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

Repeated dose toxicity: dermal

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

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

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

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)

Examinations

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

Results and discussion

Results of examinations

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

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

2.625 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: systemic toxicity

2

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

0.105 mg/kg bw/day

Sex:

male

Basis for effect level:

other: local skin reactions

3

Dose descriptor:

LOAEL

Remarks:

(local)

Effect level:

0.525 mg/kg bw/day

Sex:

male

Basis for effect level:

other: skin reactions

4

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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