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

Repeated dose toxicity: oral

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.