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

Endpoint summary

Description of key information

C(M)IT/MIT was tested in several oral repeated dose toxicity studies in rabbits, rats and dogs for 4 weeks and 3 months.

Two 90-day dermal repeated dose toxicity studies were performed with C(M)IT/MIT in rabbit and rat.

One 90 Day inhalation study in the rat was performed with CMIT/MIT.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-01-23 till 1994-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

GLP compliance:

yes

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products Inc, US
- Age at study initiation: about 7 months
- Weight at study initiation: 7.2 kg –10.9 kg (males) 7.05 kg – 9.4 kg (females)
- Fasting period before study:
- Housing: single sex room, over day individually, overnight together
- Diet (e.g. ad libitum): 400g SQC diet A (Speciel Diets Services Ltd., Witham) in the morning; amount not ingested weighed in afternoon or left overnight for access; due to low palatibility of diet, 200g extra meat offered at several incidences to animals with poor condition
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 3 months (several vaccinations)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22
- Humidity (%): 40-80
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): twice a week
- Mixing appropriate amounts with (Type of food): Special Diet (see Details on test animals)
- Storage temperature of food: RT in sealed container

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

none given; "analytical method HE 1154/57-01F"

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

7 d per week

Remarks:

Doses / Concentrations:
101 mg a.i./kg diet (nominal 150 ppm a.i.)
Basis:
other: recalculated from actual consumption, worst-case recovery

Remarks:

Doses / Concentrations:
363 mg a.i./kg diet (nominal 500 ppm a.i.)
Basis:
other: recalculated from actual consumption, worst-case recovery

Remarks:

Doses / Concentrations:
555 mg a.i./kg diet (nominal 750 ppm a.i.)
Basis:
other: recalculated from actual consumption, worst-case recovery

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

- Post-exposure period: none
- Dose selection rationale: prestudy CHE 1154/57

Positive control:

none

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily. Additional a detailed physical examination in weekly intervals.


BODY WEIGHT: Yes
- Time schedule for examinations:Individual body weights were recorded once weekly and before necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:pre-treatment and in week 12
- Dose groups that were examined:on all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on 2 occasions pre-dose and in weeks 6 and 13
- Animals fasted: Yes overnight
- How many animals: all
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:on 2 occasions pre-dose and in weeks 6 and 13
- Animals fasted: Yes overnight
- How many animals: all
- Parameters checked in table 1 were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table) / No / No data
HISTOPATHOLOGY: Yes (see table) / No / No data

Statistics:

The Body weight gains, food consumption intervals, and haematology and clinical chemistry variables were analysed using 2-way analysis of variance (ANOVA). Pairwise comparisons against the control, for each sex separately, were made using Dunnett's test.
For each variable and for each sex separately a regression test was performed to determine whether there was a linear relationship between increasing dose and response. Where this showed a significant result (P < 0.05) and any of the pairwise comparisons were also significant then the regression result was not reported.
Levene's test for equality of variances across groups, between sexes and for any interaction was also performed. When these tests showed evidence of group effects or a sex-group interaction (P<0.01), either the data were re-analysed using the same methods after applying a log-transformation, or using non-parametric methods for each sex separately. The methods used were the Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a dose-related trend and the Wilcoxon rank sum test for pairwise comparisons.
Where Levene's test showed evidence of heterogeneous variances between the sexes only (P<0.01), then a one-way ANOVA, regression test and Dunnett's test were performed for each sex separately.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY

There were no deaths during the study and the only clinical signs were as a result of reduced food consumption in certain animals
BODY WEIGHT AND WEIGHT GAIN

In both sexes there was a significant, dose related reduction in body weight gain over the first 4 weeks of treatment. In the Group 4 animals this led to a dose-related decrease in body weight gain which resulted in losses in absolute body weight
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

Group 2 (150 mg/kg/day) animals ate similar amounts of food to both the controls and pre-dose values over the treatment period.
In Group 3 (500 mg/kg/day) and Group 4 (750 mg/kg/day) there was a statistically significant reduction in food consumption, which was apparent by Week 1 and resulted in individual animals having food available overnight and having food supplementation. Over the remaining study period animals were closely monitored for food consumption and body condition, so by Week 11 no animals were receiving supplementation and all were an a normal feeding regime.
However, over the complete treatment period the Group 3 and 4 consumptions were still reduced by up to 25 % when compared to controls the reduction was statistically significant in the Group 4 animals.
The calculation for dietary test substance intake expressed as mg/kg bodyweight/day based around the worst case scenario noted in week 2, show ranges of dietary intake of 4-5, 9-20 and 12-31 mg/kg bodyweight/day for the treated groups in ascending order. The variation noted in Groups 3 and 4 relate to the changes observed in the food consumption values.
Mean test substance intake calculated on nominal dose level:
0, 6, 20, 30 mg a.i./kg bw./day
Test substance intake corrected for recovery*:
0, 4, 15, 22 mg a.i./kg bw./day
*: worst case recovery in week two diets.


OPHTHALMOSCOPIC EXAMINATION

no effects

HAEMATOLOGY

Although some inter-animal variation was apparent there were no changes of toxicological significance
CLINICAL CHEMISTRY

no effects


ORGAN WEIGHTS

Although some inter-animal variation and minor statistical significance were apparent there were no changes of toxicological significance
GROSS PATHOLOGY/HISTOPATHOLOGY: NON-NEOPLASTIC

There were no macroscopic or microscopic findings indicative of toxicity due to test article administration
There were no abnormalities detected at the visual assessment of the Bone marrow smears

Key result

Dose descriptor:

NOAEL

Effect level:

22 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no systemic effects

Critical effects observed:

not specified

Table 3: Average body weights and body weight gains during xx days of treatment

Dose rate (ppm)	Body Weights (kg)				Total Weight Gain
	Week -1	Week 4	Week 8	Week 13	kg	% of control
Male
0	9.33	10.18	10.4	10.85	1.53	100
Low	8.83	9.45	9.39	9.74	0.91	59
Mid	8.36	8.13	8.91	9.23	0.86	56
High	8.64	7.50	8.18	8.23	-0.41 ***	(negative)
Female
0	8.16	8.89	8.91	9.29	1.13	100
Low	7.93	8.48	8.70	9.09	1.16	103
Mid	8.03	7.85	8.13	8.44	0.41 *	36
High	7.86	7.43	7.66	7.84	-0.02 ***	(negative)

^a Data obtained from pages (41 -46)  in the study report.

*  Significantly different (p 0.05) from the control.

*** Significantly different (p 0.001) from the control.

Conclusions:

Observed effects of dietary Aciticide 14 administration on body weight and food consumption were most probably the result of the poor palatability of diets rather than any systemic toxicity. The NOAEL is thus 22 mg a.i./kg bw/day.

Executive summary:

The toxic potential of a13.9 % aqueous solution of a 3:1 mixture of 5 -chloro-2 -methyl-2H-isothiazol-3 -one and 2 -methyl-2H-isothiazol-3 -one in water (named Acticide 14 in this study report) was evaluated in a 90 day repeated dose dietary toxicity study in rats according to OECD guideline 409. Male and female beagle dogs were treated with the test item by dietary administration over a period of 90 days. The animals were observed for clinical signs, alterations in body weight and food consumption throughout the study period. At selected timepoints before and during the study, blood was collected for haematology and clinical chemistry. At the end of the treatment period, the animals were sacrificed and subjected to detailed macroscopic and microscopic pathological examination.

A dose-dependent loss of bodyweight and reduction in food consumption was observed, while all other observed alterations/abnormalities could not be related to treatment and were considered incidental. The applied doses could analytically not be verified, and thus the exposure doses of the test animals were calculated from the worst-case recovered values.

The observed effects on body weight gain were only seen at the two highest doses and were probably the result of the poor palatability of the diet rather than any toxic properties of Acticide 14. This view is supported by the palatability effects noted in a dose ranging study (CHE Study No 1154/57, pilot dietary study in dog) and by the fact that manipulation of the feeding regime, together with short periods of meat supplements, improved food consumption. This improvement would not have been observed if there had been any central depression of the appetite.

In conclusion, the effects an body weight and food consumption were most probably the result of the poor palatability of diets containing Acticide 14 rather than any systemic toxicity. It can therefore be concluded that there was no evidence of organ or systemic toxicity when Acticide 14 was offered in the diet at a analysed dose level up to 555 ppm (nominal concentration 750 ppm) which is equivalent to 22 mg ai/kg body weight/day (30  mg ai/kg body weight/day) to the laboratory beagle for up to 13 weeks.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

22 mg/kg bw/day

Study duration:

subchronic

Species:

dog

System:

gastrointestinal tract

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. SW 82/0169

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

OTHER SPECIFICS: Purity of test material was 14% (11% of CMIT and 3% of MIT).

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River-Kingston, Stone Ridge, New York, USA.
- Age at study initiation: Approximately 6 weeks old.
- Weight at study initiation: 135-169 g for males and 117 to 147 g for females.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

1.3 µm

Geometric standard deviation (GSD):

1.9

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2000-L stainless steel and glass chamber
- System of generating particulates/aerosols: all glass Laskin-type nebulizer

TEST ATMOSPHERE
- Brief description of analytical method used: pairs of impingers collected both the vapor and aerosol phases.

VEHICLE (if applicable)
- Concentration of test material in vehicle: 0.34, 1.15 and 2.64 mg a.i./m3

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The Kathon™ 886 concentrations were determined by sampling the chamber atmosphere using pairs of impingers which collected both the vapor and aerosol phases of Kathon™ 886.

Duration of treatment / exposure:

90 days

Frequency of treatment:

5 days per week, 6 hours per day

Dose / conc.:

0.34 mg/m³ air

Remarks:

Based on a.i. concentration

Dose / conc.:

1.15 mg/m³ air

Remarks:

Based on a.i. concentration

Dose / conc.:

2.64 mg/m³ air

Remarks:

Based on a.i. concentration

No. of animals per sex per dose:

16

Control animals:

yes

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily. Before, during and after each exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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: Yes
- Time schedule for examinations: Prior to study start and during the last week of exposure.
- Dose groups that were examined: All groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 13 week necropsy.
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- How many animals: 16/sex/group
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 13 week necropsy.
- How many animals: 16/sex/group
- Parameters checked in table [No.?] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No

Sacrifice and pathology:

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

Other examinations:

None

Statistics:

Body weight, body weight gain, clinical chemistry, hemaglobin and hematocrit were inspected for normality and homogeneity of variance by residual plots and then analyzed with a two-way ANOVA. If a significant p < 0.05 treatment group by sex effect were observed, group means were compared utilizing Dunnett’s t-test.
Hematology and differentials were inspected for normality and homogeneity of variance by stem leaf, boxplot and normal probability plots. If the ANOVA assumptions were satisfied, then the statistical analysis described above for the continuous data was utilized. When ANOVA assumptions were not satisfied, chi-square and Jonckheere tests were applied.
Pathology incidences were compared by Fischer’s Exact Test.
For all comparisons, the null hypothesis was rejected at a probability of 0.05 or less.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

rhinitis

Mortality:

mortality observed, treatment-related

Description (incidence):

rhinitis

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased body weight gains

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased serum protein

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Decreased spleen weights

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

eosinophilic droplets in the anterior respiratory mucosa of the nasal turbinates and slight rhinitis in the lining of the anterior portion of the nasal cavity

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Rats exposed to 2.64 mg/m3 exhibited signs resulting from exposure consistent with those produced by a sensory irritant (chromorhinorrhea, rhinorrhea, eye squint, bradypnea, dyspnea).

BODY WEIGHT AND WEIGHT GAIN
Decreased body weight gains at 2.64 mg/m3.

CLINICAL CHEMISTRY
Decreased serum protein in females at 2.64 mg/m3.

ORGAN WEIGHTS
Decreased male spleen weights at 2.64 mg/m3.

HISTOPATHOLOGY: NON-NEOPLASTIC
Slight to moderate incidences of eosinophilic droplets in the anterior respiratory mucosa of the nasal turbinates and slight rhinitis in the lining of the anterior portion of the nasal cavity were observed in the 2.64 mg/m3 treated animals. All the histopathologic changes were very minor, potentially reversible, and generally reflective of minimal tissue responses to a very mild, low-grade respiratory irritant. No adverse effects were seen on the histopathology of any tissues/organs distant from the site of dosing.

Key result

Dose descriptor:

NOAEL

Effect level:

0.34 mg/m³ air (analytical)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

1.15 mg/m³ air (analytical)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: overall effects clinical signs; gross pathology

Key result

Critical effects observed:

no

Conclusions:

NO(A)EL = 0.34 mg a.i./m3 (0.00034 mg/L). There were no systemic effects in this study. LOEL = 1.15 mg a.i./m3based on slight, treatment-related rhinitis. Rats at the highest dose (2.64 mg/m3) exhibited very mild, low grade respiratory irritation. No adverse effects on the histopathology of any tissues/organs distant from the site of dosing.

Executive summary:

OECD 413, subchronic inhalation toxicity, 90-day study with analytical confirmation of concentrations of Kathon™886.

LOEL = 1.15 mg a.i./m³based on slight, treatment-related rhinitis. There were no systemic effects in this study. Rats at the highest dose (2.64 mg/m³) exhibited very mild, low grade respiratory irritation. No adverse effects on the histopathology of any tissues/organs distant from the site of dosing.

NO(A)EL = 0.34 mg a.i./m3 (0.00034 mg/L).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2.36 mg/m³

Study duration:

subchronic

Species:

rat

System:

respiratory system: upper respiratory tract

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. SW 82/0169

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

OTHER SPECIFICS: Purity of test material was 14% (11% of CMIT and 3% of MIT).

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River-Kingston, Stone Ridge, New York, USA.
- Age at study initiation: Approximately 6 weeks old.
- Weight at study initiation: 135-169 g for males and 117 to 147 g for females.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

1.3 µm

Geometric standard deviation (GSD):

1.9

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2000-L stainless steel and glass chamber
- System of generating particulates/aerosols: all glass Laskin-type nebulizer

TEST ATMOSPHERE
- Brief description of analytical method used: pairs of impingers collected both the vapor and aerosol phases.

VEHICLE (if applicable)
- Concentration of test material in vehicle: 0.34, 1.15 and 2.64 mg a.i./m3

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The Kathon™ 886 concentrations were determined by sampling the chamber atmosphere using pairs of impingers which collected both the vapor and aerosol phases of Kathon™ 886.

Duration of treatment / exposure:

90 days

Frequency of treatment:

5 days per week, 6 hours per day

Dose / conc.:

0.34 mg/m³ air

Remarks:

Based on a.i. concentration

Dose / conc.:

1.15 mg/m³ air

Remarks:

Based on a.i. concentration

Dose / conc.:

2.64 mg/m³ air

Remarks:

Based on a.i. concentration

No. of animals per sex per dose:

16

Control animals:

yes

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily. Before, during and after each exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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: Yes
- Time schedule for examinations: Prior to study start and during the last week of exposure.
- Dose groups that were examined: All groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 13 week necropsy.
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- How many animals: 16/sex/group
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 13 week necropsy.
- How many animals: 16/sex/group
- Parameters checked in table [No.?] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No

Sacrifice and pathology:

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

Other examinations:

None

Statistics:

Body weight, body weight gain, clinical chemistry, hemaglobin and hematocrit were inspected for normality and homogeneity of variance by residual plots and then analyzed with a two-way ANOVA. If a significant p < 0.05 treatment group by sex effect were observed, group means were compared utilizing Dunnett’s t-test.
Hematology and differentials were inspected for normality and homogeneity of variance by stem leaf, boxplot and normal probability plots. If the ANOVA assumptions were satisfied, then the statistical analysis described above for the continuous data was utilized. When ANOVA assumptions were not satisfied, chi-square and Jonckheere tests were applied.
Pathology incidences were compared by Fischer’s Exact Test.
For all comparisons, the null hypothesis was rejected at a probability of 0.05 or less.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

rhinitis

Mortality:

mortality observed, treatment-related

Description (incidence):

rhinitis

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased body weight gains

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased serum protein

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Decreased spleen weights

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

eosinophilic droplets in the anterior respiratory mucosa of the nasal turbinates and slight rhinitis in the lining of the anterior portion of the nasal cavity

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Rats exposed to 2.64 mg/m3 exhibited signs resulting from exposure consistent with those produced by a sensory irritant (chromorhinorrhea, rhinorrhea, eye squint, bradypnea, dyspnea).

BODY WEIGHT AND WEIGHT GAIN
Decreased body weight gains at 2.64 mg/m3.

CLINICAL CHEMISTRY
Decreased serum protein in females at 2.64 mg/m3.

ORGAN WEIGHTS
Decreased male spleen weights at 2.64 mg/m3.

HISTOPATHOLOGY: NON-NEOPLASTIC
Slight to moderate incidences of eosinophilic droplets in the anterior respiratory mucosa of the nasal turbinates and slight rhinitis in the lining of the anterior portion of the nasal cavity were observed in the 2.64 mg/m3 treated animals. All the histopathologic changes were very minor, potentially reversible, and generally reflective of minimal tissue responses to a very mild, low-grade respiratory irritant. No adverse effects were seen on the histopathology of any tissues/organs distant from the site of dosing.

Key result

Dose descriptor:

NOAEL

Effect level:

0.34 mg/m³ air (analytical)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

1.15 mg/m³ air (analytical)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: overall effects clinical signs; gross pathology

Key result

Critical effects observed:

no

Conclusions:

NO(A)EL = 0.34 mg a.i./m3 (0.00034 mg/L). There were no systemic effects in this study. LOEL = 1.15 mg a.i./m3based on slight, treatment-related rhinitis. Rats at the highest dose (2.64 mg/m3) exhibited very mild, low grade respiratory irritation. No adverse effects on the histopathology of any tissues/organs distant from the site of dosing.

Executive summary:

OECD 413, subchronic inhalation toxicity, 90-day study with analytical confirmation of concentrations of Kathon™886.

LOEL = 1.15 mg a.i./m³based on slight, treatment-related rhinitis. There were no systemic effects in this study. Rats at the highest dose (2.64 mg/m³) exhibited very mild, low grade respiratory irritation. No adverse effects on the histopathology of any tissues/organs distant from the site of dosing.

NO(A)EL = 0.34 mg a.i./m3 (0.00034 mg/L).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2.36 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-09-02 till 1994-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 131-203 g (males) and 137-183 g (females)
- Housing: individually on autoclaved sawdust
- Diet (e.g. ad libitum): Ssniff R10 pelleted diet ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-25°C
- Humidity (%): 30-75 %
- Air changes (per hr): appr. 10
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: 5x7 cm on back and flanks
- Type of wrap if used: semiocclusive (Idealhaft, Paul Hartmann AG, 7920 Heidenheim, Germany)
- Time intervals for shavings or clipplings: weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done): warm water (no detergent)
- Time after start of exposure: 6 h


TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
1738, 8688 , 43438 ppm a.i.
- Concentration (if solution): 13.9%
- Constant volume or concentration used: yes



VEHICLE aqua bidest
- Justification for use and choice of vehicle (if other than water): Since the scheduled dose volumes were too small to be administered, the following dilutions of Acticide 14 (13.9%) were prepared once weekly throughout the study: 1:80; 1:16; 1:3.2
- Amount(s) applied (volume or weight with unit):60 µl/kg bw.


USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Duration of treatment / exposure:

90 days

Frequency of treatment:

once a day, 6h/d, semi-occlusive dressing

Remarks:

Doses / Concentrations:
0.75 mg Acticide 14/kg bw/day (=0.105 mg a.i./kg bw/day)
Basis:
nominal per unit body weight

Remarks:

Doses / Concentrations:
3.75mg Acticide 14/kg bw/day (=0.525 mg a.i./kg bw/day)
Basis:

Remarks:

Doses / Concentrations:
18.75mg Acticide 14/kg bw/day (=2.625 mg a.i./kg bw/day)
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure period: none
- Dose selection rationale: The dose levels were selected on the basis of a 14-day dose range-finding study in the rat (HD Project No. 1154-001)

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: All animals were examined twice daily at the beginning and end of the working day for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined at least once daily for signs of ill health or overt signs of toxicity and each finding was recorded


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations:For all animals, evaluation of cutaneous reactions was performed once a week in the morning before dosing according to the scale in appendix I of the study protocol


BODY WEIGHT: Yes
- Time schedule for examinations:The body weight of the male and female animals was recorded once a week during the treatment period and on the day of necropsy


FOOD CONSUMPTION:
- The food consumption of the males and females was recorded twice weekly during the treatment period and evaluated on a weekly basis


FOOD EFFICIENCY: No


WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:The eyes of all animals of each dose group were examined during the final week of treatment
Before the examination, a mydriatic agent (mydriaticum "Roche"®,Hoffmann-La Roche Aktiengesellschaft, 79639 Grenzach-Wyhlen, Germany) was instilled into the eyes. The following parameters were examined: ocular fundus with macula lutea, papilla and ocular vessels.


HAEMATOLOGY: Yes
- Time schedule for collection of blood:at the end of the treatment before necropsy
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes overnight fast (about 16 hours)
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:at the end of the treatment before necropsy
- Animals fasted: Yes overnight fast (about 16 hours)
- Parameters checked in table 1 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine:at the end of the treatment before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes overnight fast (about 16 hours)
- Parameters checked in table 1 were examined.


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Necropsies: Animals were killed by an intraperitoneal injection of pentobarbitone sodium (Eutha 77®,Coopers Tierarzneimittel GmbH, 30938 Burgwedel, Germany) followed by immediate exsanguination. The necropsies were conducted an 1 day. Animals were sacrificed in randomized order according to the cage plan. All animals were examined externally including all orifices. A full macroscopic examination of all tissues and organs in situ was then performed.

GROSS PATHOLOGY: Yes (see table2)
HISTOPATHOLOGY: Yes (see table2)

Statistics:

body weight, body weight change and food consumption: Levene's testl for homogeneity of variances , followed by a rank transformation and the Levene's test in the case of heterogeneity only (p 5_ 0.05, equivalent to 95% confidence level) and the one-way Analysis of Variancel,(ANOVA). If significant results for the ANOVA (p 5_ 0.05 and p 0.01), Dunnett's two-tailed t-test was used to compare each group against the control group.
organ weights: Bartlett's test for homogeneity of variances was performed, followed by a rank transformation and the Bartlett's testl in the case of heterogeneity only (p 5_0.05, equivalent to 95 per cent probability). For homogeneous data, the one-way Analysis of Variancel, (ANOVA) was performed. In the event of significant results for the ANOVAI, (p 0.05, equivalent to 95 per cent probability), by the Dunnett's1,3 two-tailed t-test was used to compare each treated group against the control group. In the case of heterogeneity of the rank-transformed data, the Kruskal-Wallis testl,2,4 was performed together with the Wilcoxon rank-sum test to compare each treated group against the control group.
hematology data, clinical chemistry and organ/body weight ratio: rank transformation followed by the Bartlett's test for homogeneity of variances. For homogeneous data, the one-way Analysis of Variancel, (ANOVA) was performed, followed in the case of significant results (p 5_0.05, equivalent to 95 per cent probability), by the Dunnett's two-tailed t-test was used to compare each treated group against the control group. In the case of heterogeneity of the rank-transformed data, the Kruskal-Wallis test was performed together with the Wilcoxon rank-sum test to compare each treated group against the control group.
Results of the ANOVA and all significances found (at least p <_ 0.05 or p<0.01) are presented in the respective tables.
The statistical evaluation was performed with the standard package SAS(Statistical Analysis System) release 6.042

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY

Two control animals (4M, 44F)) and one high-dose animal (33M) were found dead an days 12,3 and 3 respectively. These mortalities are considered to be incidental and not related to the application of the test material. The high-dose animal and the control female were found dead when the bandages were removed and it is thought that the cause of death was that the bandage was bound too tightly. The cause of death in the control male was probably a focal pneumonitis
No treatment-related clinical changes throughout the treatment period that could be ascribed to the test article.
On a few occasions, kinked tail, incrusted eyes, pinched off teeth, injuries at skull or ears, fur staining, lesions of skin and red fluid in the bedding material were recorded. These findings are considered to be unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN

There were no adverse effects on body weight gain in animals of either sex.

FOOD CONSUMPTION

There were no adverse effects on overall food consumption throughout the experimental period in male and female animals

OPHTHALMOSCOPIC EXAMINATION

There were no treatment-related findings

HAEMATOLOGY

Although statistical evaluation revealed a few significantly different minimal changes, there were no treatment-related findings observed at the end of the experimental period

CLINICAL CHEMISTRY

Although statistical evaluation revealed a few significantly different minimal changes, there were no treatment-related findings observed at the end of the experimental period

URINALYSIS

There were no treatment-related urine analysis findings at the end of the experimental period


ORGAN WEIGHTS

Although there were a few significant values for single organs in different groups, there were no apparent organ weight changes in animals of either sex when compared with the control group.

GROSS PATHOLOGY

Skin observations: see table 3
Male animals
There were no skin lesions in group 2, minimal individual reactions of single animals in group 3 and numerous individual reactions in the majority of all high-dose males throughout the experimental period.
Cutaneous reactions observed at the high dose level included, above all, slight to moderate erythema and desquamation, slight edema and atonia as well as eschar formation. Fissures and exfoliation were not observed in any animal.Group mean total score for local reactions revealed only slight cutaneous reactions (grade: 0.3) for high-dose males.
Female animals
There were minimal individual skin reactions in single group 2 animals, few reactions in some group 3 animals and numerous individual reactions in the majority of all high-dose females throughout the experimental period.
Cutaneous reactions observed included, above all, dose-related slight to moderate erythema and desquamation, slight edema and atonia as well as eschar formation. Fissures and exfoliation were not observed in any animal.
Group mean total score for local reactions revealed only minimal changes for group 3 (grade: 0.1) and slight changes for high-dose females (grade: 0.4).
General Pathology:
There were no macroscopic lesions in any of the organs or tissues examined that could be ascribed to the test article Acticide 14.
The only treatment-related findings were compound-related lesions such as inflammation, parakeratosis and acanthosis in the treated skin sites of males and females.
There were no histopathological lesions in the other organs and tissues suggestive of systemic target organ toxicity due to the test article.
No treatment-related changes were found in decedents and a relationship between the causes of death and test article toxicity could not be established. The cause of death of the male group 1 (4M) was probably a focal pneumonitis. In the other two animals (44F, group 1 and 33M, group 4) the cause of death was probably due to stress or other unspecified causes.



HISTOPATHOLOGY: NON-NEOPLASTIC


HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

2.625 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

0.105 mg/kg bw/day

Sex:

male

Basis for effect level:

other: local skin reactions

Dose descriptor:

LOAEL

Remarks:

(local)

Effect level:

0.525 mg/kg bw/day

Sex:

male

Basis for effect level:

other: skin reactions

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

other: none observed

Sex:

female

Basis for effect level:

other: skin reactions

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

table 3: skin abnormalities

	Males				Females
mg/kg bw/d	0	0.75	3.75	18.75	0	0.75	3.75	18.75
Erythema	0.0	0.0	0.0	0.6	0.0	0.1	0.4	1.0
Edema	0.0	0.0	0.0	0.2	0.0	0.0	0.0	0.3
Atonia	0.0	0.0	0.0	0.1	0.0	0.0	0.0	0.1
Desquamation	0.0	0.0	0.0	0.7	0.0	0.0	0.1	0.8
Fissures	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
% Eschar	0	0	0	60	0	2	7	75
% Exfoliation	0	0	0	0	0	0	0	0
Group mean total score	0.0	0.0	0.0	0.3	0.0	0.0	0.1	0.4

Conclusions:

In the absence of any organ or systemic toxicity, the dermal treatment-related effects of Acticide 14 were limited to local skin reactions.
NOAEL (systemic toxicity): 18.75 mg/kg bw/day (=2.625 mg a.i./kg bw/day)
NOAEL (local irritation): 0.75 mg/kg bw/day (males; =0.105 mg a.i./kg bw/day)/none observed (females)

Executive summary:

The toxic potential of a13.9 % aqueous solution of a 3:1 mixture of 5 -chloro-2 -methyl-2H-isothiazol-3 -one and 2 -methyl-2H-isothiazol-3 -one in water (named Acticide 14 in this study report) was evaluated in a 90 day repeated dose dermal toxicity study in rats according to EPA OPP 82 -3 guideline. Male and female Sprague-Dawley rats were treated with the test item on exposed skin daily for 6 hours over a period of 90 days. The test article was kept in place and prevented from oral ingestion by means of a semi-occlusive dressing for exposure and remainders of the test item were then removed with water. The animals were observed for mortality, clinical signs, body weight gain and food consumption. At the end of the tretament period, blood and urine were collected for haematology and clinical chemistry. The animals were subjected to detailed macroscopic and microscopic pathological evaluation, including scoring of observed skin abnormalities.

Mortalities observed in two control animals and one high-dose male are considered to be incidental and not related to the application of the test material. Treatment with the test article Acticide 14 applied dermally to intact skin produced skin reactions (slight to moderate erythema and desquamation, slight edema and atonia as well as eschar formation) with dose-dependent grades of severity. Females appeared to be more sensitive than males. There were no other effects at the end of the treatment period that could be attributed to ACTICIDE 14.

NOAEL (local, male rat): 0.75 mg Acticide 14/kg bw/day

NOAEL (local, female rat): none observed

NOAEL (systemic): 18.75 mg Acticide 14/kg bw/day

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.1 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

other: Skin

Organ:

skin

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-09-02 till 1994-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 131-203 g (males) and 137-183 g (females)
- Housing: individually on autoclaved sawdust
- Diet (e.g. ad libitum): Ssniff R10 pelleted diet ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-25°C
- Humidity (%): 30-75 %
- Air changes (per hr): appr. 10
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: 5x7 cm on back and flanks
- Type of wrap if used: semiocclusive (Idealhaft, Paul Hartmann AG, 7920 Heidenheim, Germany)
- Time intervals for shavings or clipplings: weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done): warm water (no detergent)
- Time after start of exposure: 6 h


TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
1738, 8688 , 43438 ppm a.i.
- Concentration (if solution): 13.9%
- Constant volume or concentration used: yes



VEHICLE aqua bidest
- Justification for use and choice of vehicle (if other than water): Since the scheduled dose volumes were too small to be administered, the following dilutions of Acticide 14 (13.9%) were prepared once weekly throughout the study: 1:80; 1:16; 1:3.2
- Amount(s) applied (volume or weight with unit):60 µl/kg bw.


USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Duration of treatment / exposure:

90 days

Frequency of treatment:

once a day, 6h/d, semi-occlusive dressing

Remarks:

Doses / Concentrations:
0.75 mg Acticide 14/kg bw/day (=0.105 mg a.i./kg bw/day)
Basis:
nominal per unit body weight

Remarks:

Doses / Concentrations:
3.75mg Acticide 14/kg bw/day (=0.525 mg a.i./kg bw/day)
Basis:

Remarks:

Doses / Concentrations:
18.75mg Acticide 14/kg bw/day (=2.625 mg a.i./kg bw/day)
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure period: none
- Dose selection rationale: The dose levels were selected on the basis of a 14-day dose range-finding study in the rat (HD Project No. 1154-001)

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: All animals were examined twice daily at the beginning and end of the working day for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined at least once daily for signs of ill health or overt signs of toxicity and each finding was recorded


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations:For all animals, evaluation of cutaneous reactions was performed once a week in the morning before dosing according to the scale in appendix I of the study protocol


BODY WEIGHT: Yes
- Time schedule for examinations:The body weight of the male and female animals was recorded once a week during the treatment period and on the day of necropsy


FOOD CONSUMPTION:
- The food consumption of the males and females was recorded twice weekly during the treatment period and evaluated on a weekly basis


FOOD EFFICIENCY: No


WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:The eyes of all animals of each dose group were examined during the final week of treatment
Before the examination, a mydriatic agent (mydriaticum "Roche"®,Hoffmann-La Roche Aktiengesellschaft, 79639 Grenzach-Wyhlen, Germany) was instilled into the eyes. The following parameters were examined: ocular fundus with macula lutea, papilla and ocular vessels.


HAEMATOLOGY: Yes
- Time schedule for collection of blood:at the end of the treatment before necropsy
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes overnight fast (about 16 hours)
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:at the end of the treatment before necropsy
- Animals fasted: Yes overnight fast (about 16 hours)
- Parameters checked in table 1 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine:at the end of the treatment before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes overnight fast (about 16 hours)
- Parameters checked in table 1 were examined.


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Necropsies: Animals were killed by an intraperitoneal injection of pentobarbitone sodium (Eutha 77®,Coopers Tierarzneimittel GmbH, 30938 Burgwedel, Germany) followed by immediate exsanguination. The necropsies were conducted an 1 day. Animals were sacrificed in randomized order according to the cage plan. All animals were examined externally including all orifices. A full macroscopic examination of all tissues and organs in situ was then performed.

GROSS PATHOLOGY: Yes (see table2)
HISTOPATHOLOGY: Yes (see table2)

Statistics:

body weight, body weight change and food consumption: Levene's testl for homogeneity of variances , followed by a rank transformation and the Levene's test in the case of heterogeneity only (p 5_ 0.05, equivalent to 95% confidence level) and the one-way Analysis of Variancel,(ANOVA). If significant results for the ANOVA (p 5_ 0.05 and p 0.01), Dunnett's two-tailed t-test was used to compare each group against the control group.
organ weights: Bartlett's test for homogeneity of variances was performed, followed by a rank transformation and the Bartlett's testl in the case of heterogeneity only (p 5_0.05, equivalent to 95 per cent probability). For homogeneous data, the one-way Analysis of Variancel, (ANOVA) was performed. In the event of significant results for the ANOVAI, (p 0.05, equivalent to 95 per cent probability), by the Dunnett's1,3 two-tailed t-test was used to compare each treated group against the control group. In the case of heterogeneity of the rank-transformed data, the Kruskal-Wallis testl,2,4 was performed together with the Wilcoxon rank-sum test to compare each treated group against the control group.
hematology data, clinical chemistry and organ/body weight ratio: rank transformation followed by the Bartlett's test for homogeneity of variances. For homogeneous data, the one-way Analysis of Variancel, (ANOVA) was performed, followed in the case of significant results (p 5_0.05, equivalent to 95 per cent probability), by the Dunnett's two-tailed t-test was used to compare each treated group against the control group. In the case of heterogeneity of the rank-transformed data, the Kruskal-Wallis test was performed together with the Wilcoxon rank-sum test to compare each treated group against the control group.
Results of the ANOVA and all significances found (at least p <_ 0.05 or p<0.01) are presented in the respective tables.
The statistical evaluation was performed with the standard package SAS(Statistical Analysis System) release 6.042

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY

Two control animals (4M, 44F)) and one high-dose animal (33M) were found dead an days 12,3 and 3 respectively. These mortalities are considered to be incidental and not related to the application of the test material. The high-dose animal and the control female were found dead when the bandages were removed and it is thought that the cause of death was that the bandage was bound too tightly. The cause of death in the control male was probably a focal pneumonitis
No treatment-related clinical changes throughout the treatment period that could be ascribed to the test article.
On a few occasions, kinked tail, incrusted eyes, pinched off teeth, injuries at skull or ears, fur staining, lesions of skin and red fluid in the bedding material were recorded. These findings are considered to be unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN

There were no adverse effects on body weight gain in animals of either sex.

FOOD CONSUMPTION

There were no adverse effects on overall food consumption throughout the experimental period in male and female animals

OPHTHALMOSCOPIC EXAMINATION

There were no treatment-related findings

HAEMATOLOGY

Although statistical evaluation revealed a few significantly different minimal changes, there were no treatment-related findings observed at the end of the experimental period

CLINICAL CHEMISTRY

Although statistical evaluation revealed a few significantly different minimal changes, there were no treatment-related findings observed at the end of the experimental period

URINALYSIS

There were no treatment-related urine analysis findings at the end of the experimental period


ORGAN WEIGHTS

Although there were a few significant values for single organs in different groups, there were no apparent organ weight changes in animals of either sex when compared with the control group.

GROSS PATHOLOGY

Skin observations: see table 3
Male animals
There were no skin lesions in group 2, minimal individual reactions of single animals in group 3 and numerous individual reactions in the majority of all high-dose males throughout the experimental period.
Cutaneous reactions observed at the high dose level included, above all, slight to moderate erythema and desquamation, slight edema and atonia as well as eschar formation. Fissures and exfoliation were not observed in any animal.Group mean total score for local reactions revealed only slight cutaneous reactions (grade: 0.3) for high-dose males.
Female animals
There were minimal individual skin reactions in single group 2 animals, few reactions in some group 3 animals and numerous individual reactions in the majority of all high-dose females throughout the experimental period.
Cutaneous reactions observed included, above all, dose-related slight to moderate erythema and desquamation, slight edema and atonia as well as eschar formation. Fissures and exfoliation were not observed in any animal.
Group mean total score for local reactions revealed only minimal changes for group 3 (grade: 0.1) and slight changes for high-dose females (grade: 0.4).
General Pathology:
There were no macroscopic lesions in any of the organs or tissues examined that could be ascribed to the test article Acticide 14.
The only treatment-related findings were compound-related lesions such as inflammation, parakeratosis and acanthosis in the treated skin sites of males and females.
There were no histopathological lesions in the other organs and tissues suggestive of systemic target organ toxicity due to the test article.
No treatment-related changes were found in decedents and a relationship between the causes of death and test article toxicity could not be established. The cause of death of the male group 1 (4M) was probably a focal pneumonitis. In the other two animals (44F, group 1 and 33M, group 4) the cause of death was probably due to stress or other unspecified causes.



HISTOPATHOLOGY: NON-NEOPLASTIC


HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

2.625 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

0.105 mg/kg bw/day

Sex:

male

Basis for effect level:

other: local skin reactions

Dose descriptor:

LOAEL

Remarks:

(local)

Effect level:

0.525 mg/kg bw/day

Sex:

male

Basis for effect level:

other: skin reactions

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

other: none observed

Sex:

female

Basis for effect level:

other: skin reactions

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

table 3: skin abnormalities

	Males				Females
mg/kg bw/d	0	0.75	3.75	18.75	0	0.75	3.75	18.75
Erythema	0.0	0.0	0.0	0.6	0.0	0.1	0.4	1.0
Edema	0.0	0.0	0.0	0.2	0.0	0.0	0.0	0.3
Atonia	0.0	0.0	0.0	0.1	0.0	0.0	0.0	0.1
Desquamation	0.0	0.0	0.0	0.7	0.0	0.0	0.1	0.8
Fissures	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
% Eschar	0	0	0	60	0	2	7	75
% Exfoliation	0	0	0	0	0	0	0	0
Group mean total score	0.0	0.0	0.0	0.3	0.0	0.0	0.1	0.4

Conclusions:

In the absence of any organ or systemic toxicity, the dermal treatment-related effects of Acticide 14 were limited to local skin reactions.
NOAEL (systemic toxicity): 18.75 mg/kg bw/day (=2.625 mg a.i./kg bw/day)
NOAEL (local irritation): 0.75 mg/kg bw/day (males; =0.105 mg a.i./kg bw/day)/none observed (females)

Executive summary:

The toxic potential of a13.9 % aqueous solution of a 3:1 mixture of 5 -chloro-2 -methyl-2H-isothiazol-3 -one and 2 -methyl-2H-isothiazol-3 -one in water (named Acticide 14 in this study report) was evaluated in a 90 day repeated dose dermal toxicity study in rats according to EPA OPP 82 -3 guideline. Male and female Sprague-Dawley rats were treated with the test item on exposed skin daily for 6 hours over a period of 90 days. The test article was kept in place and prevented from oral ingestion by means of a semi-occlusive dressing for exposure and remainders of the test item were then removed with water. The animals were observed for mortality, clinical signs, body weight gain and food consumption. At the end of the tretament period, blood and urine were collected for haematology and clinical chemistry. The animals were subjected to detailed macroscopic and microscopic pathological evaluation, including scoring of observed skin abnormalities.

Mortalities observed in two control animals and one high-dose male are considered to be incidental and not related to the application of the test material. Treatment with the test article Acticide 14 applied dermally to intact skin produced skin reactions (slight to moderate erythema and desquamation, slight edema and atonia as well as eschar formation) with dose-dependent grades of severity. Females appeared to be more sensitive than males. There were no other effects at the end of the treatment period that could be attributed to ACTICIDE 14.

NOAEL (local, male rat): 0.75 mg Acticide 14/kg bw/day

NOAEL (local, female rat): none observed

NOAEL (systemic): 18.75 mg Acticide 14/kg bw/day

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.1

Study duration:

subchronic

Species:

rat

Mode of Action Analysis / Human Relevance Framework

CMIT/MIT causes local toxicity at the site of contact e.g. irritation/corrosion of the forestomach, skin or upper respiratory tract following repeated exposure via oral, dermal or inhalation routes. Seconndary toxicityies may be observed which are typically bodyweight reductions and effects on haematological parameters as a result of primary local toxicity.

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 repeat dose effects.