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

Carcinogenicity

Description of key information

C(M)IT/MIT was tested in two chronic/carcinogenicity tests by either the oral route (rat) or dermal route (mouse).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. 48014

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilution in water
- Final dilution of a dissolved solid, stock liquid or gel: 30, 100, 300 ppm a.i.

OTHER SPECIFICS: Purity of test material was 14.2%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage Facility, Portage, Michigan, USA
- Age at study initiation: approximately 3 weeks old
- Weight at study initiation: Males: 162 - 282 grams; Females: 122 – 207 grams
- Fasting period before study: Not described
- Housing: Not described
- Diet (e.g. ad libitum): Not described
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not described

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

n/a

Duration of treatment / exposure:

24 months

Frequency of treatment:

Continuous

Post exposure period:

n/a

Dose / conc.:

30 ppm

Remarks:

Nominal based on a.i.

Dose / conc.:

100 ppm

Remarks:

Nominal based on a.i.

Dose / conc.:

300 ppm

Remarks:

Nominal based on a.i.

No. of animals per sex per dose:

90 males and 80 females/group

Control animals:

yes, concurrent vehicle

other: inorganic stabilizer salt control

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical signs, daily and physical examinations, weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Yes, prior to study initiation and during the twenty-fourth month of dosing
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 20/sex/group
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months
- Animals fasted: No data
- How many animals: 20/sex/group
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 3, 6, 12, 18, 24 months of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Other examinations:

Health monitoring (Sentinel animal program) and kidney ultrasonography (conducted on all males during the 6th and 12th months of the study)

Statistics:

Distributions of the following parameters were inspected for normality and homogeneity of variance across treatment groups and sampling times: body weights, cumulative body weight changes, feed consumption, water consumption, organ weights, urinalysis, hematology, and clinical chemistry. Square root transformations were used for parameters of the white blood cell differential prior to further analysis. Analysis of variance (or covariance if pre-test scores were available) was used to determine whether or not statistically significant differences existed among the various treatment group means. When significant treatment effects were found, pairwise comparisons of least square means were made between each Kathon biocide-treated group and control using Dunnett's t-test. Statistical significance was indicated when a p-value of 0.05 or less (Dunnett's comparison) was obtained.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreases in body weight and body weight gain at 300 ppm a.i.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slight decrease in food consumption was seen in 300 ppm male animals

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Decreased water consumption in all dose levels

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gastric irritation

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Gastric irritation, Adverse effects were seen in kidneys and in adrenal gland of male and female

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related effects on body weight or body weight gain at doses up to and including 100 ppm.
Decreases in body weight and body weight gain were seen in 300 ppm animals throughout the study and were considered most likely secondary to decreases in water consumption.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no treatment-related effects on food consumption at doses up to and including 100 ppm. A slight decrease in food consumption was seen in 300 ppm male animals, likely associated with decreased water consumption in this group. No treatment-related effects on food consumption were seen in females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
A treatment-related and concentration-dependent decrease in water consumption was seen in both sexes in all Kathon biocide-treated groups throughout the study. These decreases ranged from 0-22 % at 30 ppm, 3-30 % at 100 ppm and 15-40 % at 300 ppm ai. These decreases appear to be due to the unpalatability of the Kathon biocide and not its inorganic stabilizer salts since the water consumption in Group 2 (salt control) was comparable to the tap water control throughout the study. Based on the average daily water consumption, the 300 ppm dose was judged to be a maximum tolerated dose.

URINALYSIS
No changes in urinary parameters were seen which were considered indicative of treatment-related systemic toxicity. Increases in urinary specific gravity were observed sporadically in mid- and high-dose rats at 3 and 6 months only. These increases are not unexpected, given decreased water consumption seen in these groups. No treatment related effects were seen via ultrasound in the renal pelvis of rats at any dose level.

GROSS PATHOLOGY
Gastric irritation at 100 and 300 ppm.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related morphologic changes were observed in the stomach and occurred in both sexes at 100 and 300 ppm. The primary effect noted was gastric irritation which was reflected by thickening of the forestomach mucosa due to hyperplasia and hyperkeratosis of the squamous mucosa. Focal necrosis of the superficial glandular mucosa and edema and inflammatory cell infiltration in the forestomach submucosa were seen in the 300 ppm males.
Treatment-related morphologic changes were observed in both sexes in mid and high dose groups, in kidneys (from 30 ppm ai to 300 ppm ai) and in the adrenal gland of both sexes at the higher dose (300ppm ai).

OTHER FINDINGS
There were no treatment-related effects at any dose at any time point seen via ultrasound of the kidneys of male rats.

Relevance of carcinogenic effects / potential:

n/a

Dose descriptor:

NOEL

Effect level:

300 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOEL

Effect level:

30 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: gross pathology

Remarks on result:

other: Effect type: toxicity (migrated information)

Results of carcinogenicity study in rats via drinking water

Parameter	Control		Salt control		Low dose    (30 ppm)		Medium dose (100 ppm)		High dose  (300 ppm)
	m^a	f^a	m^a	f^a	m^a	f^a	m^a	f^a	m^a	f^a
Number of animals examined	90	80	90	80	90	80	90	80	90	80
MortalityAdjustedsurvival, 24 months	36 %	35 %	41 %	35 %	30 %	33 %	37 %	38 %	46 %	32 %
Body weight	--	--	--	--	--	--	--	--	dec.	dec.
Food consumption	--	--	--	--	--	--	--	--	dec.	dec.
Decreasewater consumption	--	--	--	--	0-22 %		3-30 %		15-40 %
Organ: forestomach
Gross pathology* Organ: forestomach gastric irritation Organ : kidneys Glomerulonephrosis Organ : adrenal gland, hypertrophy	--	--	--	--	--   inc    inc.	--   inc   --	inc.   inc.   inc.	inc.   inc.   --	inc.   inc.   inc.	inc.   inc.    inc.
Microscopic pathology* Hyperplasia	1/24	0/20	3/27	0/20	1/19	0/20	3/25	5/21	18/30	8/19

* specify effects; for different organs give special findings in the order organ weight, gross pathology and microscopic pathology if there are effects

^a give number of animals affected/total number of animals, percentage

-- no change in controls versus dosed group

Conclusions:

Chloro-methylisothiazolone/methylisothiazolone produced no evidence of carcinogenicity at doses up to and including the highest dose tested (300 ppm a.i or 17 to 27 mg a.i./kg body weight /day).

Executive summary:

OECD 453, US EPA 83-5, Combined chronic/carcinogenicity study in rats with analytical confirmation of drinking water concentrations. Administration of Kathon biocide to male and female rats in the drinking water for 24 months at concentrations up to and including 300 ppm active substance showed no effects on the type or incidence of neoplasms in any group. No systemic effects. Treatment-related morphologic changes were observed only in the stomach of both sexes in mid and high dose groups. Gastric irritation was the primary effect observed. No adverse effects on the histopathology of any tissues/organs distant from the site of dosing.No treatment-related signs of toxicity were seen at 30 ppm active ingredient (2.0 mg a.i./kg b.w./day in males and 3.1 mg a.i./kg b.w./day in females), the no-observed-effect level (NOEL) in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

17.2 mg/kg bw/day

Study duration:

chronic

Species:

rat

System:

gastrointestinal tract

Organ:

other: forestomach

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

only male mice were used and only one dose of TS

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. MH31:9E

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilution in water
- Final dilution of a dissolved solid, stock liquid or gel: 400 ppm a.i.

OTHER SPECIFICS: Purity of test material was 1.5% (of which, 75 % CMIT, 25 % MIT)

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Portage, Michigan, USA
- Age at study initiation: Approximately one month
- Weight at study initiation: 23-31 g
- Fasting period before study:
- Housing: Not described
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not described

Route of administration:

dermal

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: 2 x 3 cm
- % coverage: Not described
- Type of wrap if used: Not described
- Time intervals for shavings or clipplings: Not described

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not described
- Time after start of exposure: Not described

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL/animal (10 µg/animal) a.i.
- Concentration (if solution): 400 ppm a.i.
- Constant volume or concentration used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not described

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

30 months

Frequency of treatment:

3 days/week

Post exposure period:

No

Dose / conc.:

10 other: µg/animal (nominal)

No. of animals per sex per dose:

40

Control animals:

yes, concurrent vehicle

Positive control:

3-methylcholanthrene (1000 ppm a.i.)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

DERMAL IRRITATION (if dermal study): Yes / No / No data
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: Yes, weekly for first 13 weeks and monthly thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Other examinations:

No

Statistics:

Overall survival distribution among the groups was compared using both the generalized Wilcoxon test and the log rank test. Thirty-month survival was analysed by a chi-square test (water control group versus Kathon™ CG-treated group) or Fisher’s exact test (3-MC group versus water control group or the Kathon™ CG-treated group).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brown staining, eschar and/or dessication, flaking of skin at application site of Kathon™ CG treated mice

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Brown staining, eschar and/or dessication, flaking of skin at application site of Kathon™ CG treated mice

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Brown staining, eschar and/or dessication, flaking of skin at application site of Kathon™ CG treated mice

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

HISTOPATHOLOGY: NON-NEOPLASTIC
The treated skin of the Kathon™ CG animals did show focal or multifocal epidermal necrosis, hyperplasia, hyperkeratosis, eschar, dermal inflammation, and increased dermal collagen.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment-related findings were evident at necropsy in the Kathon™ CG-treated animals. Histopathologic evaluation revealed no indications of a treatment-related increased incidence of neoplasms either at the application site or systemically in the Kathon™ CG-treated animals. The incidence of systemic neoplasms was similar in the water control and Kathon™ CG-treated group.

Key result

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Conclusions:

No adverse effects were seen on the histopathology of any tissues/organs distant from the site of dosing.

Executive summary:

The mouse skin painting carcinogenicity study was initiated prior to the adoption of carcinogenicity study guidelines. However, the principles of OECD Guideline 451, in general, were followed with the deviations as noted, below. Kathon™CG, when applied dermally to the closely clipped skin on the backs of male CD-1 mice at a concentration of 400 ppm active substance and at a dose of 25 µL 3 times per week for 30 months, showed no local or systemic tumorigenic potential. No adverse effects were seen on the histopathology of any tissues/organs distant from the site of dosing.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

mouse

Mode of Action Analysis / Human Relevance Framework

C(M)IT/MIT produced no evidence of carcinogenicity (ie., no treatmentrelated increase in the type or incidence of neoplasms in any group) up to the highest tested doses in these studies.

Justification for classification or non-classification

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