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Genetic toxicity: in vivo

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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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Light yellow transparent liquid
Details on test material:
2-methyl-4-isothiazolin-3-one (RH-573), Lot No. 800IJ123 (TD # 01-119) was used. All test article preparations in this report are adjusted for a purity of 51.4%.

All though not stated in the report, the remainder of the test substance composition was water.

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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.

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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%.

Applicant's summary and conclusion

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.