.5 to 25 ug/mL, the number of mutants per 1x10(6) surviving cells ranged from 4 to 19.

In the activated system, the number of mutants per 1x106 surviving cells in the solvent controls, water and acetone ranged from 2 to 26. In the test article-treated cultures, the number of mutants per 1x10(6) surviving cells ranged from 3 to 17 at concentrations of 0.5 to 25 ug/mL. There was no sign of a dose dependent response in either system.

The positive controls, EMS and DMBA, caused a significant increase in the number of mutants per 1x10(6) surviving cells (774 and 515 for EMS replicates A and B, respectively, and 444 and 350 for DMBA replicates A and B, respectively).

CONFIRMATORY MUTATION ASSAY (B-4)
The confirmatory Mutation Assays B-2 and B-3 were lost due to lack of surviving colonies in the Parallel Toxicity plates. The following test article concentrations were tested in the confirmatory Mutation Assay:
With and Without Activation: 5.0, 10, 15, 25 and 40 ug/mL.

The results of the Parallel Toxicity Test from the confirmatory assay are summarized and presented in Table 5.

The results of the confirmatory assay are summarized and presented in Tables 6 (nonactivated system) and 7 (activated system).

The RCEs ranged from 91 % to 5.0 % for the test article concentrations of 5.0 to 25 ug/mL in the non-activated portion of the assay. The highest test article concentration of 40 ug /mL had no surviving colonies and could not be cloned for mutant selection. In the activated system, the test article concentrations of 5.0 to 40 ug/mL had RCEs ranging from 104% to 20%.

In the non-activated system, the number of mutants per 1x10(6) surviving cells in the concurrent solvent controls, water and DMSO, ranged from 1 to 3. Among the test article treated cultures, the number of mutants per 1x10(6) surviving cells ranged from 0 to 5.

In the activated system, the number of mutants per 1x10(6) surviving cells in the solvent controls, water and acetone, ranged from 1 to 4. Among the test article-treated cultures, the number of mutants per 1x10(6) surviving cells ranged from 0 to 4.

The positive controls, EMS and DMBA, caused significant increases in the number of mutants per 1x10(6) surviving cells (178 and 157 for EMS, and 84 and 154 for DMBA, replicates A and B, respectively).

None of the test article doses showed a significant inrease in the number of mutants per 1x10(6) surviving cells and there was no sign of a dose dependent response in either systems. All of the criteria for a valid assay were met.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

pH AND OSMOLALITY TESTS

There was a change in the pH of the culture medium, as indicated by change in color of the phenol red indicator, after the addition of the test article dosing solutions to the culture medium in the Range Finding Test. The pH was slightly acidic (culture medium turned light orange) in the 5000 ug/mL test article concentration. Since the change in pH was not very significant, it was not necessary to adjust it back to normal.

The osmolality readings of the culture medium with water and 5000 ug/mL of the test article in the Range Finding Test are given below:

    Osmolality (mOsmol/kg H2O )
 Concentration  Without Activation  With Activation
 Solvent (Water)  307  332
 5000 ug/mL  314  334

The osmolality readings were below the upper limit for the CHO/HGPRT Gene Mutation Assay (500 mOsmol/kg). Therefore, there was no significant effect on osmolality caused by the addition of the test article at the maximum concentration tested.

ANALYSIS OF DOSING SOLUTIONS

The dosing solutions from both the definitive (B-1) and confirmatory (B-4) assays were analyzed by the Toxicology Department at Rohm and Haas Company, 727 Norristown Road, Spring House, Pennsylvania 19477-0904. The levels of KordekB 573T in the dosing solutions ranged from 91.3-108 % of the target concentrations. The dosing solutions were therefore, adequately prepared for

this study.

TABLE 1

CHO/HGPRT GENE MUTATION ASSAY RANGE FINDING TEST RESULTS

 Test Art. Conc.     Without Activation  Test Art. Conc.  With S9 Activation   
 ug/mL  Ave. No. Colonies/Dose (S.D.)  RCE*  ug/mL  Ave. No. Colonies/Dose (S.D.)  RCE*
 Solvent  136 (10.2)  100%  Solvent  139 (8.0)  100%
 0.1  123 (7.1)  90%  0.1  127 (6.6)  91%
 0.5  109 (3.2)  80%  0.5  140 (15.9)  101%
 1.0  109 (15.0)  80%  1.0  115 (11.3)  83%
 5.0  108 (12.7)  79%  5.0  100 (1.7)  72%
 10  53 (23.5)  39%  10  70 (6.4)  50%
 50  0 (0.0)  0%  50  0 (0.0)  0%
 100  0 (0.0)  0%  100  0 (0.0)  0%
 500  NA  NA  500  0 (0.0)  0%
 1000  NA  NA  1000  NA  NA
 5000  NA  NA  5000  NA  NA

The number of cells seeded per plate was 200.

*RCE = [(Average No. of Colonies in Test Plates)/(Average No. of Colonies in Solvent Control Plates)] x 100

NA = Not applicable. Too toxic to seed for colony formation

TABLE 2

CHOIHGPRT GENE MUTATION ASSAY PARALLEL TOXICITY TEST RESULTS

DEFINITIVE

Test Art. Conc.     Without Activation  Test Art. Conc.  With S9 Activation   
 ug/mL  Ave. No. Colonies/Dose (S.D.)  RCE*  ug/mL  Ave. No. Colonies/Dose (S.D.)  RCE*
 Solvent  142 (0.7)  100%  Solvent  133 (5.7)  100%
 0.5  113 (4.9)  79%  0.5  106 (1.4)  80%
 1.0  103 (2.1)  72%  1.0  93 (7.1)  70%
 5.0  105 (9.9)  74%  5.0  90 (13.4)  67%
 10  82 (12.0)  57%  10  103 (3.5)  77%
 15  83 (3.5)  58%  15  75 (4.9)  56%
 25  41 (6.4)  29%  25  56 (1.4)  42%
 EMS Solvent Control  132 (0.7)  100%  DMBA SolventControl  131 (0.0)  100%
 EMS (0.5 uL/mL)  72 (3.5)  54%  DMBA (5.0 ug/mL)  82 (2.1)  62%

*RCE = [(Average No. of Colonies in Test Plates)/(Average No. of Colonies in Solvent Control Plates)] x 100

The solvents for EMS and DMBA are DMSO and acetone, respectively.

TABLE 3

CHO/HGPRT GENE MUTATION ASSAY RESULTS - WITHOUT ACTIVATION

DEFINITIVE

 Test Art. Conc. ug/mL  Ave. No. of C.E. Colonies/Plate (S.D.)  C.E.  Total Mutants Counted  No. of Plates Seeded  No. of Plates Counted  Mutants/106 Surviving Cells
 Solvent A  151 (6.2)  75.5%  25  12  12  14
 Solvent B  158 (6.8)  79.0%  8  12  12  4
 0.5 A  133 (12.7)  66.5%  11  12  11  8
 0.5 B  253 (2.9)  126.5%  14  12  12  5
 1.0 A  111 (9.3)  55.5%  14  12  11  11
 1.0 B  131 (9.6)  65.5%  28  12  11  19
 5.0 A  142 (14.2)  71.0%  15  12  12   9
 5.0 B  196 (13.4)  98.0%  37  12  12  16
 10 A  111 (8.5)  55.5%  15  12  12  11
 10 B  114 (18.2)  57.0%  17  12  11  14
 15 A  121 (2.3)  60.5%  7  12  12  5
 15 B  123 (17.4)  61.5%  5  12  11  4
 25 A  103 (12.8)  51.5%  8  12  12  6
 25 B  208 (24.8)  104.0%  9  12  10  4
 EMS Sol. Control A  201 (4.0)  100.5%  12  12  12  5
 EMS Sol. Control B  117 (5.0)  58.5%  51  12  12  36
 EMS (0.5 uL/mL) A  97 (11.0)  48.5%  826  12  11  774
 EMS (0.5 uL/mL) B  96 (11.0)  48.0%  593  12  12  515

* C.E. = Cloning Efficiency = Avg..No. of C.E. Colonies per Plate/200 Colonies Seeded

**R.M. = Medium with 6-TG

Mutants/106 Survivors = [100/(C.E. x 2.4)] x (Total Mutants Counted per Dose) x [(Mutatnt Plates Seeded per Dose)/(Mutant Plates Counted per Dose)]

The solvent for EMS is DMSO

TABLE 4

CHO/HGPRT GENE MUTATION ASSAY RESULTS - WITH ACTIVATION

DEFINITIVE

 Test Art. Conc. ug/mL  Ave. No. of C.E. Colonies/Plate (S.D.)  C.E.  Total Mutants Counted  No. of Plates Seeded  No. of Plates Counted  Mutants/106 Surviving Cells
 Solvent A  132 (6.7)  66.0%  16  12  12  10
 Solvent B  137 (20.9)  68.5%  4  12  12  2
 0.5 A  190 (14.6)  95.0%  6  12  11  3
 0.5 B  98 (9.1)  49.0%  5  12  12  4
 1.0 A  118 (4.7)  59.0%  16  12  8  17
 1.0 B  114 (6.0)  57.0%  9  12  9  9
 5.0 A  134 (17.0)  67.0%  15  12  10  11
 5.0 B  116 (35.4)  58.0%  10  12  6  14
 10 A  143 (22.4)  71.5%  7  12  9  5
 10 B  119 (10.8)  59.5%  4  12  6  6
 15 A  119 (15.1)  59.5%  5  12  10  4
 15 B  129 (14.5)  64.5%  9  12  12  6
 25 A  151 (16.0)  75.5%  15  12  10  10
 25 B  115 (2.5)  57.5%  20  12  12  14
 DMBA Sol. Control A  149 (32.6)  74.5%  14  12  11  9
 DMBA Sol. Control B  122 (7.6)  61.0%  38  12  12  26
 DMBA (5.0 ug/mL) A  91 (7.2)  45.5%  485  12  12  444
 DMBA (5.0 ug/mL) B  106 (2.6)  53.0%  445  12  12  350

* C.E. = Cloning Efficiency = [(Avg. No. of C.E. Colonies per Plate)/(200 Colonies Seeded)]

**R.M. = Medium with 6-TG

Mutants/106 Survivors = [100/(C.E. x 2.4)] x (Total Mutants Counted per Dose) x [(Mutant Plates Seeded per Dose)/(Mutant Plates Counted per Dose)]

The solvent for DMBA is acetone

TABLE 5

CHOIHGPRT GENE MUTATION ASSAY PARALLEL TOXICITY TEST RESULTS

CONFIRMATORY

Art. Conc.     Without Activation  Test Art. Conc.  With S9 Activation   
 ug/mL  Ave. No. Colonies/Dose (S.D.)  RCE*  ug/mL  Ave. No. Colonies/Dose (S.D.)  RCE*
 Solvent  217 (18.4)  100%  Solvent  162 (2.1)  100%
 5.0  198 (33.9)  91%  5.0  166 (5.7)  102%
 10  160 (13.4)  74%  10  169 (7.1)  104%
 15  122 (12.7)  56%  15  141 (36.8)  87%
 25  11 (8.5)  5%  25  103 (6.4)  63%
 40  0 (0.0)  0%  40  33 (0.7)  20%
 EMS Sol. Control  211 (8.5)  100%  DMBA Sol. Control  150 (4.9)  100%
 EMS (0.5 uL/mL)  98 (12.0)  46%  DMBA (5.0 ug/mL)  114 (33.9)  76%

*RCE = [(Average No. of Colonies in Test Plates)/(Average No. of Colonies in Solvent Control Plates)] x 100

The solvents for EMS and DMBA are DMSO and acetone, respectively.

TABLE 6

CHO/HGPRT GENE MUTATION ASSAY RESULTS - WITHOUT ACTIVATION

CONFIRMATORY

 Test Art. Conc. ug/mL  Ave. No. of C.E. Colonies/Plate (S.D.)  C.E.  Total Mutants Counted  No. of Plates Seeded  No. of Plates Counted  Mutants/106 Surviving Cells
 Solvent A  175 (7.4)  87.5%  2  12  12  1
 Solvent B  180 (11.5)  90.0%  6  12  12  3
 5.0 A  171 (2.0)  85.5%  1  12  12  0
 5.0 B  182 (23.1)  91.0%  0  12  12  0
 10 A  182 (16.5)  91.0%  4  12  12  2
 10 B  157 (7.1)  78.5%  2  12  12  1
 15 A  157 (11.0)  78.5%  10  12  12  5
 15 B  156 (9.6)  78.0%  0  12  12  0
 25 A  142 (8.5)  71.0%  0  12  12  0
 25 B  136 (6.8)  68.0%  0  12  12  0
 40 A  NA  NA  NA  12  NA  NA
 40 B  NA  NA  NA  12  NA  NA
 EMS Sol. Control A  166 (4.4)  83.0%  2  12  11  1
 EMS Sol. Control B  176 (7.0)  88.0%  2  12  12  1
 EMS (0.5 uL/mL) A  111 (8.3)  55.5%  237  12  12  178
 EMS (0.5 uL/mL) B  117 (8.5)  58.5%  220  12  12  157

* C.E. = Cloning Efficiency = [(Avg. No. of C.E. Colonies per Plate)/(200 Colonies Seeded)]

NA = Not applicable. Culture was not cloned due to toxicity.

**R.M. = Medium with 6-TG

Mutants/106 Survivors = [100/(C.E. x 2.4)] x (Total Mutants Counted per Dose) x [(Mutant Plates Seeded per Dose)/(Mutant Plates Counted per Dose)]

The solvent for EMS is DMSO.

TABLE 7

CHO/HGPRT GENE MUTATION ASSAY RESULTS - WITH ACTIVATION

CONFIRMATORY

 Test Art. Conc. ug/mL  Ave. No. of C.E. Colonies/Plate (S.D.)  C.E.  Total Mutants Counted  No. of Plates Seeded  No. of Plates Counted  Mutants/106 Surviving Cells
 Solvent A  151 (9.0)  75.5%  1  12  12  1
 Solvent B  162 (1.7)  81.0%  5  12  12  3
 5.0 A  156 (5.5)  78.0%  5  12  12  3
 5.0 B  167 (21.5)  83.5%  0  12  12  0
 10 A  151 (1.5)  75.5%  3  12  12  2
 10 B  159 (6.8)  79.5%  6  12  12  3
 15 A  154 (11.8)  77.0%  4  12  12  2
 15 B  168 (18.2)  84.0%  4  12  12  2
 25 A  156 (6.6)  78.0%  4  12  12  2
 25 B  178 (6.1)  89.0%  5  12  12  2
 40 A  154 (14.6)  77.0%  2  12  12  1
 40 B  188 (13.5)  94.0%  10  12  12  4
 DMBA Sol. Control A  178 (7.0)  89.0%  2  12  12  1
 DMBA Sol. Control B  160 (14.8)  80.0%  8  12  12  4
 DMBA (5.0 ug/mL) A  115 (4.2)  57.5%  97  12  10  84
 DMBA (5.0 ug/mL) B  92 (21.2)  46.0%  156  12  11  154

* C.E. = Cloning Efficiency = [(Avg. No. of C.E. Colonies per Plate)/(200 Colonies Seeded)]

**R.M. = Medium with 6-TG

Mutants/106 Survivors = [100/(C.E. x 2.4)] x (Total Mutants Counted per Dose) x [(Mutant Plates Seeded per Dose)/(Mutant Plates Counted per Dose)]

The solvent for DMBA is acetone.

Conclusions:
Interpretation of results (migrated information):
negative

The test article, Kordek® 573T, did not cause a significant increase in the mutant frequency at the HGPRT locus among the test article-treated cultures in the presence and absence of exogenous metabolic activation. There was no dose-dependent response in the test article treated cultures. The mutant frequencies of the test article solvent and the solvent for the positive controls were within SITEK's historical negative control values. The positive controls caused a significant increase in the mutant frequencies. All criteria for a valid assay were met. Thus, Kordek® 573T (Sample No. TD99-087, Lot No. B1103, 97.5 % active ingredient) was considered to be non-mutagenic in CHO cells when tested under the conditions of this study.
Executive summary:

Kordek® 573T (Sample No. TD99-087, Lot No. B1103, 97.5 % active ingredient) was tested for its potential to cause gene mutations at the HGPRT locus in cultured CHO cells with and without exogenous metabolic activation.

A Range Finding Test (RFT) was performed to assess the toxicity of the test article to CHO cells within this test system. Ten concentrations ranging from 0.1 to 5000 μg/mL were tested with and without metabolic activation. Toxicity was assessed by measuring the reduction in the Relative Cloning Efficiencies (RCEs). In the non-activated system, the concentrations of 0.1 to 10 μg/mL had RCEs ranging from 90% to 39%. The concentrations of 500, 1000 and 5000 μpg/mL were not seeded for colony formation due to toxicity, and the concentrations of 50 and 100 μg/mL had no surviving colonies. In the activated system, the test article concentrations of 0.1 to 10 μg/mL had RCEs ranging from 101% to 50%. The concentrations of 1000 and 5000 μg/mL were not seeded for colony formation due to toxicity, and the concentrations of 50, 100, and 500 μg/mL had no surviving colonies in this system.

The doses selected for the Mutation Assays were based on the results of the RFT. The Mutation Assay was performed in two independent trials: a definitive (B-1) and a confirmatory assay (B-4). The first two confirmatory assays (B-2 and B-3) were lost due to the lack of surviving colonies in the Parallel Toxicity plates. The cultures in the definitive Assay were treated at the following concentrations:

With and Without Activation: 0.5, 1.0, 5.0, 10, 15 and 25 μg/mL.

After a 9-day period of expression, the cultures were cloned. The RCEs among the test article-treated cultures ranged from 29 % to 79% and 42% to 80%, for the test article concentrations of 0.5 to 25 μg/mL with and without exogenous metabolic activation.

The confirmatory assay was performed at the following test article concentrations:

With and Without Activation: 5.0, 10, 15, 25 and 40 μg/mL.

The test article concentrations tested in the confirmatory Mutation Assay (B-4) were based on the parallel toxicity results.

After an 8-day period of expression, the cultures were cloned. The RCEs ranged from 91 % to 5.0 % in the test article concentrations ranging from 5.0 to 25 μg/mL in the nonactivated portion of the assay. The highest test article concentration of 40 μg/mL had no surviving colonies and could not be cloned for mutant selection. In the activated system, the test article concentrations ranging from 5.0 to 40 μg/mL had RCEs ranging from 104% to 20%.

The osmolality of the highest concentration tested (5000 μg/mL) in the definitive Mutation Assay was less than 500 mOsmol/kg in the culture medium. Thus, there was no effect on osmolality caused by the addition of the test article at the maximum concentration.

At the conclusion of the expression period for each assay, the cultures were cloned in medium containing 6-thioguanine to select HGPRT enzyme-deficient mutants. These cultures were then incubated for 7-8 days. After the incubation period, the Percent Clonable Cells (Cloning Efficiency) and mutant frequencies were calculated for each culture by counting the number of colonies.

The test article, Kordek® 573T, did not cause a significant increase in the mutant frequency at the HGPRT locus among the test article-treated cultures in the presence and absence of exogenous metabolic activation. There was no dose-dependent response in the test article treated cultures. The mutant frequencies of the test article solvent and the solvent for the positive controls were within SITEK's historical negative control values. The positive controls caused a significant increase in the mutant frequencies. All criteria for a valid assay were met. Thus, Kordek® 573T (Sample No. TD99-087, Lot No. B1103, 97.5 % active ingredient) was considered to be non-mutagenic in CHO cells when tested under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The following 2 studies were identified as key studies -

Unscheduled DNA synthesis in rat hepatocytes - 2-methyl-4-isothiazolin-3-one (RH-573) did not cause any significant changes in the degree of nuclear labeling of cultured hepatocytes after treatment of male rats at doses of 103,206 and 308 mg a.i./kg, whether assayed at 2 to 4 hours after treatment or at 14 to 16 hours. Therefore, 2-methyl-4-isothiazolin-3 -one (RH-573) was evaluated as negative in the in vivo/in vitro assay for unscheduled DNA synthesis (UDS) in the livers of male Crl:CD (SD)IGR BS rats under the conditions of this study.

Micronucleus study in mouse - Kordek® 573T, Sample Number TD99-087, Lot Number B1103, 97.5% active ingredient, was evaluated for its potential to induce chromosomal damage in vivo, as assessed by the micronucleus assay with mouse bone marrow cells. The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle controls. An increase in the

frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, MMC. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to

detect induced cytogenetic damage. Under the conditions of this study, Kordek®573T was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.

Link to relevant study records

Referenceopen allclose all

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
Study period:
22 April - 3 July 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
other: CD (SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
The indicator cells for this assay were hepatocytes obtained from healthy young adult Crl:CD (SD)IGS BR rats. Animals purchased from Charles River Laboratories, Raleigh, North Carolina, were used for the assay. The animals were approximately 8-9 weeks of age at the initiation of dosing.

Animals were housed up to 2 per cage during acclimation and singly after randomization in suspended stainless-steel cages measuring 24.2 cm x 22.0 cm x 17.3 cm (DxWxH). PMI Certified Rodent Diet 5002, and tap water were supplied ad libitum. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed on a retrospective basis for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens. The temperature and relative humidity of the animal room were set at 22 +/- 4C (64.4 to 78.g°F) and 55 +/- 15% respectively. Temperature and humidity were recorded at least once daily. The lighting controls were set to maintain a 12-hour light:2-hour dark cycle (lights on approximately 0600 to 1800 hours), which was interrupted during animal dosing of the 14- to 16-hour timepoint. The air handling controls were set for ten or greater air changeslhour in the study room.

Animals were acclimated for at least 5 days prior to the initiation of dosing. They were identified by eartag after computer-generated random assignment to treatment groups according to Covance-Vienna SOPS. Treatment groups were identified by cage label. Animals were weighed prior to dosing and were dosed based upon the individual animal weights. Animals were anesthetized prior to surgery to obtain the hepatocytes (Ketarnine:Xylazine at about 100 mg/kg:13.4 mg/kg by intraperitoneal injection) and exsanguinated during the procedure.
Route of administration:
oral: gavage
Vehicle:
The vehicle control was sterile water for injection, USP (Baxter, Lot No. C541854). The vehicle control animals were dosed with the same lot of water used to dilute the test article and dosed by the same route as, and concurrently with, the test article in amounts equal to the maximum volume of dosing formulations administered to the experimental animals. The dosing volume was 10 mL/kg. Four control rats at each timepoint in the UDS study were treated by oral gavage (no control articles were used in the dose range finding assay). Vehicle control hepatocytes were subjected to all of the manipulations used for the hepatocytes derived from test article-treated animals.
Details on exposure:
Dose Selection
The highest dose selected for the UDS assay was 300 mg a.i./kg, based on the results of the dose range finding assay. Toxicokinetic studies in rodents have shown that 2-methyl-4-isothiazolin-3-one is distributed to the liver following oral exposure (Dow Chemical Company, 1997; Dow Chemical Company, 2003). Two additional dose levels were selected, using dilutions of the highest dose. The test article was therefore tested at 103, 206 and 308 mg a.i./kg.

References:
Dow Chemical Company. (1997). 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one [14C-Kathon Biocide] Toxicokinetic Study in Rats, Rohm and Haas Company Report No. 97-1058.

Dow Chemical Company. (2003). Tissue Distribution of 2-methyl-4-isothiazolin-3-one [14C-RH-5731 in the Mouse, Rohm and Haas Company Report No. 03RC-042.
Duration of treatment / exposure:
Two timepoints for UDS analysis were employed, one at 2 to 4 hours after administration of a single dose of the test article and another at 14 to 16 hours after administration. The group of animals for each analysis was dosed on different dates, independent of the ordering of the timepoint.

For the 2- to 4-hour timepoint (dosing date of May 08,2003), the animals ranged in weight from 258-309 grams. For the 14- to 16-hour timepoint (dosing date of May 15,2003), the weight range of the animals used was 297-337 grams. At the initiation of dosing, the weight variation of animals did not exceed f 20% of the mean weight at each timepoint.

An acute dosing regimen (single administration) was used, and the route of administration for test article and the vehicle control groups was oral gavage. The dosing volume was kept constant at 10 &kg. The positive control was prepared fresh for each timepoint and administered by IP injection at a dosing volume of 1 &kg. Delivery volumes were calculated on the basis of the most recent animal weight. The animals were observed for toxic signs and mortality within 0.5 hours of dosing and just prior to perfusion for hepatocyte collection.
Frequency of treatment:
A single dose was administered by oral gavage.
Post exposure period:
One group was sacrificed 2-4 hours after dosing while the other group was sacrificed 14 - 16 hours after dosing.
Remarks:
Doses / Concentrations:
0, 103, 206 and 308 mg a.i./kg.
Basis:
actual ingested
No. of animals per sex per dose:
4/dose group for each sacrifice time except for the high dose which had 6 rats/sacrifice time (extra animals included in case some died).
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control article, N-dimethylnitrosarnine (DMN: CAS No. 62-75-9, Sigma Chemical Co., Lot No. 101K0182), is known to induce UDS in rat hepatocytes in vivo and was included in the UDS assay. DMN was dissolved in sterile deionized water and administered at a dosing volume of about 1 mWkg. DMN was administered at approximately 10 mg/kg and 15 mg/kg for the 2- to 4-hour and 14- to 16-hour timepoints, respectively. The positive control was prepared fresh for each timepoint and administered by intraperitoneal injection to four rats per timepoint.
Tissues and cell types examined:
Cell Collection and Culture
This assay was based on the procedures described by Butterworth et al. (1987). The hepatocytes were obtained by perfusion of livers from 4 animals per group in situ with HBSS/EGTA followed by WMEC. The hepatocytes were obtained by mechanical dispersion of excised liver tissue in a sterile culture dish containing WMEC. The suspended tissues and cells were allowed to settle to remove cell clumps and debris prior to collection. The collected cell suspension was centrifuged and the cell pellet resuspended in WME+. After obtaining a viable cell count, a series of culture dishes was inoculated with approximately 0.5 x 10(6) viable cells in 3 mL of WME+. Culture dishes that were used for the UDS assay contained plastic coverslips. Dishes used to assess attachment efficiency had no coverslips. Cultures were identified with the animal eartag number.

Details of tissue and slide preparation:
An attachment period of 1.5 to 2 hours at 35 to 38°C in an atmosphere of 4 to 6% C02 in air was used to establish the cell cultures as monolayers. Unattached cells were then removed, the cultures washed twice, and labeling was initiated by refeeding the cultures with 2.5 mL of WME-treat. Three of the replicate cultures from each animal were used for the UDS assay, and one culture was used to assess cell attachment. Any remaining cultures were kept for analysis in the event of technical problems.

Attachment efficiency, an estimate of the number and viability of cells attaching to the dishes, was determined for one culture from each animal using trypan blue dye exclusion and in situ analysis.

After a labeling period of about 4 hours, the labeled cell cultures were washed twice, refed with WMEI containing 0.25 rnM thymidine, and returned to the incubator for 16 to 20 hours.

Termination
The nuclei were swollen by addition of 1% sodium citrate to the cultures (containing cell monolayers) for 10 minutes. Next, the cells were fixed in acetic acid:ethanol(1:3) and dried at least overnight. The coverslips were mounted on glass slides, dipped in an emulsion of Kodak NTB2 and water, and air-dried. The emulsion-coated slides were stored for 8 days at >0-10°C in light-tight boxes containing a desiccant. The emulsions were developed in Kodak D19, fixed with Kodak Rapid Fixer, and stained with a modified hematoxylin and eosin procedure.

Slide Analysis
After autoradiography, all slides were reviewed for quality before analysis. The quality of the autoradiography, the number and distribution of cells on the slides, and cellular morphology were considered in the evaluation. Three treatment groups from each timepoint were analyzed for nuclear labeling. Three animals from the vehicle, positive control and test article dose groups were analyzed, beginning with the lowest numbered animal having cells acceptable for analysis.

The cells were examined microscopically at approximately 1500x magnification under oil immersion and the field was displayed on the video screen of an automatic counter. Only normally-appearing nuclei were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred rather than repair synthesis. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (cytoplasmic count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting.

The net nuclear grain count was routinely determined for 50 randomly selected cells on triplicate coverslips (150 total nuclei) for each animal. The average mean net nuclear grain count (k standard deviation) was determined from the triplicate coverslips (150 total nuclei) for each animal and averaged for each treatment condition. In some instances, the counts were inconsistent and slides were analyzed independently by another technician. When the counts were similar, the data was averaged and when the data differed, the consistent data was included.
Evaluation criteria:
Assay Acceptance Criteria
An assay normally is considered acceptable for evaluation of the test results only if all of the criteria listed below are satisfied. This listing may not encompass all test situations, thus the study director must exercise scientific judgment in modifying the criteria or considering other causes that might affect reliability and acceptance.

Cell Culture Conditions
The viability of the vehicle control hepatocytes collected from the perfusion process must be at least 50%. Normally the perfusion viability exceeds 70%, but a variety of factors can affect cell yield and viability, so values below 70% are not uncommon nor necessarily detrimental. The toxicity of the treatment with the test article may be reflected in perfusion viability; therefore, a lower limit is not set for cultures from the test article-treated animals.

Acceptable Controls
The average net nuclear labeling in the vehicle control cultures is typically in the range of -5.00 to 1.00. In addition, no more than 10% of the cells should be in repair (containing five or more net nuclear grains). If the analyzed vehicle control animals fail to meet these criteria, the assay is normally considered to be invalid.

The positive control is used to demonstrate that the cell population employed was responsive and the methodology was adequate for the detection of UDS. The average response to the positive control treatments must exceed either criteria used to indicate UDS.

Acceptable Number of Doses
A minimum of three dose levels is analyzed for nuclear grain counts at each timepoint. Repeat trials need only augment the number of analyzed dose levels in the first trial to achieve a total of three acceptable dose levels.

Grain count data obtained per animal is acceptable as part of the evaluation if obtained from at least two replicate cultures and at least 100 cells per animal. Grain count data should be available from three of the four animals.

Continued below.
Statistics:
No additional information available.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
hypoactive at the two highest doses
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Analyses of 2-methyl-4-isothiazolin-3-one (RH-573) dose formulation samples for concentration confirmation were within the required target range (+/-10%). This information confirms that the rats received very accurate final in vivo doses of the test article in this UDS study.

Probe Study
Within 1 hour of dosing, all males in the 308 and 411 mg a.i./kg groups and all females in the 257, 308 and 411 mg a.i./kg groups were slightly hypoactive. Additional toxic signs within 1 hour of dosing included irregular respiration, salivation, audible respiration and squinted eyes (257 mg a.i./kg and higher). During the next two days of observations, toxic signs included the observations noted previously, plus piloerection. cold to touch, clear discharge (anal-genital), pale extremities, ataxia, sternal recumbency, few or no feces and recumbency. One female at 257 mg a.i./kg was found dead 1 day after dosing, and 2 females at 308 mg a.i./kg and 2 at 31 1 mg a.i./kg were also found dead 1 day after dosing. One male in the 411 mg a.i./kg dose group was sacrificed in a moribund condition 1 day after dosing. Based on toxic signs over a small concentration range, it was decided that both sexes behaved similarly, which justified the use of males only for the UDS assay.

UDS Assay
For the UDS assay, the test article was administered at 103, 206 and 308 mg a.i./kg in water, based on the results of the dose range finding study. The test material was administered by oral gavage in volumes of 10 mL/kg to 4 rats per dose group at each timepoint (two additional rats were dosed in the high dose group). Four animals from each group were perfused and hepatocytes were seeded for attachment. UDS was determined from three animals per group.

At the 2- to 4-hour timepoint, one animal dosed at 308 mg a.i./kg was observed with slight hypoactivity prior to perfusion. All other animals were normal after dosing and prior to perfusion.

At the 14- to 16-hour timepoint (Table7), one animal dosed at 308 mg a.i./kg was observed with salivation and slight hypoactivity after dosing. All other animals were normal after dosing. Prior to perfusion, the animal with observations after dosing was hypoactive. One additional animal in the 308 mg a.i./kg group was slightly hypoactive. All other animals were normal prior to perfusion.

For the early UDS timepoint, perfusions were initiated 3.2 to 3.4 hours after dose administration. The hepatocytes ranged in viability (determined by trypan blue dye exclusion) from 63.2% to 93.2% of the total cells collected in the perfusate. The attachment efficiency varied from 30.8% to 99.1%, and the viability of the attached cells was good, ranging from 81.4% to 98.1%.

For the 14- to 16-hour timepoint, perfusions were initiated 14.3 to 14.6 hours after dose administration. The hepatocytes ranged in viability from 72.6% to 90.6% of the total cells collected in the perfusate. The attachment efficiency varied from 49.6% to 93.3%, and the viability of the attached cells was good, ranging from 85.2% to 98.2%.

All three test article treatment groups (103, 206 and 308 mg a.i./kg) were analyzed for nuclear labeling at both timepoints. The minimum criteria for a UDS response was determined using the group averages of the concurrent vehicle control treatments. A positive response was indicated by an increase in the group average of the mean net nuclear grain count to at least three grains per nucleus above the concurrent vehicle control average (and leading to a positive number) or by an increase in the group average of the percent of nuclei with five or more net grains such that the percentage of these nuclei in the test cultures was incremented by 10% above the percentage observed for the group average of the concurrent vehicle controls.

The UDS data for both timepoints is summarized in Table 1.

For the 2- to 4-hour timepoint, the mean net nuclear grain count for the vehicle control animals was -0.19, and the average percent of cells containing five or more net nuclear grains was 6.00%. Therefore, the criteria for a positive response in the treated groups were a mean net nuclear grain count exceeding 2.81 or at least 16.00% of the nuclei containing five or more grains. None of test article treatment groups yielded a positive mean net nuclear grain count, and the highest percent cells with >/= 5grains was only 6.44%, well below the criterion for a positive response. Thus, no evidence for UDS was obtained at the early timepoint of 2 to 4 hours after treatment of the animals.

For the 14- to 16-hour timepoint, the mean net nuclear grain count for the vehicle control animals was 0.67, and the average percent of cells containing five or more net nuclear grains was 8.44% (Table 1). The criteria for a positive response were a mean net nuclear grain count exceeding 3.67 or at least 18.44% of the nuclei containing five or more grains. None of the test article treatment groups yielded a positive mean net nuclear grain count, and the highest percent cells with 5 grains was 14.21%, which was below the criterion for a positive response. Thus, no evidence for UDS was obtained at the later timepoint of 14 to 16 hours after treatment of the animals.

Heavily-labeled nuclei (blackened with numerous grains) represent cells in S-phase as opposed to DNA repair. The number observed for each animal for both UDS timepoints was low and did not interfere with the detection of UDS.

The vehicle control results were well within the acceptable criteria for this study The DMN positive control treatments induced large increases in nuclear labeling that clearly exceeded both criteria used to indicate UDS. Since the positive control animals were responsive, the test results were considered to provide conclusive evidence for the lack of UDS induction by the test article.

TABLE 1. SUMMARY OF UDS SLIDE DATA

   Dose (mg a.i./kg)  Time(hr)  MeanNuclear Grainsa + SD  Mean Net Nuclear Grainsb + SD  Mean Cytoplasmic Grainsc + SD  % Cells with > NNGd Grains + SD  % Cells in S-phasee
 Vehicle Control  0  2 -4  6.35 + 1.27  -0.19 + 0.96  6.54 + 1.92  6.00 + 4.47  1.38%
   0  14 - 16  6.78 + 1.04  0.67 + 0.87  6.12 + 1.67  8.44 + 4.67  0.41%
 Positive Control  10  2 -4  33.28 + 9.76  27.11 + 8.11  6.17 + 1.68  96.89 + 3.89  0.71%
   15  14 - 16  32.89 + 9.63  26.97 + 9.56  5.92 + 0.70  99.11 + 1.45  0.29%
 Test Article  103  2 - 4  6.33 + 0.52  0.17 + 0.52  6.16 + 0.87  6.44 + 3.43  1.07%
     14 - 16  6.72 + 0.54  1.19 + 1.08  5.53 + 1.08  14.21 + 6.56  0.64%
   206  2 - 4  7.17 + 0.85  -0.59 + 0.70  7.77 + 1.19  5.56 + 3.13  0.67%
     14 - 16  6.51 + 1.13  0.38 + 1.02  6.13 + 1.38  8.23 + 4.19  0.11%
   308  2 -4  6.78 + 1.10  -0.38 + 0.88  7.17 + 1.27  6.00 + 5.20  0.38%
     14 - 16  7.28 + 1.44  0.23 + 1.05  7.05 + 1.51  7.78 + 7.38  0.13%

a Average nuclear grain count.

b Average of net nuclear grain count with standard deviation (SD) between coverslips.

Net nuclear grains (NNG) = Nuclear grain count - Average cytoplasmic grain count.

cAverage of cytoplasmic grain count.

dAverage percentage of cells with greater than or equal to 5 net nuclear grains.

eThe percentage of heavily labeled cells observed.

Vehicle control article = Sterile water for injection, USP, 10 rnL/kg.

Positive control article = Dimethylnitrosamine, 1 mL/kg.

Test article = 2-methyl-4-isothiazolin-3-one (RH-573), 10 rnL/kg.

Criteria for a positive response:

2-4 hr timepoint - mean net nuclear grain counts > 2.81 or nuclei containing > 5 NNG > 16.00%.

14-16 hr timepoint - mean net nuclear grain counts > 3.67 or nuclei containing > 5 NNG > 18.44%.

Conclusions:
Interpretation of results (migrated information): negative
2-methyl-4-isothiazolin-3-one (RH-573) was therefore evaluated as negative in the in vivo/in vitro assay for UDS in the livers of male Crl:CD®(SD)IGS BR rats under the conditions of this study.
Executive summary:

The objective of this assay was to detect in vivo liver DNA damage in Crl:CD®(SD)IGS BR rats caused by 2-methyl-4-

isothiazolin-3-one (MI; RH-573), or an active metabolite, by measuring unscheduled DNA synthesis (UDS) in hepatocytes collected by perfusion and cultured in vitro with 3H-thymidine.

A preliminary dose range finding study was performed to select doses for the UDS study. Three rats/sex/dose group were dosed by oral gavage with the test article in water at 103, 206, 257, 308 and 411 mg a.i./kg. Within 1 hour of dosing, all males in the 308 and 411 mg a.i./kg groups and all females in the 257, 308 and 411 mg a.i./kg groups were slightly hypoactive. Additional toxic signs within 1 hour of dosing included irregular respiration, salivation, audible respiration and squinted eyes (257 mg a.i./kg and higher). During the next two days of observations, toxic signs included the observations previously noted, plus piloerection, cold to touch, clear discharge (anal-genital), pale extremities, ataxia, sternal recumbency, few or no feces and recumbency. One female at 257 mg a.i./kg was found dead 1 day after dosing, and 2 females at 308 mg a.i./kg plus 2 at 411 mg a.i./kg were also found dead 1 day after dosing. One male in the 411 mg a.i./kg dose group was sacrificed in a moribund condition 1 day after dosing. Based on toxic signs over a small concentration range, it was decided that both sexes behaved similarly, which justified the use of males only for the UDS assay. Based on these observations, the maximum dose of 300 mg a.i./kg was selected for the UDS assay, and the mid-dose and low dose selections were 200 and 100 mg a.i./kg.

2-methyl-4-isothiazolin-3-one (RH-573) was administered by oral gavage at a volume of 10 mL/kg to male rats at doses of 103, 206 and 308 mg a.i./kg in water for the UDS assay. Hepatocytes were subsequently harvested at two time-points: 2 to 4 hours and 14 to 16 hours after dosing. The vehicle control animals were dosed concurrently with water by oral gavage at 10 mL/kg and were harvested at the same two time-points. Two positive control groups, dosed intraperitoneally with dimethylnitrosamine (DMN) were included. The dose was 10 mg DMN/kg for the 2 to 4 hour harvest and 15 mg DMN/kg for the 14 to 16 hour harvest. Four male Crl:CD®(SD)IGS BR rats were treated per group, except for the high dose where two extra animals were included.

Toxic signs observed in the UDS assay were salivation and some slight hypoactivity. No deaths occurred. All animals from each group were perfused for the collection of hepatocytes and establishment of cultures and cultures from three animals per group were evaluated for UDS. After attachment of the cells, the cultures were labeled with 10 μCi/rnL 3H-TdR for 4 hours. The cultures were prepared for analysis of nuclear labeling by autoradiography after washing out the unincorporated label. The nuclear labeling, measured as the mean net nuclear grain count or the percent of nuclei with five or more net nuclear grains, in the test article-treated groups remained similar to that obtained for the vehicle control animals for both harvest times. The criteria for indicating a positive response were not approached. In contrast, the DMN positive control induced large increases in nuclear labeling.

2-methyl-4-isothiazolin-3-one (RH-573) was therefore evaluated as negative in the in vivo/in vitro assay for UDS in the livers of male Crl:CD®(SD)IGS BR rats under the conditions of this study.

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
Study period:
3-29 November 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Male and female CD-1 mice, approximately 8 weeks old (weighing approximately 23-29 g) were used for this study. The animals were obtained from Charles River Laboratories (Raleigh, N.C.) and acclimatized in animal facilities at Rohm and Haas Company for a 7 day quarantine period. Animals were ear tagged prior to the initiation of the study. Purina Certified Rodent Chow 5002 C and water filtered through a reverse osmosis system were available ad libitum. The animals were housed in an environmentally controlled room with temperature, relative humidity, and light cycle set according to Rohm and Haas Toxicology Department Standard Operating Procedures (SOP) (section 10). Daily temperatures averaged 22 degrees C with relative humidity averages between 43-60%, which were within the acceptable range as defined in the SOP. Animals were assigned to treatment groups through the use of a computerized randomization procedure according to sex and body weight prior to administration of the test articles. Animals were within the acceptable weight range of plus or minus 20% from the mean. Animals were fasted for approximately 3 hours prior to dose administration.
Route of administration:
oral: gavage
Vehicle:
Distilled water
Details on exposure:
The dose levels for this study were selected after evaluating the results of an acute oral toxicity study in male and female mice (see Rohm and Haas Report No. 99R- 131). The maximum concentration for this study was 100 mg/kg (OECD Guideline 474). In addition to the maximum dose level, 50 and 10 mg/kg of KordekB 573T were tested.

Animals were treated with the test article (at the above mentioned concentrations), distilled water (negative control), or Mitomycin-C (at a dose of 2.0 mg/kg of the positive control substance). The test article and the negative control were administered by gavage, in a single oral dose. The positive control was administered in a single dose by intraperitoneal (i.p.) injection since this is the accepted route for this substance. For each treatment and control group, 5 male and 5 female animals were dosed per time point, with a volume of 10 ml/kg. Animals were euthanized by cervical dislocation at approximately 24 and 48 hours after dosing. In the high dose group, 2 additional animals per time point were dosed to account for the possibility of unexpected deaths

The positive control groups were euthanized 24 hours after dosing. Animals were observed for the presence of clinical signs during the treatment period and prior to sacrifice
Duration of treatment / exposure:
Animals were sacrificed 24 or 48 hours post dosing
Frequency of treatment:
Mice received a single oral gavage treatment of test material
Post exposure period:
24 and 48 hour post exposure period
Remarks:
Doses / Concentrations:
10, 50 and 100 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
For each treatment and control group, 5 male and 5 female animals were dosed per time point
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin-C
Tissues and cell types examined:
Animals were prepared for micronucleus evaluation as follows: The groin area was wetted with 70% ethanol in water. Both femurs were removed by making incisions at the hip joint and below the knee cap, and the knee cap was removed. The bone marrow was flushed into a 15-ml centrifuge tube, containing approximately 1 ml of Fetal Bovine Serum (FBS) using a 1-cc syringe fitted with a 25-gauge needle. The tubes were centrifuged at 120 x g for 5 minutes, and the supernatant was removed, leaving approximately 0.1 ml above the cell pellet. The cell pellet was re-suspended in the remaining serum until a homogeneous suspension was observed.
Details of tissue and slide preparation:
A small drop of the cell suspension (approximately 10 ul) was placed on the unfrosted end of a clean microscope slide and spread along the length of the slide. The slides were air dried for at least 1 hour, and then fixed in methanol for 15 minutes and allowed to dry. The slides were stained with Acridine Orange staining solution.

Slides from at least five animals per sex/dose group were observed when possible. Three slides were prepared per animal. Slides were coded and read blind in order to avoid bias on the part of the scorer. The slides were read using an epifluorescence microscope to illuminate the acridine orange stain (Hayashi et al., 1983).

Slides were scanned for regions of suitable technical quality, where the cells were well spread, undamaged and well stained. These regions were normally located in a zone close to the middle of the smear. Staining was tan to faint grey in normochromatic erythrocytes (NCE) and bright orange in polychromatic erythrocytes (PCE). Micronuclei appeared bright green against an orange background in PCE and generally were round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei have sharp borders and were usually between 1/20 and 115 the size of the PCE. The end point to be scored was the number of cells containing micronuclei (not the number of micronuclei per cell).

For each animal, a total of at least 1000 erythrocytes (polychromatic, referred to as PCE 1 and normochromatic) were recorded to calculate the PCE/NCE ratio. For each animal, the remaining number of polychromatic erythrocytes were recorded to total at least 2000 (referred to as PCE 2) and were scored for the presence or absence of micronuclei. The frequency of micronucleated polychromatic erythrocytes (MN-PCE) and the PCE/NCE ratio were calculated on the basis of these data.

Cell counting was accomplished using the Xybion PathITox computer software system, G Module (GICELL program version 4.21) which captures data from the cell counter keyboard and provides an audit trail for quality assurance.

Reference:
Hayashi, M., T. Sofuni and M. Ishidate, Jr. (1983). An application of acridine orange fluorescent staining to the micronucleus test, Mutat. Res., 105,253-256.
Evaluation criteria:
The test article is considered positive in this assay if it elicits a dose-response or a statistically significant increase in the number of micronucleated cells over that of the concurrent vehicle control at one or more dose levels. In the event that the test article elicits a significant increase in the number of MN-PCE due to an unusually low number of MN-PCE in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data.

A test article is considered negative in this assay if: No indication of a dose-response is observed and the treatment groups do not show a statistically significant increase in the number of MN-PCE when compared to the vehicle control.

The above criteria are to be used as a guideline for evaluating the assay results. The study director may take other appropriate factors into consideration for evaluating the test results.
Statistics:
Data were analyzed separately for male and female animals using a Statistical Analysis System (SAS), version 6.09 enhanced. An arcsine square root transformation was applied to the percent of micronucleated PCE's; all subsequent analyses for this parameter were conducted on transformed data. Initially, a three-way analysis of variance model was applied to the data to determine the significance of each main effect (sex, group, and day) and all two-way and three-way interaction effects. If significant interaction effects were identified, then the data were analyzed separately for each sex and/or day. Three independent single degree of freedom contrasts of the group means were used to test for trends in the group means and included an assessment of: 1) an overall effect of Kordek 573T treatment relative to control, and 2) a linear dose-response trend among the Kordek 573T groups, and 3) a quadratic dose-response trend among the KordekB 573T. Additionally, pairwise comparisons between each of the three Kordek 573T groups and the control group were made using Dunnett's t-test (Kirkland, 1989).

Reference:
Kirkland, D.J. (Editor), Statistical Evaluation of Mutagenicity Test Data. United Kingdom Environmental Mutagen Society, Cambridge University Press, 1989.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Two female mice died 24 hours after dosing.
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Clinical Signs
No visible abnormalities were observed in any of the male or female mice treated with either the negative control or with 50 mg/kg or 10 mg/kg Kordek 573T.

Two female mice were ataxic, passive, and exhibited labored breathing approximately 4 hours after dosing with 100 mg/kg Kordelt 573T: Approximately 24 hours after dosing, these two female mice were found dead. No other signs were observed in any of the female mice treated with 100 mg/kg Kordek 573T. No clinical signs were observed in any of the male mice treated with 100 mg/kg Kordek 573T.

Micronucleus Evaluation
The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle control values. This was true for both the 24 and 48 hour time points. There was no statistically significant change in the polychromaticl normochromatic ratio at either 24 or 48 hours, which is indicative of the absence of cytotoxicity.

An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, mitomycin-C. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage.

Dosing Solution Analysis

Samples of the dosing solutions were submitted for independent chemical analysis of the test article concentration. The results indicate that the concentration of the Kordek 573T dosing solutions ranged from 85.1 to 98.8 percent of target value, which is within an acceptable range of the expected target concentration.

Table 1 - Micronucleus Evaluation

Mean Summary Data

Male Animals

 Group  Dosemg/kg  NCE (S.D.)  PCE1 (S.D.)  MN-PCE (S.D.)  PCE2 (S.D.)  PCE Total (S.D.)  PCE/NCE Ratio (S.D.)  MN-PCE % (S.D.)
 Day 1                
 Control  0  443 (26)  628 (18)  4 (1)  1471 (24)  2099 (21)  1.42 (0.08)  0.18 (0.05)
 Kordek 573T  10  446 (45)  632 (44)  3 (2)  1511 (80)  2143 (65)  1.43 (0.24)  0.12 (0.11)
   50  473 (30)  626 (21)  2 (1)  1448 (42)  2074 (44)  1.33 (0.06)  0.09 (0.07)
   100  460 (77)  621 (77)  2 (1)  1479 (87)  2101 (55)  1.42 (0.49)  0.09 (0.06)
 Mitomycin-C  2  559 (63)  561 (93)  105 (17)  1498 (94)  2059 (42)  1.03 (0.29)  5.09 (0.83)#
 Day 2                
 Control  0  474 (73)  616 (82)  2 (1)  1478 (78)  2094 (31)  1.34 (0.38)  0.10 (0.05)
 Korted 573T  10  468 (59)  629 (50)  3 (2)  1473 (70)  2102 (46)  1.37 (0.25)  0.14 (0.10)
   50  450 (71)  616 (80)  3 (1)  1479 (68)  2095 (39)  1.43 (0.47)  0.13 (0.05)
   100  478 (77)  585 (66)  2 (2)  1496 (90)  2081 (27)  1.26 (0.32)  0.10 (0.08)

SD=Standard Deviation

N=Number of Observations (N = 5 for all groups except for the high dose males on day 1 and 2 where N = 7).

NCE=Normochromatic Erythrocytes

MN-PCE=Micronucleated Polychrornatic Erythrocytes

PCE=Polychrornatic Erythrocytes

PCE TOTAL=PCEl + PCE2

PCE/NCE RATIO=PCEl/NCE

MN-PCE%=MN-PCE/ (PCEl+PCE2)*100

CALCULATIONS BASED ON INDIVIDUAL ANIMAL VALUES.

Statistical Methods: Analysis of Variance Followed by Dunnett's T-Test on Least Square Means.

For All Statistical Mgthods: ' * ' Indicates a Statistically Significant Difference from Control (p<0.05).

# GREATER THAN 2 FOLD INCREASE OVER CONTROL VALUES

Table 1 - Micronucleus Evaluation

Mean Summary Data

Female Animals

Group  Dose mg/kg  NCE (S.D.)  PCE1 (S.D.)  MN-PCE (S.D.)  PCE2 (S.D.)  PCE Total (S.D.)  PCE/NCE Ratio (S.D.)  MN-PCE % (S.D.)
 Day 1                
 Control  0  457 (72)  635 (74)  3 (1)  1477 (103)  2112 (52)  1.44 (0.37)  0.14 (0.06)
 Kordek 573T  10  432 (80)  642 (75)  2 (1)  1446 (119)  2088 (45)  1.55 (0.47)  0.09 (0.06)
 Kordek 573T  50  486 (99)  594 (95)  2 (2)  1507 (70)  2102 (50)  1.29 (0.46)  0.12 (0.08)
 Kordek 573T  100  423 (60)  680 (71)  3 (1)  1417 (59)  2097 (56)  1.64 (0.38)  0.13 (0.05)
 Mitomycin -C  2  597 (93)  523 (112)  94 (18)  1546 (148)  2069 (48)  0.91 (0.32)  4.54 (0.84)#
 Day 2                
 Control  0  426 (66)  665 (55)  2 (1)  1384 (66)  2049 (25)  1.60 (0.36)  0.10 (0.07)
 Kordek 573T  10  460 (52)  643 (77)  2 (1)  1455 (46)  2098 (90)  1.42 (0.33)  0.11 (0.04)
 Kordek 573T  50  422 (72)  647 (93)  2 (2)  1443 (86)  2090 (39)  1.59 (0.43)  0.11 (0.07)
 Kordek 573T  100  442 (49)  614 (70)  2 (1)  1471 (111)  2085 (54)  1.42 (0.31)  0.11 (0.07)

SD=Standard Deviation

N=Number of Observations (N = 5 for all groups except for high dose females sacrificed on day 2 where N = 7. Two females died 24 hours after dosing apparently from the 24 hr sacrifice group)

NCE=Normochromatic Erythrocytes

MN-PCE=Micronucleated Polychrornatic Erythrocytes

PCE=Polychrornatic Erythrocytes

PCE TOTAL=PCEl + PCE2

PCE/NCE RATIO=PCEl/NCE

MN-PCE%=MN-PCE/ (PCEl+PCE2)*100

CALCULATIONS BASED ON INDIVIDUAL ANIMAL VALUES.

Statistical Methods: Analysis of Variance Followed by Dunnett's T-Test on Least Square Means.

For All Statistical Mgthods: ' * ' Indicates a Statistically Significant Difference from Control (p<0.05).

# GREATER THAN 2 FOLD INCREASE OVER CONTROL VALUES

 

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, Kordek 573T (Lot Number B1103, 97.5% active ingredient) was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.
Executive summary:

Kordek® 573T, Sample Number TD99-087, Lot Number B1103, 97.5% active ingredient, was evaluated for its potential to induce chromosomal damage in vivo, as assessed by the micronucleus assay with mouse bone marrow cells. Adult CD-1 male and female mice (5 male and 5 female animals per group, except for the high dose group, which had 2 additional animals per time point) received a single oral dose of the test article at concentrations of 100, 50 or 10 mg/g. Control animals received a single oral dose of distilled water (vehicle control), or an intraperitoneal injection of 2.0 mg/kg Mitomycin-C (positive control) (MMC). Animals from test article and vehicle control groups were euthanized at 24 or 48 hours after treatment. Animals from the positive control group were euthanized 24 hours after treatment. Bone marrow slides were prepared and the frequency of micronucleated polychromatic erythrocytes was measured as an indicator of cytogenetic damage. For each animal, a total of at least 2000 polychromatic erythrocytes were scored for the presence or absence of micronuclei. In addition, the polychromatic erythrocyte1 normochromatic erythrocyte (PCE/NCE) ratio was measured to evaluate the cytotoxicity of the test agent.

The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle controls. An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, MMC. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage.

Under the conditions of this study, Kordek®573T was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on the results of both the in-vitro and in-vivo studies, this test material will not be classified for genotoxicity.