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:

13 January - 31 January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

other: Clear liquid, colorless to brown

Details on test material:

The test article, 2-methyl-4-isothiazolin-3-one (supplied as a 50% solution in water known as Kordek 573 Industrial Microbiocide), was received from Rohm and Haas Company, Spring House, PA on November 13, 2001. In a GLP certificate of analysis, the purity was found to be 51.4%.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

One hundred twenty six sexually mature, virgin female Crl:CD (SD)IGS BR rats were received in good health from Charles River Laboratories, Inc., Raleigh, North Carolina, on 23 December 2002. The animals were approximately 70 days old. Upon receipt, each female was examined by a qualified technician. The rats were initially weighed on 23 December 2002. Each rat was uniquely identified by a Monel metal eartag displaying the animal number and housed for 15 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and moribundity. Body weights were recorded prior to initiation of breeding.

Upon arrival and until pairing, all rats were individually housed in clean, wire-mesh cages (27 cm L x 25 cm W x 18 cm H) suspended above cage-board. The cage-board was changed at least three times per week. The rats were paired for mating in the home cage of the male. Following positive identification of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required as the females were euthanized prior to the date of expected parturition.

The basal died used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research Laboratories, Inc. Municipal water supplying the facility is sampled for contaminants according to standard operating procedures. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse-osmosis-purified (on-site) drinking water, delivered by an automatice watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study.

Environmental Conditions
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain daily averages of 71 +/-5F (22 +/- 3C) and 50 +/-20% relative humidity. Room temperature and relative humidity were monitored using the Metasys DDC Electronic Environmental control system and were recorded approximately hourly. Average mean daily temperature ranged from 70.6F to 71.9F (21.4 to 22.2C) and mean daily relative humidity ranged from 40.0 to 49.1% during the study. Light timers were calibrated to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod. Air handling units were set to provide approximately 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test mixtures were administered orally by gavage, via an appropriate-sized flexible, Teflon-shafted, stainless steel ball-tipped dosing cannula (Natume, Tokyo, Japan), once daily during gestation days 6-19. A dosage volume of 5 ml/kg was used. The concurrent control animals received deionized water at 5 ml/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

Table 1 presents the study group assignment

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One 10 ml sample from the control formulation and one 10 ml sample from the top, middle and bottom strata of each of the text article formulations prepared on January 10, 2003, were collected for homogeneity analysis. Eight-day stability of the test article in the vehicle at a concentration range of 0.5 - 8.0 mg/ml was established in a previous study (WIL-91004; Rohm and Haas Report No. 01RC-269A). One 10 ml sample from the middle stratum of each dosing formulation prepared on January 16 and 24, 2003, was collected to verify the concentration of the test article dosing formulations.

All anlayses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, Inc. The test article formulations were found to be homogeneous, contained the amounts of test article specified in the protocol and were stable for at least eight days.

Details on mating procedure:

At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the study director, each animal judged to be in good health and meeting acceptable body weight requirements (a minimum of 220 g) was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was prepared. The selected females were approximately 12 weeks old when paired for breeding.

Positive evidence of mating was confirmed by the presence of a copulatory plug or the presence of sperm following vaginal lavage. Each mating pair was examined daily. The day on which evidence of mating was identified was termed day 0 of gestation and the animals were separated.

The bred females were assigned to groups using a WIL Toxicology Data Management System (WTDMS) computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Body weight values ranged from 199 g to 293 g on day 0 of gestation.

Duration of treatment / exposure:

Gestation Days 6-19

Frequency of treatment:

Daily

Duration of test:

Gestation Days 6-19

No. of animals per sex per dose:

25 bred females/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

The test article, 2-methyl-4-isothiazolin-3-one (supplied as a 50% solution in water known as Kordek 573F Industrial Microbiocide), was administered orally by gavage to three groups of 25 bred female Crl:CD (SD)IGS BR rats once daily from gestation days 6 through 19. Initial dosage levels were 0, 5, 20 and 60 mg/kg/day administered at a dosage volume of 5 ml/kg. Based on the excessive toxicity (moribunity, adverse clinical signs and body weight loss) observed in the high-dose group, the 60 mg/kg/day dose level was determined to exceed the maximum tolerated dose. Beginning on January 16, 2003 (gestation days 6-9), the Group 4 dosage level was lowered to 40 mg/kg/day; for reporting purposes, this group is designated as 60/40 mg/kg/day. Therefore, the 40 mg/kg/day dose level was used for final interpretation, conclusions and for determination of the NOAEL. All animals were observed twice daily for appearance and behavior. Clinical observations, body weights and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving female. The uteri and placentae were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

Examinations

Maternal examinations:

Clinical Observations and Survival
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical observations were recorded from days 0 through 20 of gestation (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately one hour following dose administration. All significant findings were recorded.

A gross necropsy was performed on females that died or were euthanized in extremis during the course of the study. The head, tongue, trachea, esophagus, lungs and stomach from these females were retained in 10% neutral buffered formalin for possible future microscopic evaluation. The number and location of implantation sites, corpora lutea and number of viable fetuses were recorded. All carcasses were discarded.

Body Weights and Gravid Uterine Weights
Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-18, 18-20, 6-20 and 0-20.

Gravid uterine weight was collected and net body weight (the day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Food Consumption
Individual food consumption was recorded on gestation days 0, 6, 9, 12, 15, 18 and 20. Food intake was reported as g/animal/day for the corresponding body weight change intervals. On the occasions when food intake could not be measured for one of the days

Ovaries and uterine content:

Gestation Day 20 Laparohysterectomy
All surviving maternal rats were euthanized by carbon dioxide inhalation on gestation day 20. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the organs were examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded. The uterus and ovaries were excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all in utero fetuses, early and late resorptions and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. The carcass of each female was then discarded.

Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss as described by Salewski.


References:
Salewski, E. Farbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte (Staining Method for a macroscopic test for implantation points in the uterus of the rat). Naunyn - Schmiedebergs Archiv fur experimentelle Pathologie und Pharmakologi 1964, 247,367.

Fetal examinations:

Fetal Morphological Examination
Each viable fetus was individually sexed, weighed, euthanized by an intrathoracic injection of sodium pentobarbital, and tagged for identification. Fetal tags contained the WIL study number, the female number and the fetus number. A detailed external examination of each fetus was conducted to include, but was not limited to, an examination of the eyes, palate and external orifices, and each finding was recorded. Late resorptions (advanced degree of autolysis) were examined externally, the degrees of autolysis were recorded and the tissues were discarded. Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination.

Heads from approximately one-half of the fetuses in each litter were placed in Bouin's fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a mid-coronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue by a method similar to that described by Dawson and Inouye. External, visceral and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have significant biological effect on animal health or body conformity, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with body function, or may be incompatible with life).

Statistics:

All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for a significance level of only 5%, comparing each test article-treated group to the control group. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by +/- 1 in the last significant figure.

Mean maternal body weights (absolute and net), body weight changes (absolute and net), food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, and feal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and fetal sex distribution) were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, the Mann-Whitney U-test was used to compare the test article-treated groups to the control group. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particulr external, visceral and skeletal malformation or variation were analyzed by the Kruskal-Wallis nonparametric ANOVA test followed by the Mann-Whitney U-test (if appropriate) as described above.

Indices:

Intrauterine data were summarized using two methods of calculation. An example of each method of calculation follows:

1. Group Mean Litter Basis:
Post-Implantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group)/(No. Gravid Females/Group)

2. Proportional Litter Basis:
Summation Per Group (%) = [Sum Post-Implantation Loss/Litter (%)]/(No Litters/Group)

Where:
Post-implantation Loss/Litter (%) = [No. Dead Fetuses, Resorptions (Early/Late)/Litter]/(No. Implantation Sites/Litter) x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = [Sum Viable Fetuses Affected/Litter (%)]/ (No. Litters/Group)

Where:
Viable Fetuses Affected/Litter (%) = [No. Viable Fetuses Affected/Litter]/[No. Viable Fetuses/Litter} x 100

Historical control data:

No data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: 60/40 mg/kg/day exhibited clinical effects and body weight effects

Details on maternal toxic effects:
Maternal Clinical Observations and Survival
In the 60/40 mg/kg/day group, female nos. 17240, 17286 and 17327 died on gestation days 8, 10, and 15, respectively and female nos. 17286 and 17285 were euthanized in extremis on gestation days 8 and 9, respectively. Female nos 17240 and 17264 were administered the test article at 60 mg/kg/day for two days. Female no. 17285 received the test article at 60 mg/kg/day for three days. Female nos. 17286 and 17327 were only dosed at 40 mg/kg/day. On the day of death or one day prior to death, two or more of these females were observed with rocking, lurching or swaying while ambulating, hypoactivity, rales, gasping, labored respiration, red material around the nose, mouth and/or eye(s) and/or decreased defecation at the daily observations and/or one hour following dose administration. In addition, female no. 17240 had partial closure of both eyes, hypothermia (body cool to the touch), paleness and small feces, and female no. 17285 had salivation at the daily observations. The onset of these findings generally coincided to when the animals were in a moribund state. Based on the excessive maternal toxicity (moribundity, adverse clinical signs and body weight loss) observed in the high dose group, a dose level of 60 mg/kg/day was determined to exceed the maximum tolerated dose. In an attempt to ensure that a sufficient number of high-dose-group females survied to the scheduled laparohysterectomies, beginning on January 16, 2003 (gestation days 6-9) the Group 4 dosage level was lowered to 40 mg/kg/day. All other Group 4 females survived to the scheduled necropsy. Of the surviving females, a majority of the females received the test article at 60 mg/kg/day for 1-3 doses; four females received only 40 mg/kg/day.

Test article-related clinical findings noted in the 60/40 mg/kg/day group consisted of the following. Increased occurrences of rales, gasping and labored respiration were noted in this group at the daily observations and one hour following dose administration (at 60 and 40 mg/kg/day). These findings were noted beginning on the first day of dosing. Rales and labored respiration noted sporadically at the daily observations and/or one hour following dose administration throughout the remainder of gestation. Gasping was noted sporadically in a limited number of animals through gestation day 13. Female no. 17259 in this group was noted with rocking, lurching or swaying while ambulating, hypoactivity and head tilt on multiple days at the daily observations and one hour following dose administration. Female no. 17244 was noted with hypoactivity, rales, gaspong and labored respiration on gestation day 13. In addition, slightly increased incidences (one to three animals) of red material around the nose, mouth and/or eye(s) were noted in the 60/40 mg/kg/day group.

Clinical findings in the 5 and 20 mg/kg/day groups occurred similarly in the control group, were noted in a limited number of animals, were present prior to the initiation of dosing and/or did not occur in a dose-related manner.

Maternal Body Weights and Gravid Uterine Weights.
A lack of mean maternal body weight gain was noted in the 60/40 mg/kg/day group during gestation days 6-9 (majority of the animals dosed at 60 mg/kg/day), compared to a mean body weight gain in the control group; the difference was statistically significant. Following the reduction of the dosage level to 40 mg/kg/day, mean body weight gains in the 60/40 mg/kg/day group were similar to the control group throughout the remainder of gestation. When the entire treatment period (gestation days 6-20) was evaluated in this group, a statistically significant decrease in mean body weight gain was observed. The decreases at both time periods were attributed to the test article. Although mean body weights in the 60/40 mg/kg/day group were not statistically significant during gestation days 8-20, they were decreased (3.9 - 5.4%) when compared to the control group values.

The effects on body weight during gestation days 8-20 and on body weight gain during the entire treatment period were the result of the lack of body weight in this group during gestation days 6-9 when the majority of the animals received 60 mg/kg/day of the test article. Mean net body weight gain was statistically significantly reduced in the 60/40 mg/kg/day group when compared to the control group. Mean net body weight was also decreased (not statistically significant) when compared to the control group value. No test article-related effects was observed for gravid uterine weight in the 60/40 mg/kg/day group.

Mean body weight, body weight gain, net body weight, net body weight change and gravid uterine weight in the 5 and 20 mg/kg/day groups were similar to the control group values; none of the differences were statistically significant.

Maternal Food Consumption
Food consumption, evaluated as g/animal/day, in the 60/40 mg/kg/day group was statistically significantly decreased when compared to the control group value on gestation days 6-9. The reduction was considered to be test article-related. Food consumption in this group was similar to the control group values during gestation days 9-12, 12-15, 15-18 and 18-20 and when the entire treatment period (gestation days 6-20) was evaluated.

Food consumption in the 5 and 20 mg/kg/day groups was similar to that in the control group throughout the treatment period (gestation days 6-20). None of the differences from the control group were statistically significant.

Maternal Necropsy Data
In the 60/40 mg/kg/day group female nos. 17240, 17286 and 17327 died on gestation days 8, 10 and 15, respectively, and female nos. 17264 and 17285 were euthanized in extremis on gestation days 8 and 9, respectively. With the exception of female no. 17327, all of these females had dark red areas in the glandular portion of the stomach. This finding was attributed to the irritant properties of the test article. Other evidence of test article-related irritation included dark red discoloration and/or dark red areas in the lungs of female nos. 17285 and 17286. In addition, female nos. 17240 and 17327 had lungs that were not fully collapsed. These findings in the lungs correspond to the clinical findings of rales, gasping and labored respiration noted for these females prior to death. With the exception of female no. 17286, which was nongravid, all of these females had 12 to 16 normally developed implantations in utero. In addition, female no. 17327 had three early resorptions in utero.

All other females survived to the scheduled necropsy. No internal findings were noted at the scheduled necropsy. One female each in the 20 and 60/40 mg/kg/day groups was determined to be nongravid via ammonium-sulfide staining; all other females were gravid.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Gestation Day 20 Laparohysterectomy Data
Intrauterine parameters (post-implantation loss, live litter size, fetal body weight and fetal sex ratio) were unaffected by test article administration at all dose levels. No statistically significant differences from the control group were noted. The mean numbers of corpora lutea and implantation sites were similar in all groups, including the control group.

Fetal Morphological Data
The number of fetuses (litters) available. for morphological evaluation were 374 (25), 336 (25), 348 (24) and 284 (19) in the control, 5, 20 and 60/40 mg/kg/day groups, respectively. Malformations were observed in 3 (3) and 1 (1) fetuses (litters) in the control and 60/40 mg/kg/day groups, respectively.

External Malformations and Variations
No external malformations and external developmental variations were noted for any fetus in this study.

Visceral Malformations and Variations
There were no test article-related soft tissue malformations observed in fetuses in this study. Soft tissue malformations were noted in 2 (2) and 1 (1) fetuses (litters) in the control and 60/40 mg/kg/day groups, respectively. Fetus no. 17248-15 in the 60/40 mg/kg/day group had situs inversus (the heart, lungs, great and major vessels, trachea, esophagus, stomach, pancreas, spleen, liver and intestine were laterally transposed). This soft tissue malformation was not attributed to the test article because it occurred in a single fetus and has been known to occur in low incidence in the rat based on the WIL historical control data sets. Fetus no. 17282-01 in the control group had an interrupted aortic arch (the brachiocephalic trunk and left carotid artery arose from the ascending aorta, the left subclavian artery arose from the descending aorta; the ductus arteriosus communicated with the descending aorta). Fetus no. 17308-08 in the control group had an absent right testis and right epididymis. No other soft tissue malformations were noted in this study.

The only soft tissue developmental variation observed in the test article-treated groups was a hemorrhagic ring around the righ iris observed in a single fetus (no. 17287-11) in the 60/40 mg/kg/day group. This finding was not considered test article-related, because it occurred in a single fetus.

Dark red areas in the lungs were observed in fetus no. 17280-01 in the 20 mg/kg/day group. This finding was not classified as a visceral malformation or developmental variation, and was not included in any summary data tabulation. Because this finding was observed in a single fetus in the low-dose group, it was not attributed to administration of the test article.

Skeletal Malformations and Variations
No skeletal malformations were noted in fetuses in the 5, 20 and 60/40 mg/kg/day groups. The only skeletal malformation observed in this study was a rib anomaly in fetus no. 17295-03 in the control group; the anomaly consisted of only 12 pairs of ribs present.

There were no test article-related skeletal developmental variations observed in fetuses in this study. The only statistically significant difference from the control group was an increase in the mean litter proportion of hyoid unossified (1.0% per litter) observed in the 5 mg/kg/day group when compared to the concurrent control group value (0.0% per litter0. This value was within the WIL historical control data range (0.0-3.4% per litter) and similar increases were not observed in the 20 and 60/40 mg/kg/day groups. In addition, no other signs of developmental delay (such as reduced fetal weight, reduced mineral ization of other skeletal structures) were noted in fetuses in the 5 mg/kg/day group. Therefore, the slight increase in the incidence of hyoid unossified was not attributed to the test article. Other skeletal developmental variations noted in the treated groups occurred similarly in the control group, occurred in single animals, were within the WIL historical control data range and/or did not occur in a dose-related manner.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Summary of External, Visceral and Skeletal Examinations

There were no test article-related external, visceral and skeletal malformations observed in fetuses in this study. Fetal external, visceral and skeletal malformations were observed in 3 (3) and 1 (1) fetuses (litters) in the control and 60/40 mg/kg/day groups, respectively. When evaluated on a proportional basis (% per litter), no statistically significant differences from the control group were noted. All malformations in this study were considered spontaneous in origin. Fetal developmental variations in the test article-treated groups occurred infrequently, were observed at a similar frequency in the concurrent control group and/or were within the WIL historical control data range.

Table 1 Summary of Fetal Data at Scheduled Necropsy (% Per Litter)

	Mean (S.D.
Group Number	1	2	3	4
Dose Level	0	5	20	60/40
Corpora Lutea	16.4 (1.73)	15.4 (3.47)	17.0 (4.10)	17.3 (2.70)
Implantation Sites	15.4 (1.73)	14.2 (3.83)	15.5 (4.31)	15.9 (2.17)
Viable Fetuses (%)	96.7 (5.87)	94.7 (6.70)	92.9 (11.67)	93.3 (11.02)
Dead Fetuses (%)	0.0 (0.00)	0.0 (0.00)	0.0 (0.00)	0.0 (0.00)
Early Resorptions (%)	3.3 (5.87)	5.3 (6.70)	7.1 (11.67)	6.4 (11.12)
Late Resorptions (%)	0.0 (0.00)	0.0 (0.00)	0.0 (0.00)	0.3 (1.15)
Total Resorptions (%)	3.3 (5.87)	5.3 (6.70)	7.1 (11.67)	6.7 (11.02)
Pre-Implantation Loss (%)	5.8 (7.76)	9.1 (14.18)	10.1 (15.39)	7.0 (7.75)
Post-Implantation Loss (%)	3.3 (5.87)	5.3 (6.70)	7.1 (11.67)	6.7 (11.02)
Males (%)	56.0 (11.23)	55.5 (20.18)	52.4 (15.26)	45.4 (16.66)
Females (%)	44.0 (11.23)	44.5 (20.18)	47.6 (15.26)	54.6 (16.66)
Male Fetal Weights (g)	3.6 (0.30)	3.8 (0.27)	3.6 (0.29)	3.5 (0.35)
Female Fetal Weights (g)	3.4 ( 0.30)	3.5 (0.30)	3.4 (0.32)	3.3 (0.36)
Combined Fetal Weights (g)	3.5 (0.30)	3.7 (0.29)	3.5 (0.31)	3.4 (0.37)

Proportional (%) data compared using the Kruskal-Wallis Test

Fetal weights compared using Dunnett's test

None significantly different from control group.

Table 2 Summary of Fetuses and Litters with Malformations (Absolute No.)

	Fetuses				Litters
Dose Group	1	2	3	4	1	2	3	4
Dose Level (mg/kg/day)	0	5	20	60/40	0	5	20	60/40
Number Examined Externally	374	336	348	284	25	25	24	19
Number with Findings	0	0	0	0	0	0	0	0
Number Examined Viscerally	374	336	348	284	25	25	24	19
......Interrupted Aortic Arch	1	0	0	0	1	0	0	0
......Situs Inversus	0	0	0	1	0	0	0	1
......Testis - Absent	1	0	0	0	1	0	0	0
......Epididymis - Absent	1	0	0	0	1	0	0	0
Number Examined Skeletally	374	336	347 -A	284	25	25	24	19
.....Rib Anomaly	1	0	0	0	1	0	0	0
Total Number with Malformations
.....External	0	0	0	0	0	0	0	0
.....Soft Tissue	2	0	0	1	2	0	0	1
.....Skeletal	1	0	0	0	1	0	0	0
......Combined	3	0	0	1	3	0	0	1

A = The eviscerated carcass of Fetus No. 17297 -05 was inadvertently placed in Bouin's Fixative; therefore, no skeletal examination was performed on this fetus.

Applicant's summary and conclusion

Conclusions:

Due to the excessive toxicity (moribundity, adverse clinical signs and body weight loss) observed in the high-dose group, a dose level of 60 mg/kg/day was determined to exceed the maximum tolerated dose. Following the reduction of the dosage level to 40 mg/kg/day, deaths and clinical findings of rales, gasping and labored respiration were noted. Based on the results of this study, a dose level of 20 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. A dose level of 40 mg/kg/day was considered to be the NOAEL for developmental toxicity.

Executive summary:

Objective

The objective of the study was to determine the potential of the test article, 2 -methyl-4 -isothiazolin-3 -one, to induce developmental toxicity after maternal exposure on days 6 -19 of gestation, to characterize maternal toxicity at the exposure levels tested and to determine a NOAEL (no-observed-adverse-effect level) for maternal toxicity and developmental toxicity.

Study Design

The test article, 2-methyl-4-isothiazolin-3-one (supplied as a 50% solution in water known as Kordek 573F Industrial Microbiocide), was administered orally by gavage to three groups of 25 bred female Crl:CD (SD)IGS BR rats once daily from gestation days 6 through 19. Initial dosage levels were 0, 5, 20 and 60 mg/kg/day administered at a dosage volume of 5 ml/kg. Based on the excessive toxicity (moribunity, adverse clinical signs and body weight loss) observed in the high-dose group, the 60 mg/kg/day dose level was determined to exceed the maximum tolerated dose. Beginning on January 16, 2003 (gestation days 6-9), the Group 4 dosage level was lowered to 40 mg/kg/day; for reporting purposes, this group is designated as 60/40 mg/kg/day. Therefore, the 40 mg/kg/day dose level was used for final interpretation, conclusions and for determination of the NOAEL. All animals were observed twice daily for appearance and behavior. Clinical observations, body weights and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving female. The uteri and placentae were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

Results

Three females in the 60/40 mg/kg/day group were found dead between gestation days 8 and 15 (one of these females received the test article at only 60 mg/kg/day, and the other two females received the test article at only 40 mg/kg/day). Two females in this group were euthanized in extremis between gestation days 8 and 9 (both females received the test article at only 60 mg/kg/day). On the day of death or one day prior to death, two, three or four of the moribund females or females found dead were observed with the following clinical findings; rocking, lurching or swaying while ambulating, hypoactivity, rales, gasping, labored respiration, decreased defecation, red material around the nose, mouth and/or eyes. The onset of these findings generally coincided to when the animals were in a moribund state. Test article-related findings at necropsy for these females included red areas in the glandular portion of the stomach and lung findings (dark red discoloration of the lungs, dark read areas in the lungs and/or lungs not fully collapsed). All other females in all test article groups survived to the scheduled necropsy. Test article-related clinical findings observed in the surviving females included rales, gasping and labored respiration in the 60/40 mg/kg/day group.

A test article-related lack of mean body weight gain (gestation days 6 -9) and reductions in mean net body weight (not statistically significant), net body weight gain and food consumption (gestation days 6 -9) were noted in the 60/40 mg/kg/day group. The majority of the animals in this group were dosed at 60 mg/kg/day during gestation days 6 -9; once the dose level was reduced to 40 mg/kg/day, no effects on body weight gain or food consumption were observed. Mean gravid uterine weight for the 60/40 mg/kg/day group was unaffected by test article administration. No test article-related effects on mean body weight, body weight gain, net body weight, net body weight change, gravid uterine weight and food consumption were noted in the 5 and 20 mg/kg/day groups.

At the scheduled necropsy on gestation day 20, no test article-related internal findings were observed at any dose level.

Intrauterine growth and survival were unaffected by test article administration.

The numbers of fetuses (litters) available for morphological evaluation were 374 (25), 336 (25), 348 (24) and 284 (19) in the control, 5, 20 and 60/40 mg/kg/day groups, respectively. Fetal external, visceral or skeletal malformations were observed in 3 (3) and 1 (1) fetuses (litters) in the control and 60/40 mg/kg/day groups, respectively. All malformations and developmental variations in this study were considered spontaneous in origin.

Conclusion

Due to the excessive toxicity (moribundity, adverse clinical signs and body weight loss) observed in the high-dose group, a dose level of 60 mg/kg/day was determined to exceed the maximum tolerated dose. Following the reduction of the dosage level to 40 mg/kg/day, deaths and clinical findings of rales, gasping and labored respiration were noted. Based on the results of this study, a dose level of 20 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. A dose level of 40 mg/kg/day was considered to be the NOAEL for developmental toxicity.