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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-11-03 till 1994-01-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: .

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 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

One non-pregnant female inadvertently sacrified on day 17 p.c. On day 10 p.c. 2 animals incorrectly dosed (dosage appr. 4 % higher).One female rat of the control group, which littered, had a body weight of 262 g due to pregnancy.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Sprague Dawley Crl: CD (SD)BR, from “specific pathogen free" colony
- Source: Charles River Wiga GmbH, 97633 Sulzfeld, Germany
following data only for females; males were not treated with the test article and were used for mating only
- Age at study initiation: : 8 to 12 weeks
- Weight at study initiation: 180 to 238 g
- Housing: individually in macrolon III-cages on autoclaved sawdust
- Diet (e.g. ad libitum): ad lib. Ssniff R10 standard diet
- Water (e.g. ad libitum): ad lib. tap water
- Acclimation period: 7 days prior to mating


ENVIRONMENTAL CONDITIONS
- Temperature (°C): (17) 19-25°C
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: daily


VEHICLE

aqua bidestillata

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

first and last week of treatment; analysed by HPLC-UV;
result: analytical conc. =92-98% of nominal dose

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:4
- Length of cohabitation: during the night
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation

Duration of treatment / exposure:

10 days, from day 6 - 15 post-coitum; 5 days postexposure-period;

Frequency of treatment:

daily for 10 consecutive days

Duration of test:

(23 d in total)

No. of animals per sex per dose:

25 inseminated females per group

Control animals:

yes, concurrent vehicle

Details on study design:

day 20 post-coitum: sacrificed

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations:Individually for days 0, 6, 9, 12, 16 and 20 post-coitum

FOOD CONSUMPTION: Yes Individually for the intervals from day 0-6, 6-9, 9-12, 12-16 and 16-20 post-coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: only macroscopically for changes

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of life/dead fetuses: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Intra-uterine deaths were classified as follows:
Early resorptions showed decidual or placental tissues only.
Late resorptions showed embryonic or fetal tissue in addition to placental tissue but did not include fetuses dying in utero within approximately 2 days prior to the terminal kill.
Dead fetuses included only the fetuses dying in utero within approximately the last 2 days.
The uteri of apparently non-pregnant females were immersed in a 10 per cent solution of ammonium sulphide to reveal evidence of implantation (Salewski technique).

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No data
- Sex

Approximately half of the fetuses from each litter were eviscerated and the carcasses processed for skeletal examination (Alizarin staining technique).
The remaining half was fixed in ethanol and examined for visceral abnormalities using a modified Wilson-Barrow technique.
Dead fetuses were evaluated separately, if applicable. Structural deviations were classified as follows:
Malformation: rare and/or probably lethal, e.g. hydrocephaly.
Variation: changes which regularly occur also in control groups and which are not of functional significance.
In one litter each from group 2 (animal 45) and 4 (animal 94) all fetusses were determined for visceral examination were inadvertently eviscerated. Therefore only visceral examination of the heads was possible.

Statistics:

body weight, body weight change and food consumption: Levene's test for homogeneity of variances followed by rank transformation and the Levene's test in the case of heterogeneity only (p < 0.05, equivalent to 95 per cent confidence level) and ANOVA with Dunnett's two-tailed t-test to compare each treated group against the control group.
litter weight: the Bartlett's test for homogeneity of variance followed by rank transformation andBartlett's test in the case of heterogeneity only (p < 0.05, equivalent to 95 per cent confidence level) ;IF homogen: ANOVAwith Dunnett's two-tailed t-test was used to compare each treated group against the control group; IF heterogen Kruskal-Wallis test with Wilcoxon rank-sum test to compare each treated group against the control group.
number of corpora lutea, number of implantations, pre-implantation loss, total intra-uterine deaths, post-implantation loss, number of live fetuses and proportion of male fetuses: rank transformation followed by the Bartlett's test for homogeneity of variances. IF homogeneous: ANOVA, followed in the case of significant results (p < 0.05, equivalent to 95 per cent confidence level) by the Dunnett's two-tailed t-test to compare each treated group against the control group. IF heterogen: rank transformed data the Kruskal-Wallis test together with the Wilcoxon rank-sum test to compare each treated group against the control group.
mean fetal: rank transformation followed by the Bartlett's test for homogeneity of variances ANCOVA (covariate: number of fetuses). with Dunnett's two-tailed t-test was used to compare each treated group against the control group.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No mortalities were observed in this study.
CLINICAL OBSERVATIONS
Treatment-related clinical signs were observed in groups 3 (70 mg/kg) and 4 (139 mg/kg).
In group 3 (70 mg/kg), two animals showed gasping and/or wheezing respiration shortly after dosing an 1 or 2 days at the beginning of the treatment period. During the further course of treatment no compound-related clinical signs were observed.
In group 4 (139 mg/kg), six animals showed gasping and/or wheezing respiration shortly after dosing for 1 to 5 days. Mostly these signs were observed at the beginning of treatment but two animals showed them also during the second half of the treatment period.
In group 2 (28 mg/kg), no treatment-related clinical signs were observed.
BODY WEIGHT
Group mean body weight gain was slightly affected by treatment with Acticide 14 in groups 2 (28 mg/kg) and 3 (70 mg/kg), and moderately affected in group 4 (139 mg/kg).
In groups 2 (28 mg/kg) and 3 (70 mg/kg), mean body weight gain was slightly reduced from day 6 to 9 post-coitum. During the further course of treatment it was comparable to that in the control group. Evaluation for the entire treatment-period as well as for the entire study revealed a slight reduction of mean body weight gain in these groups.
In group 4 (139 mg/kg), mean body weight gain was moderately and statistically significantly reduced from day 6 to 9 post-coitum and slightly reduced from day 12 to 16 post-coitum. In consequence, mean body weight gain was also reduced from day 6 to 16 post-coitum and from day 0 to 20 post-coitum.
FOOD CONSUMPTION
A minimal to slight effect of treatment with Acticide 14 an mean daily food consumption was observed in all dose groups.
In groups 2 (28 mg/kg) and 3 (70 mg/kg), mean daily food consumption was minimally lower than the control group from day 6 to 9 post-coitum, which is considered to be treatment related. During the further course of the study food consumption in these groups was comparable to that in the control group.
In group 4 (139 mg/kg), mean daily food consumption was slightly reduced from day 6 to 9 post-coitum and slightly and statistically significantly reduced from day 12 to 16 post-coitum. Consequently, evaluation for the entire treatment-period as well as the entire study revealed a slight reduction of food consumption in this group.
NECROPSY FINDINGS
Necropsy did not reveal any treatment-related findings. In one animal each from group 3 (70 mg/kg) and 4 (139 mg/kg) findings in the kidneys and/or liver were detected. These single observations are considered to be incidental and not attributable to treatment with Acticide 14.

Effect levels (maternal animals)

Dose descriptor:

LOAEL

Effect level:

28 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
IMPLANTATION
No effect of treatment with Acticide 14 on implantation data was observed. The pre-implantation loss was comparable and within the normal range in all groups.
POST-IMPLANTATION LOSS
The post-implantation loss was not affected by treatment with Acticide 14. The observed inter-group differences are considered to be incidental.
NUMBER, SEX AND WEICHT OF THE FETUSES
No effect of treatment with Acticide 14 was observed on number, sex and weight of the fetuses.
The mean number of fetuses per litter was comparable in all groups.
The fetal sex distribution in the dose groups was comparable to that in the control group.
The mean fetal weight was similar in all groups.
FETAL DEFECTS
Fetal examination did not reveal any treatment-related malformations or variations. External, visceral and skeletal malformations were found in all groups including the control group.
In group 2 (28 mg/kg), two malformed fetuses were found in two different litters. In one fetus a single common ventricle of the heart was detected at visceral examination. The second fetus showed skeletally absent rib(s).
In group 3 (70 mg/kg), three fetuses with malformations were detected in three different litters. In two fetuses malformations such as asplenia or anorchism were detected at visceral examination. Externally, the third fetus showed microtia and anasarca. Visceral examination of this fetus did not reveal any further findings.
In group 4 (139 mg/kg), two malformed fetuses were found in two different litters. One fetus showed microsplenia at visceral examination. Externally, the second fetus showed agnathia. Visceral examination of this fetus revealed malformations such as anophthtalmia, aglossia and nares non-patent.
One control fetus showed anal atresia as an external malformation.
Visceral or skeletal variations were detected in fetuses of all study groups.
The type and the incidence of the observed malformations and variations did not show a dose-relationship and did not indicate any effect of treatment with Acticide 14.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

>= 19.6 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 19.6 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Administration of Acticide 14 to pregnant rats during the phase of organogenesis did not result to observation of any signs of teratogenicity or embryotoxicity.

Executive summary:

The potential of a 14% aqueous solution of 3 parts 5 -chloro- 2 -methyl-2H-isothiazol-3 -one and 1 part 2 -methyl-2H-isothiazol-3 -one (cited as Acticide 14 in the report) to induce teratogenic effects in rats was evaluated in a study according to guideline EPA OPP 83 -3. Pregnant female Sprague-Dawley rats were treated with the test substance by oral gavage during the period of organogenesis (days 6 -15 post coitum). Animals were observed for mortality, signs of toxicity, food consumption and body weight gain during the treatment and a post-exposure period of 5 days. At day 20 of gestation, animals were sacrificed and examined for macroscopic pathological abnormalities. Uterine contents were examined for signs abnormal pregnancy courses, and fetuses were examined for external, visceral and skelettal abnormalities.

Treatment with the test article resulted in maternal toxicity with clearly distingished dose-dependent grades of severity (clinical signs, moderately reduced body weight gain, slightly reduced food consumption). In spite of the observed adverse maternal effects, treatment with the test article did not have any influence on the embryonic and fetal development, as there was no embryotoxicity and no teratogenicity detected in any of the dose groups.