Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results of 2 -generation oral via drinking water study, a dose concentration of 1000 ppm (69-93 mg/kg/day for the F0 pre-mating period and 86-115 mg/kg/day for the F1 pre-mating period) was considered to be the NOAEL (no-observed-adverse-effect level) for reproductive toxicity of 2-methyl-4-isothiazolin-3-one when administered in the drinking water to rats. A dose concentration of 200 ppm (15-22 mg/kg/day for the F0 pre-mating period and 19-26 mg/kg/day for the F1 pre-mating period) was considered to be the NOAEL for parental systemic toxicity. A dose concentration of 200 ppm was considered to be the NOAEL for neonatal toxicity. The only neonatal effect observed in the F1 and F2 offspring were decreased body weights during the latter part of the pre-weaning period.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 December 2001 - 12 September 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment. A breeding record containing the male and female identification numbers and the start date of cohabitation was prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a copulatory plug or the presence of sperm in a vaginal smear. Each mating pair was examined daily. The day when evidence of mating was identified was termed day 0 of gestation. Then the animals were separated and the female was housed in an individual plastic cage with nesting material. When evidence of mating was not apparent after 14 days, the female was placed in a plastic maternity cage with nesting material. The males and females of each generation were mated once to produce one set of litters per generation (the F1 and F2 litters). Prior to the Fo pairing (week lo), male body weights ranged from 340 g to 612 g and female body weights ranged from 201 g to 333 g. The animals were approximately 16 weeks old. All animals were randomly selected for pairing.

Prior to the F1 pairing (week 28), male body weights ranged from 368 g to 629 g and female body weights ranged from 174 g to 367 g. The animals were 12 to 14 weeks old. All animals were randomly selected for pairing avoiding sibling matings. Pre-coital intervals were calculated according to the following method: rats paired over a 12-hour dark cycle were considered to have been paired for "one" day.

F0 ANIMALR ECEIPT AND ACCLIMATION
One hundred thirty-five male and 137 female Crl:CD(SD)IGS BR rats were received from Charles River Laboratories, Inc., Raleigh, North Carolina, on December 11, 2001. In order to ensure that litterrnates would not be paired with each other for mating, the male and female animals received fkom Charles River Laboratories, Inc. were from different barrier rooms (R05 and R12, respectively). The animals were 29 days old upon receipt. Each animal was examined by a qualified technician upon receipt and weighed the day following receipt. All animals were uniquely identified by a Monel metal eartag displaying the animal number. During the acclimation period (15 days), the animals were observed twice daily for mortality and moribundity.

F0, F1 AND F2 ANIMAL HOUSING
Following receipt (F0) or selection (F1) and until pairing, all F0 and F1 parental test animals were individually housed in clean, wire-mesh cages (27.31 cm x 26.67 cm x 17.78 cm) suspended above cage-board. Cage board was changed three times per week. The animals were paired for mating in the home cage (27.3 1 cm x 26.67 cm x 17.78 cm) of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy of the parental generations, and the females were transferred to plastic maternity cages (approximately 40.64 cm x 18.73 cm x 20.64 cm) with nesting material (Bed-O'COBs; The Andersons, Industrial Products Division, Maumee, Ohio). The dams were housed in these cages until weaning on lactation day 21. Following weaning of the litters in each generation (F1 and F2), the maternal females were individually housed in suspended wire-mesh cages until the scheduled necropsy, and the weaned F1 pups selected for the FI maturation and breeding phase were housed together by litter for one week. The remaining F1 pups were necropsied on PND 21. Beginning on PND 28, the F1 pups were individually housed in suspended wire-mesh cages until the start of the mating period. Maternal females for which there was no evidence of mating were placed in plastic maternity cages with nesting material upon completion of a 14-day mating period. If these animals did not deliver after 25 days, they were returned to individual suspended wire-mesh cages. Animals were housed in accordance with the "Guide for the Care and Use of Laboratory Animals."' The animal facilities at WIL Research Laboratories, Inc., are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

DIET, DRINKING WATER AND MAINTENANCE OF F0 AND F1 ANIMALS
The basal ration used in this study was PMI Nutrition International, Inc., Certified Rodent Lab Diet 5002. The diet used at WIL Research Laboratories, Inc., was a certified feed with appropriate analyses performed and provided by the manufacturer. Municipal water supplying the facility is regularly sampled for contaminants according to standard operating procedures. Contaminants were not present in animal feed or water at concentrations expected to interfere with the objectives of this study. The basal diet and reverse-osmosis-treated (on-site) drinking water, provided in water bottles, were provided ad libitum throughout the acclimation period and during the study. Water bottles were changed and sanitized once per week. Food jars were equipped with retainers to minimize spillage. Feeders were sanitized weekly.

ENVIRONMENTAL CONDITIONS
Animals were housed throughout the acclimation period and during the study in an environmentally controlled room. Controls were set to maintain a temperature of 71 +/- 5°F (22 +/- 3°C) and a relative humidity of approximately 30-70%. Prior to March 1, 2002, room temperature and relative humidity were recorded daily. During this time, the actual temperature ranged from 67.1°F to 71.8°F (19.5°C to 22.1°C) and humidity ranged from 36.0% to 64.1%. From March 1, 2002 through the end of the study, room temperature and relative humidity were monitored using the Metasys DDC Electronic Environmental control system and were recorded approximately hourly. Actual mean daily temperature during this period ranged from 70.3°F to 70.9°F (21.3°C to 21.6°C) and mean daily relative humidity ranged from 37.1% to 60.3%. 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.

ASSIGNMENT OF F0 ANIMALS TO TREATMENT GROUPS
At the conclusion of the acclimation period, all available F0 animals were weighed and examined in detail for physical abnormalities. At the discretion of the study director, animals judged to be in good health and meeting acceptable body weight requirements (200 to 300 g for males and 150 to 250 g for females) were selected for use in the computerized randomization procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into the WIL Toxicology Data Management System (WTDMSTM). A printout containing the animal numbers, corresponding body weights and individual group assignment was generated based on body weight stratification randomized in a block design. The animals were then arranged into groups according to the printout. The animals were approximately six weeks old at the initiation of test article administration. Male body weights ranged from 149 g to 221 g and female body weights ranged from 123 g to 163 g on the day of randomization (prior to study initiation). The individual randomization weights are not presented in this report but are maintained in the study records.
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on exposure:
For the control group, an appropriate volume of deionized water was dispensed into storage containers.

For preparation of drinking water for the treated groups, an appropriate amount of the test article, a 50% aqueous solution of 2-methyl-4-isothiazolin-3-one, was weighed for each group into a tared weighing vessel. Sufficient deionized water was added to a storage container for each group to bring the weight to approximately 60% of the target weight of the preparation. The test article was then quantitatively transferred to the storage container, and sufficient deionized water was added to bring the weight of each preparation to the target weight. A stir bar was added, and each preparation was mixed using a magnetic stirrer until uniform. The test drinking water solutions were prepared weekly and stored at room temperature.

The formulations were visually inspected by the study director on December 2 1, 2001, and were found to be visibly homogeneous and acceptable for dose administration.

The test drinking water was offered to F0 and F1 males and females ad libitum for a minimum of 70 days prior to mating. Administration of the test drinking water preparations continued during mating, gestation and lactation and concluded on the day of euthanasia. The test article was administered as a constant concentration (ppm) in the drinking water. The F0 and F1 parental animals were assigned to study groups as outlined in Table 1.

Dose levels were selected so that the decreases in water consumption at the high dose level (known to occur with this substance and similar substances) would not be of sufficient magnitude to compron~iset he general health of the animals. The low and mid doses were multiples of the high dose level selected to determine a no effect level.

The offspring of the Fo generation (F1 litters) were potentially exposed to the test article in utero, through nursing during PND 0-21 and via administration of test drinking water following weaning (beginning on PND 22). The F1 pups selected for breeding (30/sex/group) were provided with test drinking water for at least 70 days prior to mating and throughout mating, gestation and lactation until the day of necropsy. The offspring of the F1 generation (F2 litters) were potentially exposed to the test article in utero and through nursing during PND 0-2 1.
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment. A breeding record containing the male and female identification numbers and the start date of cohabitation was prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a copulatory plug or the presence of sperm in a vaginal smear. Each mating pair was examined daily. The day when evidence of mating was identified was termed day 0 of gestation. Then the animals were separated and the female was housed in an individual plastic cage with nesting material. When evidence of mating was not apparent after 14 days, the female was placed in a plastic maternity cage with nesting material.

The males and females of each generation were mated once to produce one set of litters per generation (the F1 and F2 litters). Prior to the Fo pairing (week lo), male body weights ranged from 340 g to 612 g and female body weights ranged from 201 g to 333 g. The animals were approximately 16 weeks old. All animals were randomly selected for pairing.

Prior to the F1 pairing (week 28), male body weights ranged from 368 g to 629 g and female body weights ranged from 174 g to 367 g. The animals were 12 to 14 weeks old. All animals were randomly selected for pairing avoiding sibling matings.

Pre-coital intervals were calculated according to the following method: rats paired over a 12-hour dark cycle were considered to have been paired for "one" day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Instrument: Hewlett-Packard 1100 liquid chromatograph equipped with a variable wavelength UVNIS detector, autosampler, and HP ChemStation data management system, or equivalent system
Column: Supelcosil LC-ABZ; 4.6 x 150 mm, 5-um particle size
Mobile Phase: 50:50, (v/v) water/methanol
Flow Rate: 1.0 mL/minute
Detection: UV at 254 nm
Injection Volume: 5.0 uL
Retention Time: Approximately 1.8 minutes for 2-methyl-4-isothiazolin-3-one
Run Time: Approximately 3.0 minutes

Duplicate 10-mL samples were collected from the top, middle and bottom of each formulation used for the first week of test drinking water administration. One set of samples at each of the selected dosage levels was analyzed for homogeneity. The remaining set of samples from the low-, mid- and high-dose concentrations was combined and stored under laboratory conditions for 15 days. These samples were analyzed for stability. Samples (10 mL each) of test drinking water were collected weekly from preparations for each dosage level, including controls, and stored at -20°C. Samples were analyzed for test article concentration in dosing formulations prepared for the first and third weeks of dosing and once per month thereafter. The remaining samples were discarded following receipt of acceptable analytical results.

All analyses were conducted by the Analytical Chemistry Department at WIL Research Laboratories, Inc. The test drinking water preparations were homogeneous, were stable for 15 days under normal laboratory conditions, and contained the specified amounts of test article.
Duration of treatment / exposure:
The test drinking water was offered to F0 and F1 males and females ad libitum for a minimum of 70 days prior to mating, during mating and pregnancy and during the raising of the subsequent offspring.
Frequency of treatment:
Continuous.
Details on study schedule:
December 11, 2001 .................................. Experimental starting date (animal receipt)
December 26, 200 1 ................................. Initiation of F0 test article administration
March 5-19, 2002 ..................................... F0 breeding range
April 21-May 27, 2002 ............................. Evaluation of acquisition of developmental landmarks for selected F1 pups
April 30, 2002 ............................................ Last necropsy of non-selected F1 pups
May 7, 2002 ................................................ Last necropsy of F0 generation
April 18, 2002 ............................................ Initiation of F1 test article administration (first PND 22)
July 10-24, 2002 ....................................... F1 breeding range
September 1, 2002 .................................... Last necropsy of F2 pups
September 12, 2002 ...................................Last necropsy of F1 generation
March 4, 2003 .............................................Experimental termination date (last microscopic examination)
Remarks:
Doses / Concentrations:
0, 50, 200 and 1000 ppm a.i. in drinking water
Basis:
nominal in water
No. of animals per sex per dose:
30 males and 30 females/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
The test article, 2-methyl-4-isothiazolin-3-one, was administered on a continuous basis in the drinking water at dose concentrations of 50, 200 and 1000 ppm for a minimum of 70 consecutive days prior to mating. F0 animals were approximately six weeks of age at the beginning of test article administration; administration of the test article to F1 offspring selected to become the F1 generation began at weaning. The F0 and F1 males received the test article throughout mating and continuing until the day of euthanasia. The F0 and F1 females received the test article throughout mating, gestation and lactation, and continuing until the day of euthanasia.

All animals were observed twice daily for appearance and behavior. Clinical observations, body weights, and water and food consumption were recorded at appropriate intervals prior to mating and during gestation and lactation. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day 21. For both generations (F1 and F2), eight pups per litter (four per sex, when possible) were selected on postnatal day (PND) 4 to reduce the variability among the litters. Administration of the test article to the F1 animals was initiated on PND 22. Offspring (30/sex/group) from the pairing of the F0 animals were selected to constitute the F1 generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats. Unselected F1 and all F2 pups were necropsied on PND 21; selected organs were weighed. Each surviving F0 and F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups, respectively; selected organs were weighed. Spermatogenic endpoints (sperm motility, morphology and numbers) were evaluated for all F0 and F1 males, and ovarian primordial follicle counts were recorded for all F1 females in the control and high-dose groups. Designated tissues from all F0 and F1 parental animals and from all parental animals that died or were euthanized in extremis were examined microscopically. In addition, reproductive organs from females that failed to deliver in the low- and mid-dose groups, and from the males with which they were paired, were examined microscopically.
Positive control:
No data
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND MORTALITY
Detailed physical examinations were recorded weekly for all parental animals throughout the study period. All animals were observed twice daily for moribundity and mortality. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHTS
Individual F0 and F1 male body weights were recorded weekly throughout the study, beginning one week prior to the start of dosing, and prior to the scheduled necropsy. Individual F0 and F1 female body weights were recorded weekly until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire F0 or F1 generation (males only). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 1 1, 14, 17 and 20 and on lactation days 1, 4, 7, 14 and 21. Body weight changes are presented for each of these intervals and for the entire gestation and lactation intervals (days 0-20 and 1-2 1, respectively). After weaning (lactation day 21), weekly body weights were recorded for these females until the scheduled necropsy.

FOOD CONSUMPTION
Individual Fo and F1 male and female food consumption was measured weekly, beginning one week prior to the start of dosing, until pairing. Food intake was not recorded during the mating period. Male food consumption was measured after mating on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation days 0, 4, 7, 1 1, 14, 17 and 20 and lactation days 1, 4, 7, 14 and 21. Food consumption was calculated and reported as g/animal/day for the corresponding body weight change intervals. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.

WATER CONSUMPTION
Individual Fo male and female water consumption was measured twice weekly, beginning one week prior to the initiation of test article administration and continuing until pairing. Water intake was not recorded during the mating period. Male water consumption was measured after mating on a twice-weekly basis until the scheduled necropsy. Female water consumption was recorded daily during gestation and lactation. For the F1 generation, water consumption was collected beginning on PND 21. Because the F1 weanlings were gang-housed by litter from PND 21-28, daily water consumption values during this period were recorded on a per litter basis. Beginning on PND 28, water consumption for the F1 animals was recorded twice weekly until pairing; water consumption for these animals was then recorded as described for the Fo animals. Water consumption was calculated and reported as g/animal/day for the corresponding body weight change intervals.

F0 AND F1 PARTURITION
All females were allowed to deliver naturally and rear their young to weaning (PND 21). During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation lengths were calculated using the date delivery started.
Oestrous cyclicity (parental animals):
Vaginal lavages were prepared daily to determine the stage of estrus for each female, beginning 21 days prior to pairing and continuing until evidence of mating was observed. For females with no evidence of mating, lavages were continued until termination of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] beginning 21 days prior to initiation of the mating period and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of estrus.
Sperm parameters (parental animals):
Immediately upon euthanasia, the reproductive tract of each Fo and F1 male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL bovine serum albumin (BSA). After a ten-minute incubation period, a sample of sperm was loaded into a 100-um cannula for determination of sperm motility. Because sperm motility can be affected by temperature shock, all cannulas and diluents were pre-wanned in an incubator, and motility determinations were performed under constant temperature (approximately 37°C) using the Hamilton-Thome HTM-IVOS Version 10 (Version 12 for the F1 males) computer assisted sperm analysis (CASA) system. Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all dose groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:

Percent Motile (or Progressively Motile Sperm) = (Number of Motile (or Progressively Motile) Sperm)/(Total Number of Sperm Counted) x 100

Sperm morphology was evaluated by light microscopy via a modification of the wetmount evaluation technique described by Linder et al., (1992) Abnormal forms of sperm (double heads, double tails, microcephalic or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.

The left testis and epididymis from all Fo and FI males from all dose groups were stored frozen, homogenized and evaluated for determination of homogenization-resistant spermatid count and sperm production rate, using the method described by Blazak et al., (1985) and the Hamilton-Thorne CASA system.

For determination of homogenization-resistant spermatid count and sperm production rate, the samples were thawed and homogenized, and a sample was retained for subsequent analysis. An aliquot of the sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-um chamber depth. Illumination from a xenon lamp within the HTM-IVOS analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or 20 fields were counted for each sample.

References:
Blazak, W.F., Ernst, T.L., Stewart, B.E. Potential indicators of reproductive toxicity: testicular sperm production and epididymal sperm number, transit time and motility in Fischer 344 rats. Fundamental and Applied Toxicology 1985, 5, 1097- 1 103.

Linder, R.E.; Strader, L.F.; Slott, V.L.; Suarez, J.D. Endpoints of spematotoxicity in the rat after short duration exposures to fourteen reproductive toxicants. Reproductive Toxicology 1992, 6, 491-505
Litter observations:
LITTER VIABILITY AND DEATHS
Each litter was examined twice daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits on PND 0. A daily record of litter size was maintained. Intact offspring dying from PND 0 to 4 were necropsied using a fresh dissection technique described by Stuckhardt and Poppe (1984). Pups with external abnormalities which would warrant further skeletal examination were eviscerated and stained as described by Dawson (1926) for subsequent skeletal evaluation. Findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on the animal's health or body conformity, representing slight deviations from normal) or malformations (those anomalies that alter general body conformity, disrupt or interfere with body function, or may be incompatible with life). A detailed gross necropsy was performed on any pup dying after PND 4 and prior to weaning; tissues were preserved for possible future histopathological examination only as deemed necessary by the gross findings.

LITTER REDUCTION
To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. All selections were performed by computerized randomization. The remaining offspring were weighed, euthanized and discarded on PND 4. Standardization of litter size was not performed on litters with fewer than eight pups.

CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1, 4, 7, 14 and 21.

BODY WEIGHTS
Pups were individually weighed on PND 1, 4, 7, 14 and 21. Mean pup weights were presented by sex for each litter and by treatment group.

SEX DETERMINATION
Pups were individually sexed on PND 0, 4 and 21.

ANOGENITADLIS TANCE
The anogenital distance of all F2 pups was measured on PND 1 ; the absolute distance was reported for each pup. Anogenital distance was defined as the distance from the center of the anus to the center of the genital tubercle.

WEANING AND SELECTION
Each dam and litter remained housed together until weaning on lactation day 21. Thirty male and 30 female F1 pups from each group (control, 50, 200 and 1000 ppm) were randomly selected prior to weaning (lactation day 21) to comprise the F1 generation. These pups (a minimum of one male and one female per litter, when available) were administered the test drinking water beginning on PND 22.

Pups were selected for the F1 generation as follows. A computerized randomization procedure was used to select a minimum of one male and one female per litter, when available. An additional male and/or female were selected from a litter, if necessary, to obtain 30 males and 30 females for each group. Pups selected for the F1 generation also retained the dam number, followed by a hyphen "-" and the digit tattoo marking (i.e., 99999-01).

F1 DEVELOPMENTAL LANDMARKS
The following investigations were used to assess the maturation of the selected F1 pups. These procedures were concluded when the oldest pups were 50 days of age.

BALANOPREPUTSIAELP ARATION
Each male pup was observed for balanopreputial separation beginning on PND 35 as described by Korenbrot et aL., (1977). The day on which balanopreputial separation was first observed was recorded for each pup. Examination of the pups continued daily until balanopreputial separation was present. Body weights were recorded on the day of acquisition of this landmark.

VAGINAL PATENCY
Each female pup was observed for vaginal perforation beginning on PND 25 as described by Adams et al., (1985) The day on which the vaginal lumen was first observed to open was recorded for each pup. Examination of the females was continued daily until vaginal patency was present. Body weights were recorded on the day of acquisition of this landmark.



References:
Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, L. W ., Sobotka, T.J., Tilson, H.A. and Nelson, B.K. Collaborative behavioral teratology study: protocol design and testing procedure. Neurobehavioral Toxicology and Tevatology 1985, 7, 579-586.

Dawson, A.B. A note on the staining of the skeleton of cleared specimens with Alizarin Red S. Stain Technology 1926, 1, 123- 124.

Korenbrot, C.C., Huhtaniemi, I.T. and Weiner, R.W. Preputial separation as an external sign of pubertal development in the male rat. Biology of Reproduction 1977, 17, 298-303.

Stuckhardt, J.L. and Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Carcinogenesis and Mutagenesis 1984, 4, 181-188.
Postmortem examinations (parental animals):
F0 AND F1 GENERATION- ADULTS
All surviving F0 adults were euthanized following the selection of the F1 generation and completion of a detailed physical examination. All surviving F1 adults were euthanized following weaning of the F2 pups.

PATHOLOGY
MACROSCOPIC EXAMINATION
A complete necropsy was conducted on all parental animals (F0 and F1) dying spontaneously, euthanized in extremis or at termination. All animals were euthanized by C02 inhalation. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera. At the time of necropsy, the following F0 and F1 parental tissues and organs were collected and were placed in 10% neutral buffered formalin:

Adrenals (2)................................................ Seminal vesicles (2)
Cervix.......................................................... Stomach
Coagulating gland......................................Testes with epididymides(b) (1) and vas deferens
ovaries(a) and oviducts (2) ......................Uterus with vagina
Pituitary........................................................All gross lesions
Prostate

(a) = One section of each ovary was taken from all Fo females in the control and high dose groups. Five sections were taken at least 100 pm apart from the inner third of each ovary from all F1 females in the control and high dose groups.
(b) = The right testis and epididymis were fixed in Bouin's solution. Both testes and epididymides from animals that died or were euthanized in extremis were fixed in Bouin's solution.

ORGAN WEIGHTS
The following organs from all F0 and F1 parental animals euthanized at scheduled termination were weighed:

Adrenal glands.........................................Seminal vesicles with coagulating glands (with accessory fluids)
Brain ..........................................................Spleen
Epididymides(a) (total and cauda) ........Testes(a)
Kidneys ......................................................Thyroid(b)
Liver............................................................Uterus with oviducts and cervix
Ovaries
Pituitary
Prostate
(a) =These paired organs were weighed separately.
(b) = Tissue was weighed after fixation in 10% neutral-buffered formalin.

Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight ratios were reported.

HISTOLOGICAL PROCEDURES AND MICROSCOPIC EXAMINATION
Microscopic evaluations were performed on the following tissues for all F0 and F1 parental animals from the control and high dose groups and for all adult animals that died spontaneously or were euthanized in extremis. In addition, reproductive organs from females in the 50 and 200 ppm groups that failed to deliver, and from the males with which they were paired, were examined microscopically.

Adrenal glands
Cervix(c)
Coagulating gland(c)
Epididymis(a,c) (right: caput, corpus and cauda
Ovaries(b,c)
Oviducts(c)
Pituitary
Prostate(c)
Seminal vesicles(c)
Stomach
Testis(a,c)(right)
Uterus(c)
Vagina(c)
All gross (internal) lesions (all groups)
(a) = PAS and hematoxylin staining were used for the right testis and epididymis. Transverse sections of 2 to 4 microns of the testes and longitudinal sections of the epididymides were made.
(b) = A bilateral evaluation of one section of each ovary was performed for all F0 females in the control and high dose groups. A quantitative
histopathological evaluation of multiple sections was conducted on all F1 females from the control and high dose groups. This examination
included enumeration of the total number of primordial follicles according to the methods of Bolon et al., (1997) and Bucci et al., (1997).
(c) = Reproductive organs of all 50 and 200 ppm group animals suspected of reduced fertility (e.g., those that failed to mate, conceive, sire or deliver
healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected) were examined.

All preserved tissues were examined by a veterinary pathologist.

References:
Bolon, B., Bucci, T.J., Warbritton, A.R., Chen, J. J., Mattison, D .R. and Heindel, J.J. Differential follicle counts as a screen for chemically induced ovarian toxicity in mice: Results from continuous breeding bioassays. Fundamental and Applied Toxicology 1997,39, 1-1 0.

Bucci, T.J., Bolon, B., Warbritton, A.R., Chen, J.J. and Heindel, J.J. Influence of sampling on the reproducibility of ovarian follicle counts in mouse toxicity studies. Reproductive Toxicology 1997,11,689-696.
Postmortem examinations (offspring):
F1 AND F2 PUPS
Prior to weaning, 30 F1 pups/sex/group were randomly selected for the F1 parental generation and for evaluation of developmental landmarks. In addition, one F1 and one F2 pup/sex/litter (when available) were selected from the F1 and F2 weanlings for complete necropsy on PND 21; brain, spleen and thymus gland weights were recorded, and these tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. All remaining non-selected F1 and F2 weanlings were euthanized by C02 inhalation and necropsied on PND 21, with emphasis on developmental and reproductive system nlorphology. The stomachs and all gross lesions from F1 and F2 weanlings were preserved in 10% neutral-buffered formalin for possible future histopathologic examination; all other tissues were discarded.
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 minimum significance level of 5%, comparing each test article-treated group to the control group by sex. 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. Statistical analyses were not conducted if the number of animals was two or less. Data obtained from nongravid animals were excluded from statistical analyses following the mating period. Statistical analyses were not performed when weekly body weight, food consumption or water consumption data for one or more animals were not available because the animals remained in the lactation phase. The litter, rather than the pup, was used as the experimental unit.

Parental mating and fertility indices were analyzed using the Chi-square test with Yates' correction factor (Hollander and Wolfe, 1999). Mean parental (weekly, gestation and lactation) and offspring body weight data, food consumption and food efficiency data, parental water consumption data, estrous cycle data, pre-coital intervals, gestation lengths, implantation sites, live litter sizes, unaccounted sites, numbers of pups born, balanopreputial separation (day of acquisition and body weight), vaginal patency (day of acquisition and body weight), anogenital distances, absolute and relative organ weights, sperm production rate, and epididymal and testicular sperm numbers were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences.

Continued below
Reproductive indices:
Mating and fertility indices were calculated as follows:
Male (Female) Mating Index (%) = (No. of Males (Females) with Evidence of Mating)/(Total No. of Males (Females) Used for Mating) x 100

Female Fertility Index (%) = (No. of Females with Confirmed Pregnancy)/(Total No. of Females Used for Mating) x 100

Male Fertility Index (%) = (No. of Males Siring a Litter)/(Total No. of Males Used for Mating) x 100


Percent Motile (or Progressively Motile Sperm) = (Number of Motile (or Progressively Motile) Sperm)/(Total Number of Sperm Counted) x 100
Offspring viability indices:
Live Litter Size = (Total Viable Pups Day 0)/(No. Litters With Viable Pups Day 0)

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (% Per Litter) = Sum (Viable Pups Per Litter on PND 0 or PND 4mo. of Pups Born Per Litter)/(No. of Litters Per Group) X 100

Postnatal Survival for All Other Intervals (% Per Litter) = (Per Litter at Start of Interval N)/( No. of Litters Per Group) x 100

Where N = PND 0- 1, 1-4 (Pre-Selection), 4 (Post-Selection)-7, 7-1 4, 14-2 1 or 4 (Post-Selection)-2
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased body weight and food consumption in high dose animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased body weight and food consumption in high dose animals.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: Decreased water consumption was noted in F0 males at all dose levels and in the F1 males and both generations of females at the 200 and 1000 ppm dose levels during the pre-breeding period. See below.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL OBSERVATIONS AND SURVIVAL
There were no test article-related mortalities or clinical signs noted at any dose concentration. One male in the 1000 ppm group died, and one male in the 1000 ppm group and one female in the 200 ppm group were euthanized in extremis during the study. Fo male no. 83017 in the 1000 ppm group was euthanized in extremis during week 9, prior to the breeding period, and Fo male no. 83078 in this group was found dead during week 17. Fo female no. 83155 in the 200 ppm group was euthanized in extvemis on gestation day 25. On the day prior to euthanasia, male no. 83017 had red material in the urogenital area and around the nose, eyes and mouth, and black material around the mouth. In addition, this animal had malaligned upper incisors and was diagnosed by a staff veterinarian as having a possible fracture of the maxilla. Based on these findings, the moribund condition of this animal was attributed to mechanical trauma and was not considered to be test article-related. No significant clinical findings were noted for male no. 83078 near the time of death. The death of this male was the result of calculi in the kidneys and urinary bladder. Female no. 83155 was pale and had a hunched appearance and red material in the urogenital area on the day prior to euthanasia. This female was found to have a dead fetus retained in utero at the time of necropsy. The clinical findings observed for this female on the day prior to euthanasia, the retained fetus observed in this female on gestation day 25 (several days later than the expected day of parturition on gestation day 22) and the necropsy finding of red fluid contents in the abdominal cavity were suggestive that the female was moribund due to pregnancy complications. Because of the absence of similar findings in the 1000 ppm group Fo females, the moribundity of this female was not attributed to the test article.

All other Fo animals survived to the scheduled necropsies. No test-article-related clinical findings were noted at any dose concentration. Findings observed in the test article-treated groups, including hair loss, scabbing and red material on various body surfaces, soft stool and decreased defecation, were noted infrequently and/or at similar frequencies in the control group.

REPRODUCTIVE PERFORMANCE
No test article-related effects on F0 reproductive performance were observed at any dose concentration. Male mating indices were 100%, 100%, 100% and 96.6% in the control, 50, 200 and 1000 ppm groups, respectively. Female mating indices were 100%, 100%, 100% and 96.7% in the same respective groups. Male fertility indices were 93.3%, 100%, 100% and 96.6% in the control, 50, 200 and 1000 ppm groups, respectively. Female fertility indices were 93.3%, 100%, 100% and 96.7% in the same respective groups. No statistically significant differences were noted between the control and treated groups. Males that did not sire a litter numbered 2, 0, 0 and 1 in the control, 50, 200 and 1000 ppm groups, respectively. Females that had evidence of mating but did not deliver numbered 3, 0, 1 and 0 in the same respective groups.

The mean numbers of days between pairing and coitus in the test article-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were similar to the control group value. None of these differences were statistically significant.

BODY WEIGHTS
WEEKLY
No test article-related effects were noted on mean body weights or body weight gains prior to breeding at dose concentrations of 50 and 200 ppm. Mean F0 body weight gains were decreased in males and females in the 1000 ppm group beginning at week 0-1 and continuing through week 4-5 (males) or week 3-4 (females); the differences fkom the control group were statistically significant. Mean body weight gains in these animals were generally similar to the control group values for the remainder of the generation (males) or the remainder of the pre-breeding period (females). Cumulative mean body weight gains in the 1000 ppm group were decreased (statistically significant) during the pre-breeding period (weeks 0-10; females) and during the entire generation (weeks 0- 18; males). Mean weekly body weights in the 1000 ppm group F0 males were similar to the control group values for weeks 1 and 2. Mean weekly body weights in this group were decreased (statistically significant) relative to the control group values beginning at week 3 and continuing through the remainder of the generation. Mean weekly body weight in the F0 females in the 1000 ppm group was similar to the control group value for week 1 and decreased (statistically significant) for the remainder of the pre-breeding period.

Mean weekly body weight gains, cumulative body weight gains and body weights in the 50 and 200 ppm groups were generally similar to the control group values for the pre-breeding period (females) or the entire generation (males). A statistically significant decrease in mean body weight gain was observed in the 200 ppm group F0 females for week 2-3; however, because the decrease was transient, it was not attributed to the test article. Other differences from the control group were slight and were not statistically significant.

GESTATION
No test article-related effects were noted on body weight gains during gestation at dose concentrations of 50 and 200 ppm. Mean body weight gains in the 1000 ppm group F0 females were similar to the control group values during gestation days 0-4, 4-7, 7-1 1 and 11-14. For gestation days 14-17 and 17-20 and when the entire gestation period (gestation days 0-20) was evaluated, mean body weight gains in the 1000 ppm group were decreased (statistically significant) relative to the control group values. As a result of decreased mean body weight gain during the pre-mating period, mean body weights in the 1000 ppm group were decreased (statistically significant) relative to the control group values throughout gestation.

Mean body weights and body weight gains in the 50 and 200 ppm groups were similar to the control group values throughout gestation; differences were slight and were not statistically significant.

LACTATION
No test article-related effects were noted on mean body weight gains during lactation at any dose concentration. Mean body weight gains in the 1000 ppm group Fo females were similar to the control group values for lactation days 1-4 and 4-7. Mean body weight gain in the 1000 ppm group was decreased (statistically significant) relative to the control group value for lactation days 7-14. During lactation days 14-21, a mean body weight loss was observed in this group that was of smaller magnitude than the loss observed in the control group; the difference from the control group was statistically significant. When the entire lactation period (lactation days 1-21) was evaluated, a statistically significant increase in mean body weight gain was noted ill the 1000 ppm group. Because no consistent pattern of the differences in body weight change from the control group were observed in the 1000 ppm group during lactation, these differences were not considered to be test article-related.

Mean body weights and body weight gains in the 50 and 200 ppm groups were similar to the control group values throughout lactation; differences were slight and were not statistically significant. As a result of decreased mean body weight gains during the pre-mating and gestation periods, mean body weights in the 1000 ppm group were decreased (statistically significant) relative to the control group values throughout lactation.

FOOD CONSUMPTION
WEEKLY
No test article-related effects were noted on food consumption prior to breeding at dose concentrations of 50 and 200 ppm. Food consumption, evaluated as glanimallday, in F0 males and females in the 1000 ppm group was slightly decreased beginning at week 0-1 and continuing through the remainder of the pre-breeding period (females) or generation (males). The differences from the control group were generally statistically significant. Food efficiency in the 1000 ppm group was slightly decreased relative to that in the control group for weeks 0-1 through 4-5 (males) or weeks 0-1 through 3-4 (females); the differences from the control group were often statistically significant. Food efficiency in this group was unaffected by the test article for the remainder of the generation (males) or pre-breeding period (females).

Food consumption, evaluated as glanimallday, and food efficiency in the 50 and 200 ppm groups were unaffected by the test article. A statistically significant decrease in food consumption and increase in food efficiency were observed in F0 males in the 50 ppm group for week 1-2. However, the differences from the control group were transient and were not observed in the 200 ppm group; therefore, they were not attributed to the test
article.

GESTATION
No test article-related effects were noted on food consumption during gestation at dose concentrations of 50 and 200 ppm. Food consumption, evaluated as glanimallday, in F0 females in the 1000 ppm group was similar to that in the control group for gestation days 0-4, 4-7, and 7-1 1. Food consumption in this group was slightly decreased (statistically significant) relative to that in the control group for gestation days 1 1 - 14, 14- 17 and 17-20 and when the entire gestation period (gestation days 0-20) was evaluated. Food efficiency in the 1000 ppm group was generally similar to that in the control group during gestation. Differences between the control and test article-treated groups were slight and were not statistically significant.

Food consumption and food efficiency during lactation in the 50 and 200 ppm groups were unaffected by treatment with the test article. Differences from the control group were slight and were not statistically significant.

LACTATION
No test article-related effects were noted on food consumption during lactation at dose concentrations of 50 and 200 ppm. Food consumption, evaluated as glanimallday, in the 1000 ppm group was similar to that in the control group for lactation days 1-4. Food consumption in this group was decreased relative to the control group values for lactation days 4-7, 7-14 and 14-21 and when the entire lactation period (lactation days 1-21) was evaluated. Differences from the control group were statistically significant with the exception of the lactation days 4-7 interval. Food efficiency in the 1000 ppm group was similar to that in the control group for lactation days 1-4, 4-7 and 7-14. During lactation days 14-21 and when the entire lactation period (lactation days 1-21 ) was evaluated, food efficiency in the 1000 ppm group was increased (statistically significant) relative to that in the control group.

Food consumption and food efficiency during lactation in the 50 and 200 ppm groups were unaffected by the test article. The only statistically significant difference from the control group was a slight decrease in food consumption in the 200 ppm group for lactation days 14-21 ; this transient decrease was not attributed to the test article.

WATER AND TEST ARTICLE CONSUMPTION
WEEKLY
Water consumption, evaluated as glanimallday, in F0 males in the 50, 200 and 1000 ppm groups was decreased in a dose-dependent manner beginning at week 0-1 and continuing through the remainder of the generation. The differences from the control group were generally statistically significant. Water consumption in the F0 females was decreased in a dose-dependent manner in the 200 and 1000 ppm groups throughout the pre-breeding period; the differences from the control group were statistically significant. The decreases were attributed to the test article. However, the decreased water consumption was most likely due to palatability, taste andlor smell, of the test article, an irritant, and not to systemic toxicity.

Water consumption in the 50 ppm group F0 females was generally similar to that in the control group during the pre-breeding period. The only statistically significant difference from the control group was a decrease in water consumption during week 4-5; because the decrease was transient and was not accompanied by a decrease in mean body weight, it was not attributed to the test article.

Average weekly test article consumptions (mg/kg/day) are presented in Table 2.

GESTATION
Water consumption in the 50 ppm group was similar to that in the control group throughout gestation. Water consumption, evaluated as glanimallday, in F0 females in the 200 and 1000 ppm groups was decreased (statistically significant) relative to that in the control group throughout gestation. The decreases were attributed to the test article. However, similar to the pre-mating period, the decreased water consumption was most likely due to palatability, taste and/or smell, of the test article, an irritant, and not to systemic toxicity.

Average test article consumptions (mg/kg/day) during gestation are presented in Table 2.

LACTATION
Water consumption in the 50 ppm group was similar to that in the control group throughout lactation. Water consumption, evaluated as g/animal/day, in F0 females in the 200 and 1000 ppm groups was reduced (generally statistically significant) relative to that in the control group throughout lactation. The decreases were attributed to the test article. However, similar to the pre-mating and gestation periods, the decreased water consumption was most likely due to palatability, taste and/or smell, of the test article, an irritant, and not to systemic toxicity.

Average test article consumptions (mg/kg/day) during lactation are presented in Table 2.

GESTATION LENGTH AND PARTURITION
No test article-related effects were noted on mean gestation lengths or parturition at any dose concentration. Mean F0 gestation lengths in the test article-treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 50, 200 and 1000 ppm groups were 21.7, 2 1.8 and 2 1.7 days, respectively, compared to mean gestation lengths of 2 1.7 days in the concurrent control group and 21.8 days in the WIL historical control data. No signs of dystocia were noted at any dose concentration.

F0 SPERMATOGENIC ENDPOINT EVALUATIONS
No test article-related effects were observed on Fo spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dose concentration. Differences from the control group were slight and were not statistically significant.

F0 NECROPSY EXAMINATIONS
MACROSCOPIC EXAMINATION
No test article-related mortalities or internal findings were noted at any dose concentration. F0 male no. 83017 in the 1000 ppm group was euthanized in extremis during week 9, and F0 male no. 83078 in this group was found dead during week 17. F0 female no. 83155 in the 200 ppm group was euthanized in extremis on gestation day 25. Male no. 83017 was internally normal. The cause of moribundity was attributed to mechanical trauma (malaligned upper incisors, swollen face, possible fracture of maxilla) and not to the test article. Male no. 83078 had green discoloration of the prostate, green fluid contents in the urinary bladder, calculi in the kidneys and urinary bladder, a distended urinary bladder and ureters, dilated renal pelves, a white area on the left renal cortex, and depressed areas in both kidneys. The death of this male was attributed to the kidney and urinary bladder findings. Female no. 83 155 had one retained dead fetus in utero and red fluid contents in the abdominal cavity. The moribundity of this female was attributed to pregnancy complications. These mortalities were not attributed to the test article as no deaths occurred in the 1000 ppm dose concentration in the F1 generation.

At the scheduled necropsy of F0 animals, no test article-related internal findings were observed. Findings observed in the treated groups, including depressed areas on the kidneys, small seminal vesicles, dark red contents in the intestines or stomach, and small and soft testes and epididymides, were noted infrequently or at similar frequencies in the control group and/or occurred in a manner that was not dose-related. The mean numbers of implantation sites and sites unaccounted for in the test article-treated groups were similar to the control group values; no statistically significant differences were noted.

ORGAN WEIGHTS
No test article-related effects were observed on F0 organ weights at any dose concentration. Statistically significant decreases in mean absolute andlor relative (to final body weight) spleen weights were observed in F0 females at all dose concentrations. However, because the decreases were slight and did not occur in a manner that was dose related, they were not attributed to the test article. Mean absolute uterine weight was decreased (statistically significant) in females in the 200 ppm group; because of the absence of an effect in the 1000 ppm group females, this decrease was not attributed to the test article. Statistically significant increases in mean relative kidney, left testicular, epididymal and cauda epididymal, and adrenal weights were observed in F0 males in the 1000 ppm group. In addition, mean relative brain and kidney weights were increased in the 1000 ppm group F0 females (mean relative kidney weight was also increased in the 200 ppm group F0 females). However, because similar increases were not observed in the mean absolute weights in F0 males and females, they were attributed to decreased mean final body weights and not to the test article.

MICROSCOPIC EXAMINATION
There were no microscopic changes observed in the tissues specified for examination from any of the male and female F0 generation adult rats that were considered to be related to exposure at 50, 200 or 1000 ppm of the test article in the drinking water. There were several different microscopic changes observed in the various organs and tissues specified for examination, but these changes generally were of the type that occur spontaneously in laboratory rats used in reproductive studies. These changes generally occurred at low or similar frequencies between the control and high dose group rats and their incidence or severity were not influenced by exposure to the test article in the drinking water.

The most common incidental findings were diffuse cortical vacuolation in the adrenal glands; dilatation of mucosal glands in the stomach; focal atrophy, multifocal chronic inflammation or suppurative inflammation in the prostate; and lurninal distention and pigmented macrophages in the uterus. The pigmented macrophages in the uterus are typical of those that occur normally at implantation sites.

The kidneys of rats of all groups had several different miscellaneous microscopic changes that usually occurred at single or very low incidences and included focal cortical tubular basophilia, medullary cysts, foci of subcapsular depression in the cortex, focal chronic nephritis, chronic pyelitis or pyelonephritis, and pelvic dilatation or mineralization. One 1000 ppm group male with pyelonephritis also had mucosal necrosis and calculi of the
urinary bladder.

There were a few F0 generation male and female rats that were designated as reduced fertility animals (rats that failed to mate, conceive, sire or deliver healthy offspring or for which estrus cyclicity or sperm number, motility or morphology were affected). There were generally no microscopic changes related to exposure to the test article that could be associated with the reduced fertility. The incidence of reduced fertility animals was low with two males and two females of the control group and one male and one female each in the 200 and 1000 ppm groups affected. The changes that were observed in these tissues also were considered to have occurred spontaneously. Only in the 200 ppm female rat (no. 83155), which was euthanized on gestation day 25 and was found to have a dead fetus retained in utero were there histomorphologic changes considered to be related to fertility. These changes consisted of endometrial inflammation and distention of the uterus and stromal polyp in the cervix.

GENERATION F1
CLINICAL OBSERVATIONS AND SURVIVAL
There were no test article-related mortalities or clinical findings noted at any dose concentration. Female no. 83126-06 in the 200 ppm group was euthanized in extremis during study week 36. This female had a swollen urogenital area and a firm moveable mass in the left lateral abdominal area on the day of euthanasia. Because of the absence of mortalities or moribundity in the 1000 ppm group F1 females, the moribundity of this female was not attributed to the test article. Animal no. 83218-04 in the 200 ppm group was incorrectly identified as a niale at the time of sex determination. This error was discovered on PND 28, and the animal was euthanized and necropsied on PND 30. No significant clinical findings were noted in this animal at tlie time of euthanasia. All other FI animals survived to the scheduled necropsies. No test article-related clinical findings were noted. Findings observed in the test article-treated groups, including hair loss, scabbing and red material on various body surfaces and soft stool, were noted infrequently and/or at similar frequencies in the control group.

REPRODUCTIVE PERFORMANCE
No test article-related effects on F1 reproductive performance were observed at any dose concentration. Male mating indices were 96.7%, 96.7%, 96.6% and 100% in the control, 50, 200 and 1000 ppm groups, respectively. Female mating indices were 96.7%, 96.7%, 96.7% and 100% in the same respective groups. Male fertility indices were 86.7%, 96.7%, 89.7% and 93.3% in the control, 50, 200 and 1000 ppm groups, respectively. Female fertility indices were 86.7%, 96.7%, 90.0% and 93.3% in the same respective groups. No statistically significant differences were noted between the control and treated groups. Males that did not sire a litter numbered 4, 1, 3 and 2 in the control, 50, 200 and 1000 ppm groups, respectively. Females that had evidence of mating but did not deliver numbered 3, 0, 2 and 2 in the same respective groups.

The mean numbers of days between pairing and coitus in the test article-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were similar to the control group value. None of these differences were statistically significant.

BODY WEIGHTS
WEEKLY
No test article-related effects were noted on mean body weights or body weight gains prior to breeding at dose concentrations of 50 and 200 ppm. Mean body weight gains were decreased in F1 males in the 1000 ppm group beginning at week 17-18 and continuing through week 21-22. Mean F1 female body weight gain was decreased for week 17-18. These differences fi-om the control group were generally statistically significant. Mean body weight gains in these animals were generally similar to the control group values for the remainder of the generation (males) or pre-breeding period (females). Cumulative mean body weight gains in the 1000 ppm group males were decreased (statistically significant) during the pre-breeding period (weeks 18-28) and during the entire generation (weeks 18-36). Cumulative mean body weight gain for the F1 females was similar to that in the control group for the pre-breeding period. Mean weekly body weights in the 1000 ppm group F1 males and females were decreased (statistically significant) relative to the control group values throughout the generation (males) or during the pre-breeding period (females).

Mean body weight gains, cumulative mean body weight gains and body weights in the 50 and 200 ppm groups were generally similar to the control group values for the pre-breeding period (females) or the entire generation (males). A few statistically significant increases and decreases in mean body weight gain were observed in these groups during the study. However, because the differences were transient and did not occur in a dose-related manner, they were not attributed to the test article.

GESTATION
No test article-related effects were noted on mean body weights or body weight gains during gestation at dose concentrations of 50 and 200 ppm. Mean body weight gains in the 1000 ppm group F1 females were similar to the control group values during gestation days 0-4, 4-7, 7- 11 and 11-14. For gestation days 14-17 and 17-20 and when the entire gestation period (gestation days 0-20) was evaluated, mean body weight gains in the 1000 ppm group were decreased (statistically significant) relative to the control group values. As a result of decreased mean body weights during the pre-mating period, mean body weights in the 1000 pprn group were decreased (statistically significant) relative to the control group values throughout gestation.

Mean body weights and body weight gains in the 50 and 200 ppm groups were similar to the control group values throughout gestation; differences were slight and were not statistically significant.

LACTATION
No test article-related effects were noted on mean body weights or body weight gains during lactation at dose concentrations of 50 and 200 ppm. Mean body weight gains in the 1000 ppm group F1 females were similar to the control group values for lactation days 1-4 and 4-7. Mean body weight gain in this group was decreased (statistically significant) relative to the control group value for lactation days 7-14. A less substantial mean body weight loss (statistically significant) was observed in the 1000 ppm group than in the control group for lactation days 14-21. When the entire lactation period (lactation days 1-21) was evaluated, mean body weight gain in the 1000 ppm group was similar to the control group value. As a result of decreased mean body weights during the pre-mating and gestation periods, mean body weights in the 1000 ppm group were decreased (statistically significant) relative to the control group values throughout lactation.

In the 200 ppm group, mean body weight gains were generally similar to the control group values during lactation days 1-4, 4-7 and 7-14. A less substantial mean body weight loss (statistically significant) was observed in the 200 ppm group than in the control group for lactation days 14-21. When the entire lactation period (lactation days 1-21) was evaluated, mean body weight gain in the 200 ppm group was similar to the control group value. Mean body weights in the 200 ppm group were similar to the control group values throughout lactation.

Mean body weights and body weight gains in the 50 ppm group were unaffected by treatment throughout lactation. Differences from the control group were slight and were not statistically significant.

FOOD CONSUMPTION
WEEKLY
No test article-related effects were noted on food consumption prior to breeding at dose concentrations of 50 and 200 ppm. Food consumption, evaluated as g/animal/day, in F1 males and females in the 1000 ppm group was slightly decreased beginning at week 18-19 and continuing through the remainder of the pre-breeding period (females) or generation (males). The differences from the control group were generally statistically significant. Food efficiency in the 1000 ppm group males and females was increased (statistically significant for the females) relative to the control group values for week 18-19. For the remainder of the generation (males) or the pre-breeding period (females), food efficiency in the 1000 ppm group was similar to that in the control group.

Food consumption and food efficiency in the 50 and 200 ppm groups were unaffected by the test article. Several statistically significant differences between the control and treated groups were noted, but no test article-related trends were apparent.

GESTATION
No test article-related effects were noted on food consumption during gestation at dose concentrations of 50 and 200 ppm. Food consumption, evaluated as glanimallday, in F1 females in the 1000 ppm group was decreased (statistically significant) relative to that in the control group throughout gestation. Food efficiency in the 1000 ppm group was generally similar to that in the control group during gestation. Differences between the control and test article-treated groups were slight and were not statistically significant.

Food consumption and food efficiency in the 50 and 200 ppm groups were unaffected by treatment with the test article. Differences from the control group were slight and were not statistically significant.

LACTATION
No test article-related effects were noted on food consumption during lactation at dose concentrations of 50 and 200 ppm. Food consumption, evaluated as glanimallday, in the 1000 ppm group F1 females was similar to that in the control group for lactation days 1-4 and 4-7. Food consumption in this group was decreased (statistically significant) relative to that in the control group for lactation days 7- 14 and 14-21 and when the entire lactation period (lactation days 1-21) was evaluated. Food efficiency in the 1000 ppm group was similar to that in the control group for lactation days 1-4,4-7 and 7-14. During lactation days 14-2 1, food efficiency in the 1000 ppm group was increased (statistically significant) relative to that in the control group. When the entire treatment period (lactation days 1-21) was evaluated, food efficiency in the 1000 ppm group was found to be similar to
that in the control group.

In the 200 ppm group, food consumption was similar to that in the control group throughout lactation. Food efficiency in this group was unaffected by treatment for lactation days 1-4, 4-7 and 7-14. For lactation days 14-21, food efficiency was increased (statistically significant) in the 200 ppm group relative to the control group value. When the entire lactation period (lactation days 1-21) was evaluated, food efficiency in the 200 ppm group was similar to that in the control group. Food consumption and food efficiency during lactation in the 50 ppm group were unaffected by the test article. No statistically significant differences from the control group were noted.

WATER AND TEST ARTICLE CONSUMPTION
PND 21-28
During postnatal days 21 -28, water consumption, evaluated as g/litter/day, was decreased in a dose-dependent manner in the F1 pups in the 50, 200 and 1000 ppm groups. The differences from the control group were always statistically significant in the 1000 ppm group and often statistically significant in the 50 and 200 ppm groups. The decreases were attributed to the test article. However, the decreased water consumption was most likely due to palatability, taste and/or smell, of the test article, an irritant, and not to systemic toxicity.

WEEKLY
Water consumption in the 50 ppm group males and females was similar to that in the control group throughout the generation (males) or pre-breeding period (females). Water consumption, evaluated as glanimallday, in the F1 males and females in the 1000 ppm group and in the F1 females in the 200 ppm group was decreased beginning at week 17-18 and continuing through the remainder of the generation (males) or the pre-breeding period (females). In the 200 ppm group, water consumption for the males was similar to that in the control group for week 17-1 8 and decreased for the remainder of the generation. The differences in the 200 and 1000 ppm groups were statistically significant. The decreases were attributed to the test article. However, the decreased water consumption was most likely due to palatability, taste andlor smell, of the test article, an irritant, and not to systemic toxicity.

Average weekly test article consumptions (mg/kg/day) are presented in Table 3.

GESTATION
Water consumption in the 50 ppm group was similar to that in the control group throughout gestation. Water consumption, evaluated as glanimallday, in F1 females in the 200 and 1000 ppm groups was decreased (statistically significant) in a dose-dependent manner relative to that in the control group throughout gestation. The decreases were attributed to the test article. However, similar to the pre-mating period, the decreased water consumption was most likely due to palatability, taste andor smell, of the test article, an irritant, and not to systemic toxicity.

Average test article consumptions (mg/kg/day) during gestation are presented in Table 3.

Continued below
Dose descriptor:
NOAEL
Effect level:
15 - 22 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Parental Parameters - Decreased body weight and feed consumption at the high dose level.
Remarks on result:
other: Generation: P - Parental Parameters (migrated information)
Dose descriptor:
NOAEL
Effect level:
19 - 26 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Parental Parameters - Decreased body weight and feed consumption at the high dose level.
Remarks on result:
other: Generation: F1 - Parental Parameters (migrated information)
Dose descriptor:
NOAEL
Effect level:
69 - 93 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Reproductive Parameters - There were no reproductive effects noted in F0 to F1 generation at the highest concentration in the drinking water examined.
Remarks on result:
other: Generation: F1 - Reproductive Parameters (migrated information)
Dose descriptor:
NOAEL
Effect level:
86 - 115 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Reproductive Parameters - There were no reproductive effects noted in F1 to F2 generation at the highest concentration in the drinking water examined.
Remarks on result:
other: Generation: F2 - Reproductive Parameters (migrated information)
Dose descriptor:
NOAEC
Effect level:
200 mg/L drinking water
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Neonatal Toxicity - There were no neonatal toxicity effects noted in F0 to F1 generation at the highest concentration in the drinking water examined.
Remarks on result:
other: Generation: F1 Neonatal Toxicity (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased mean offspring body weights in the latter part of both the F1 pre-weaning period (PND 7-21) and the F2 pre-weaning period (PND 14-21).
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
F1 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
No test article-related effects were noted on the number of F1 pups born, live litter sizes, percentages of males per litter and postnatal survival at any dose concentration. The mean numbers of F1 pups born, live litter sizes and percentages of males per litter were similar among all groups; no statistically significant differences from the control group were noted. Postnatal survival on PND 0 (relative to number born) and during PND 0-1, 1-4, 4-7, 7- 14, 14-21, birth to PND 4 and PND 4-21 was unaffected by treatment with the test article; differences from the control group were slight and were not statistically significant.

GENERAL PHYSICAL CONDITION AND MORTALITIES
No test article-related effects were noted on pup survival or the general physical condition of the pups at any dose concentration during the pre-weaning period. The numbers of pups found dead during the lactation period were 7, 1, 6 and 16 in the control, 50, 200 and 1000 ppm groups, respectively. In addition, 3, 8, 7 and 10 pups in the same respective groups were missing and presumed to have been cannibalized. The physical condition of the F1 pups during lactation was generally similar in all groups, including the control group.

OFFSPRING BODY WEIGHTS
No test article-related effects on mean F1 offspring body weights were noted at dose concentrations of 50 and 200 ppm. Mean F1 offspring body weights in the 1000 ppm group were decreased during the later part of the pre-weaning period (PND 7-21); the differences from the control group were statistically significant and attributed to the test article. The mean male and female pup body weights on PND 1 in the 1000 ppm group were slightly reduced relative to the control group; the difference was statistically significant for the males. However, similar reductions were not observed in the F2 pups on PND 1 , the male weight reduction did not occur in a dose-related manner (the mean value in the 50 ppm group was less than the value in the 200 ppm group but was similar to the 1000 ppm group value) and no indication of growth retardation was observed in fetuses in a prenatal developmental toxicity study with 2-methyl-isothiazolin-3-one following gavage doses up to 40 mg/kg/day. Therefore, the slightly reduced mean pup body weights on PND 1 in the 1000 ppm group were not considered to be test article-related.

Mean F1 offspring body weights in the 50 and 200 ppm groups were unaffected by treatment with the test article. The only statistically significant difference from the control group was a decrease in the mean weight of F1 males in the 50 ppm group for PND 21. However, this transient decrease was not attributed to the test article.

NECROPSIES OF PUPS FOUND DEAD
No test article-related findings were observed in F1 pups that were found dead at any dose concentration during the pre-weaning period. Aside from the presence or absence of milk in the stomach, no internal findings were noted in these pups.

NECROPSIES OF WEANLINGS NOT SELECTED FOR ORGAN WEIGHTS - PND 21
No test article-related findings were observed in pups that were necropsied on PND 21 at any dose concentration. The only finding noted was an accessory spleen in control group male pup no. 83111-01.

NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS - PND 21
MACROSCOPIC EXAMINATION
No internal findings were observed at the PND 21 necropsy of pups selected for organ weights at any dose concentration.

ORGAN WEIGHTS
No test article-related effects were noted on organ weights (absolute or relative to final body weight) at any dose concentration. Mean absolute spleen and thymus weights were decreased in the 1000 ppm group F1 male and female pups that were necropsied on PND 21. Mean absolute thymus weights were also decreased in the 200 ppm group F1 females. The differences from the control group were statistically significant for the females. Statistically significant increases were observed in mean relative F1 male and female brain weights in the 1000 ppm group. However, because similar effects were not observed in the mean absolute brain weights or mean relative spleen and thymus weights, these differences were attributed to the decreased mean final body weights in these animals, and not to the test article.

F1 DEVELOPMENTAL LANDMARKS
BALANOPREPUTIAL SEPARATION
There were no test article-related effects on the mean day of acquisition of balanopreputial separation or mean body weight on that day at any dose concentration. The mean day of acquisition of balanopreputial separation was increased (statistically significant) in the 1000 ppm group F1 males relative to the control group value. This delay was not considered to be a direct result of test article treatment, but a result of the statistically significant decrease in the mean body weight in these pups on the day of acquisition. In addition, no effects on anogenital distance were observed in the F2 male pups.

The mean day of acquisition of balanopreputial separation and the mean body weight on the day of acquisition were similar to control values in the 50 and 200 ppm groups.

VAGINAL PATENCY
There were no test article-related effects on the mean day of acquisition of vaginal patency or mean body weight on that day at any dose concentration. The mean day of acquisition of vaginal patency was increased (statistically significant) in the 1000 ppm group F1 females relative to the control group value. This delay was not considered to be a direct result of test article treatment, but a result of the statistically significant decrease in the mean body weight on the day of acquisition. Because animals did not gain weight at the same rate as the control group, they were not expected to mature as quickly. In addition, no effects on anogenital distance were observed in the F2 female pups.

The mean day of acquisition of vaginal patency and the mean body weight on the day of acquisition were similar to control values in the 50 and 200 ppm groups.

F2 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
No test article-related effects were noted on the mean numbers of F2 pups born, live litter sizes, the percentages of males per litter and postnatal survival at any dose concentration. The mean numbers of F2 pups born, live litter sizes and percentages of males per litter were similar among all groups; no statistically significant differences from the control group were noted. Postnatal survival on PND 0 (relative to number born) and during PND 0-1, 1-4, 4-7, 7-14, 14-21, birth to PND 4 and PND 4-21 was unaffected by treatment with the test article; differences from the control group were slight and were not statistically significant.

GENERAL PHYSICAL CONDITIONS AND MORTALITY
No test article-related effects were noted on mortality or the general physical condition of the F2 pups at any dose concentration during the pre-weaning period. The numbers of F2 pups found dead during the lactation period were 9, 9, 16 and 8 in the control, 50, 200 and 1000 ppm groups, respectively. In addition, 1, 2, 4 and 4 pups in the same respective groups were missing and presumed to have been cannibalized. The physical condition of the F2 pups during lactation was generally similar in all groups, including the control group.

ANOGENITAL DISTANCE
No test article-related effects on F2 anogenital distances were noted at any dose concentration. Anogenital distances on PND 1 for F2 male and female pups in the treated groups were similar to the control group values; differences were slight and were not statistically significant.

OFFSPRING BODY WEIGHTS
No test article-related effects on mean F2 offspring weights were noted at dose concentrations of 50 and 200 ppm. Mean F2 male and female pup body weights in the 1000 ppm group were similar to the control group values on PND 1, 4 and 7. On PND 14 and 21, mean F2 male and female pup body weights were decreased relative to the control group values; all of the differences were statistically significant.

Mean F2 pup body weights in the 50 and 200 ppm groups were similar to the control group values throughout the postnatal period; none of the differences were statistically significant.

NECROPSIES OF PUPS FOUND DEAD
No test article-related findings were observed in F2 pups that were found dead at any dose concentration during the postnatal period. Aside from the presence or absence of milk in the stomach, no internal findings were noted in these pups.

NECROPSIES OF WEANLINGS NOT SELECTED FOR ORGAN WEIGHTS - PND 21
No test article-related findings were observed in F2 pups that were not selected for organ weights at any dose concentration. Three pups in the 1000 ppm group each had a dilated a right renal pelvis. The only other finding noted was hydrocephaly, with increased cavitation of the lateral and third ventricles, in one control group pup.

NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS - PND 21
MACROSCOPIC EXAMINATION
At the necropsy of F2 pups that were selected for organ weights, no internal findings were noted at any dose concentration.

ORGAN WEIGHTS
No test article-related effects were noted on organ weights (absolute or relative to final body weight) at any dose concentration. Mean absolute spleen and thymus weights were decreased in the 1000 ppm group F1 male and female pups that were necropsied on PND 21 ; the differences from the control group were statistically significant. Statistically significant increases were observed in mean relative F2 male and female brain weights in the 1000 ppm group. However, because similar effects were not observed in the mean absolute brain weights or mean relative spleen and thymus weights, these differences were attributed to the decreased mean final body weights in these animals, and not to the test article.
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Details on Results - Parental Animals (continued)

Lactation

Water consumption in the 50 ppm group was similar to that in the control group throughout lactation. Water consumption, evaluated as g/animal/day, in F1 females in the 200 and 1000 ppm groups was decreased (statistically significant) relative to that in the control group throughout lactation. The decreases were attributed to the test article. However, similar to the pre-mating and gestation periods, the decreased water consumption was most likely due to palatability, taste and/or smell, of the test article, an irritant, and not to systemic toxicity.

Average test article consumptions (mg/kg/day) during lactation are presented in Table 3.

GESTATION LENGTH AND PARTURITION

No test article-related effects on mean F1 gestation lengths were noted at any dose concentration. Mean F1 gestation lengths in the test article-treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation length was 21.9 days in each of the treated groups, compared to mean gestation lengths of 22.0 days in the concurrent control group and 21.8 days in the WIL historical control data. No signs of dystocia were noted at any dose concentration.

Fl SPERMATOGENIC ENDPOINT EVALUATIONS

No test article-related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dose concentration. Statistically significant increases were observed in mean sperm motility in the 50 and 1000 ppm groups. However, the increases were slight and were not attributed to the test article. Other differences from the control group were slight and were not statistically significant.

F1 NECROPSY EXAMINATION

MACROSCOPIC EXAMINATION

No test article-related mortalities or internal findings were noted at any dose concentration. Female no. 83126-06 in the 200 ppm group was euthanized in extremis during study week 36. This female had a cervix, uterus and vagina that were distended, enlarged and had thick yellow contents. In addition, the female had a distended urinary bladder and ureters, a cystic left oviduct, dilated renal pelves, and pale kidneys. The macroscopic observations noted for this animal were suggestive of kidney and urinary bladder findings contributing to the moribund condition. Because no deaths were seen in the 1000 ppm group females in either generation, the moribund condition of female no. 83216-06 in the 200 ppm group was not attributed to the test article. Animal no. 83218-04 in the 200 ppm group was incorrectly identified as a male at the time of sex determination. This error was discovered on PND 28, and the animal was euthanized and necropsied on PND 30. No significant internal findings were noted at the necropsy of this animal.

At the scheduled necropsy of F1 animals, no test article-related internal findings were observed at any dose concentration. Findings observed in the treated groups, including dilated renal pelves, depressed areas on the kidneys, soft testes and small epididymides,

were noted infrequently or at similar frequencies in the control group andlor occurred in a manner that was not dose-related. The mean numbers of implantation sites and sites unaccounted for in the test article-treated groups were similar to the control group values;

no statistically significant differences were noted.

ORGAN WEIGHTS

No test article-related effects were observed on F1 organ weights at any dose concentration. Mean absolute male liver and spleen weights were decreased (statistically significant) in the 1000 ppm group relative to the control group values. However, because of the absence of similar effects on the relative (to final body weight) values, the decreases were attributed to the decreased mean final body weight in this group. In the 1000 ppm group females, the mean absolute brain weight was slightly decreased (statistically significant) relative to the control group value, while the mean relative brain weight was increased (as a result of the decrease in mean final body weight). Because of the small magnitude of the effect on the mean absolute brain weight, it was not attributed to the test article. Mean absolute pituitary weights were decreased (statistically significant) in the 50 and 200 ppm group males; because of the absence of a dose-response relationship, these decreases were not attributed to the test article. Several statistically significant increases were observed in mean relative organ weights in males and females in the 1000 ppm group. Because of the absence of effects on the mean absolute weights, these increases were attributed to the decreased mean final body weights in these animals. The only other statistically significant differences from the control group were increased mean relative kidney weights in the 200 ppm group males

and females; however, because similar increases were not observed in the mean absolute values, these increases were not attributed to the test article.

MICROSCOPIC EXAMINATION

Microscopic examination revealed no test article-related microscopic changes in the F1 generation male and female rats exposed to 50, 200 or 1000 ppm of the test article in the drinking water. As in the F0 generation, the various microscopic changes observed were typical of those that occur spontaneously in male and female rats used in reproductive studies. The changes generally occurred at single, low or similar frequencies among the groups.

The most common incidental microscopic changes in these rats consisted of diffuse cortical vacuolation in the adrenal glands, multifocal chronic or focal suppurative inflammation in the prostate, dilatation of mucosal glands in the stomach and/or luminal distention and pigmented macrophages in the uterus.

The kidneys had multiple different types of incidental microscopic changes that generally occurred at single or low incidences among the affected groups with no evidence of test substance relationship with their type, incidence or severity. Pelvic dilatation was the most common spontaneously occurring renal change.

Microscopic examination of the few F1 generation male and female rats designated as reduced fertility animals also showed no test article-related changes that could be associated with the altered reproductive functions included in the "reduced fertility" category. Also, there was no evidence of any increased incidence of affected male or female rats in the test article-treated groups. The changes that were observed in these animals were considered to have occurred spontaneously and to have been incidental to exposure to the test article in the drinking water.

A numeric evaluation of the ovarian primordial follicles of the F1 generation 200 and 1000 ppm group females showed no evidence of an effect on the amount of primordial follicles. Based on the counts of the primordial follicles, there was no statistically

significant difference in the group means of primordial follicles between the F1 generation control and 1000 ppm groups.

Table 2 Average weekly test article consumptions (mg/kg/day) - F0 Generation

 Dosage Level     Average Test Article Consumption (mg/kg/day)      
 (ppm)  Males  Femalesa  Females - Gestationb  Females - Lactationc
 0  0  0  0  0
 50  4  7  7  13
 200  15  22  23  48
 1000  69  93  89  220

a= Values presented are for the pre-breeding period (weeks 0-1 through 9- 10)

b= Values presented are for the entire gestation period (gestation days 0-20).

c = Values presented are for the entire lactation period (lactation days 1-21).

Table 3 Average weekly test article consumptions (mg/kg/day) - F1 Generation

 Dosage level           Average Test Article Consumption (mg/kg/day)
 (ppm)  Males  Femalesa  Females - Gestationb  Females - Lactationc
 0  0  0  0  0
 50  5  8  6  12
 200  19  26  20  47
 1000  86  115  84  207

a = Values presented are for the pre-breeding period (weeks 17- 18 through 27-28).

b = Values presented are for the entire gestation period (gestation days 0-20).

c = Values presented are for the entire lactation period (lactation days 1-21).

Conclusions:
Based on the results of this study, a dose concentration of 1000 ppm (69-93 mg/kg/day for the F0 premating period and 86-115 mg/kg/day for the F1 pre-mating period) was considered to be the NOAEL (no-observed-adverse-effect level) for reproductive toxicity of 2-methyl-4-isothiazolin-3-one when administered in the drinking water to rats. A dose concentration of 200 ppm (15-22 mg/kg/day for the F0 pre-mating period and 19-26 mg/kg/day for the F1 pre-mating period) was considered to be the NOAEL for parental systemic toxicity. A dose concentration of 200 ppm was considered to be the NOAEL for neonatal toxicity. The only neonatal effect observed in the F1 and F2 offspring were decreased body weights during the latter part of the pre-weaning period.
Executive summary:

Objective

This study was conducted to evaluate the potential adverse effects of treatment of F0 and F1 parental animals with 2-methyl-4-isothiazolin-3-one administered in the drinking water on the reproductive capabilities, encompassing growth, mating, parturition and lactation, of the F0 and F1 generations and F1 and F2 neonatal survival, growth, and development. There was one mating period for both the F0 and F1 generations; one set of litters was produced in each generation.

Study Design

The test article, 2-methyl-4-isothiazolin-3-one, was administered on a continuous basis in the drinking water at dose concentrations of 50, 200 and 1000 ppm for a minimum of 70 consecutive days prior to mating. F0 animals were approximately six weeks of age at the beginning of test article administration; administration of the test article to F1 offspring selected to become the F1 generation began at weaning. The F0 and F1 males received the test article throughout mating and continuing until the day of euthanasia.

The F0 and F1 females received the test article throughout mating, gestation and lactation, and continuing until the day of euthanasia.

All animals were observed twice daily for appearance and behavior. Clinical observations, body weights, and water and food consumption were recorded at appropriate intervals prior to mating and during gestation and lactation. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day 21. For both generations (F1 and F2), eight pups per litter (four per sex, when possible) were selected on postnatal day (PND) 4 to reduce the variability among the litters. Administration of the test

article to the F1 animals was initiated on PND 22. Offspring (30/sex/group) from the pairing of the F0 animals were selected to constitute the F1 generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats. Unselected F1 and all F2 pups were necropsied on PND 21; selected organs were weighed. Each surviving F0 and F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups,

respectively; selected organs were weighed. Spermatogenic endpoints (sperm motility, morphology and numbers) were evaluated for all F0 and F1 males, and ovarian primordial follicle counts were recorded for all F1 females in the control and high-dose groups. Designated tissues from all F0 and F1 parental animals and from all parental animals that died or were euthanized in extremis were examined microscopically. In addition, reproductive organs from females that failed to deliver in the low- and mid-dose groups, and

from the males with which they were paired, were examined microscopically.

Results

No test article-related mortalities or clinical findings were observed at any dose concentration. Reproductive performance (estrous cycles, mating and fertility indices, mean numbers of days between pairing and coitus, and mean gestation length) and parturition in the F0 and F1 animals were unaffected by the test article. No test article-related macroscopic or microscopic findings or effects on mean organ weights were observed in F0 or F1 parental animals at any dose concentration. Spermatogenic endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in the F0 and F1 males were unaffected by the test article. No test article-related effects were observed on F1 and F2 pup survival or the general physical condition of the pups during the preweaning period. No test article-related macroscopic findings or effects on organ weights were observed in F1 or F2 pups at the scheduled necropsies; no test article-related macroscopic findings were noted for found dead or euthanized in extremis F1 or F2 pups. No direct effects of the test article were observed on the mean days of acquisition of balanopreputial separation and vaginal patency in the F1 pups. Anogenital distances for the F2 pups were unaffected by treatment with the test article.

Decreased water consumption was noted in F0 males at all dose levels and in the F1 males and both generations of females at the 200 and 1000 ppm dose levels during the pre-breeding period. During gestation and lactation, water consumption was decreased in both the F0 and F1 females in the 200 and 1000 ppm dose groups. In addition, water consumption was reduced at all dose levels for the F1 generation during the week following weaning (PND 21-28) when the animals were housed by litter. The decreased water consumption was not indicative of systemic toxicity, but most likely due to aversion to the taste and/or smell of the test article, an irritant. No other effects of the test article were observed on any parameters evaluated at dose concentrations of 50 and 200 ppm.

Test article-related effects were noted in the 1000 ppm group and consisted of:

  • Decreased mean body weight gains in males and females during the first one-to-five weeks of each generation and during the middle and/or late parts of gestation and lactation; decreased mean body weights beginning at week 2 or 3 and continuing throughout the remainder of the generation (F0) or throughout the generation (F1).
  • Decreased food consumption throughout each respective generation (males); decreased food consumption throughout the pre-breeding period and during middle-to-late gestation and middle-to-late lactation (F0 females); decreased food consumption throughout the pre-breeding and gestation periods and during middle-to-late lactation (F1 females); decreased food efficiency during the first four or five weeks of the study (F0 only)
  • Decreased mean offspring body weights in the latter part of both the F1 pre-weaning period (PND 7-21) and the F2 pre-weaning period (PND 14-21)

Conclusions

Based on the results of this study, a dose concentration of 1000 ppm (69-93 mg/kg/day for the F0 premating period and 86-115 mg/kg/day for the F1 pre-mating period) was considered to be the NOAEL (no-observed-adverse-effect level) for reproductive toxicity of 2-methyl-4-isothiazolin-3-one when administered in the drinking water to rats. A dose concentration of 200 ppm (15-22 mg/kg/day for the F0 pre-mating period and 19-26 mg/kg/day for the F1 pre-mating period) was considered to be the NOAEL for parental systemic toxicity. A dose concentration of 200 ppm was considered to be the NOAEL for neonatal toxicity. The only neonatal effect observed in the F1 and F2 offspring were decreased body weights during the latter part of the pre-weaning period.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
69 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

There are 2 key studies identified for this endpoint -

Developmental toxicity study (oral) in rats - 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.

Developmental toxicity study (oral) in rabbits - Based on the results of this study, a dose level of 10 mg/kg/day was considered to be the no observed- adverse-effect-level (NOAEL) for maternal toxicity and a dose level of 30 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for developmental toxicity of 2 -methyl-4-isothiazolin-3-one when administered orally by gavage to rabbits.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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.
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.
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.
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.

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity
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.
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

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.

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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 February - 1 March 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP?GUideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
One-hundred ten sexually mature, virgin female New Zealand White rabbits were received in good health from Covance Research Products, Inc., Denver, Pennsylvania, on January 8, 2002. The animals were approximately 5.0-5.5 months old. Upon receipt, each female was examined by a qualified technician. The rabbits were initially weighed on January 9, 2002. Each rabbit was uniquely identified by a color-coded plastic eartag displaying the animal number and housed for 21 days for acclimation purposes. During the acclimation period, the animals were observed twice daily for mortality and moribundity. Body weights were recorded prior to the initiation of artificial insemination.

ANIMAL HOUSING
Upon arrival, all rabbits were individually housed in clean, stainless steel wire-bottom cages suspended above ground corncob bedding (Bed-O’Cobs®; The Andersons, Industrial Products Division, Maumee, Ohio). Nesting material was not required, as the females were euthanized prior to the date of expected parturition.

DIET, DRINKING WATER AND MAINTENANCE
The basal diet used in this study, PMI Nutrition International, Inc. Certified Rabbit LabDiet® 5322, 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 the standard operating procedures. The results of the diet and water analyses are maintained at WIL Research Laboratories, Inc. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. The basal diet was restricted to approximately 150 g/animal/day during the acclimation period and throughout the study. Reverse-osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum during the study.

ENVIRONMENTAL CONDITIONS
All rabbits were housed throughout the acclimation period and during the study in an environmentally controlled room. Controls were set to maintain a temperature of 66 ± 5ºF (19 ± 3ºC) and a relative humidity of approximately 50 ± 20%. Room temperature and relative humidity were recorded daily. The actual temperature ranged from 66.2ºF to 67.9ºF (19.0ºC to 19.9ºC) and humidity ranged from 47.7% to 59.2% 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
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, animals judged to be in good health and meeting acceptable body weight requirements (3.0 to 4.5 kg) were selected for use in the computerized randomization procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into the WIL Toxicology Data Management System (WTDMSTM). A printout containing the animal numbers, corresponding body weights and individual group assignments was generated based on body weight stratification in a block design. The animals were then arranged into groups according to the printout. The experimental design for WIL-91006 consisted of three test article-treated groups and one control group composed of 25 rabbits each. The selected females were approximately six months old at insemination. Body weight values of the selected females ranged from 3189 g to 4236 g on day 0 of gestation.


Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The vehicle utilized in preparation of the test mixtures and for administration to the control group animals was deionized water, prepared on-site.

The test mixtures were administered orally by gavage, via French rubber catheters (Jorgensen Laboratories, Inc., Loveland, Colorado, or equivalent), once daily during gestation days 6-28. 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. The basal diet (150 g/day) was offered to the animals as soon as possible following dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purposes of dosage calculations, a correction factor of 2.0 was used. All doses were administered as mg of active ingredient per kg body weight per day. The test article was stored at room temperature and was considered stable under this condition.

SAMPLING AND ANALYSES
Homogeneity and eight-day stability of the test article in the vehicle at a concentration range of 0.5-8.0 mg/mL (encompassing the range of 0.6-6 mg/mL used in the current study) was established in a previous study (WIL-910041; Rohm and Haas Report No. 01RC-269A).

Samples (10 mL each) for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group). Formulations used in dosing and analyzed for concentration verification were well within 10% of the target concentrations and, therefore, met the acceptance criteria.
Details on mating procedure:
Semen was collected from 13 resident male rabbits of the same strain that were obtained from the same source as the females. The collection apparatus consisted of an artificial vagina (Holborn Surgical Instruments Company, Limited, London, England) filled with warm water (approximately 55°C) and fitted with a graduated collection tube. After collection, the gelatinous plug (if any) was removed from each sample and the ejaculate was evaluated for volume, motility and concentration. Motility was determined microscopically on a percentage basis from a drop slide preparation using low-power magnification. The concentration of sperm/mL was determined using a standard dilution in a red blood cell pipette and a hemacytometer under high-power magnification. Semen with greater than 50% motility was diluted with 3.0 mL of 0.9% USP physiological normal saline and was maintained at 34-37°C in a water bath during the insemination procedure. The final concentration obtained was greater than 3 million motile sperm/mL.

Diluted semen from one male was used to inseminate an equal number of females in each group. A 0.25- to 0.50-mL aliquot of each diluted semen sample was deposited into the anterior vagina of each female with a plastic insemination pipette. Immediately following insemination, each doe was administered an intravenous injection (100 USP Units) of Novarel™ human chorionic gonadotropin (Ferring Pharmaceuticals Inc., Tarrytown, New York) via the marginal ear vein to induce ovulation. Artificial insemination procedures were performed from January 29 to January 31, 2002. The day of insemination was designated gestation day 0.
Duration of treatment / exposure:
gestation days 6 through 28
Frequency of treatment:
daily on gestation days 6 through 28
Duration of test:
animals were sacrificed on gestation day 29
No. of animals per sex per dose:
25 bred female rabbits/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
The test article, 2-methyl-4-isothiazolin-3-one, in the vehicle, deionized water, was administered orally by gavage to three groups of 25 artificially inseminated female New Zealand White rabbits once daily from gestation days 6 through 28. Dosage levels were 3, 10 and 30 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen at 5 mL/kg. 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 29, a laparohysterectomy was performed on all surviving females. The uteri, placentae and ovaries 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. Stomachs were retained for possible histopathologic examination. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.
Maternal examinations:
CLINICAL OBSERVATIONS AND SURVIVAL
All rabbits 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 29 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 aborted during the course of the study. Gross lesions were recorded but not preserved. The number and location of implantation sites and corpora lutea and the number of viable fetuses were recorded. Recognizable fetuses were examined externally and preserved in 10% neutral-buffered formalin. Stomachs from maternal animals were saved in 10% neutral-buffered formalin for possible histopathologic examination. The carcasses were then discarded.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Individual maternal body weights were recorded on gestation days 0 and 6-29 (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, 12-21, 21-29, 6-29 and 0-29.

Gravid uterine weight was collected and net body weight (the day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the day 0-29 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 0-29 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. On the occasions when food intake could not be measured for one of the days in a given interval, values were calculated using the appropriate number of days for that interval.
Ovaries and uterine content:
All surviving maternal rabbits were euthanized on gestation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents 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. 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.

Gross lesions were recorded but were not retained. Stomachs from maternal animals were retained in 10% neutral-buffered formalin for possible histopathologic examination. The carcasses were 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 (1964).

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

1. Group Mean Litter Basis:

Postimplantation Loss/Litter = [No. Dead Fetuses, Resorptions (Early/Late)]/Group/[No. Gravid Females/Group]

2. Proportional Litter Basis:

Summation Per Group (%) = [Sum (Postimplantation Loss/Litter (%))]/[No. Litters/Group]

Where:

Postimplantation Loss/Litter (%) = [(No. Dead Fetuses, Resorptions (Early/Late)/Litter)/(No. Implantation Sites/Litter)] x 100

Reference:
Salewski, E. Färbemethode 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 für Experimentelle Pathologie und Pharmakologi 1964, 247, 367.
Fetal examinations:
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. Each fetus was weighed, tagged and euthanized by an intrathoracic injection of sodium pentobarbital (if necessary). Nonviable fetuses (the degree of autolysis was minimal or absent) were examined, the crown-rump length measured, weighed and tagged individually. Crownrump measurements and degrees of autolysis were recorded for late resorptions and the tissues were discarded. Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe (1984) fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development by a method described in Woo and Hoar (1972).

The heads from approximately one-half of the fetuses in each litter were removed from the carcasses and placed in Bouin’s solution for subsequent processing and soft-tissue examination using the Wilson (1965) sectioning technique. Folded retinas were observed in a large number of fetuses in all groups following Bouin’s fixation and free-hand razor sectioning. As a result, sections of one affected fetus per litter were embedded in paraffin, stained with hematoxylin and eosin, and examined microscopically by John T. Yarrington, D.V.M., Ph.D., D.A.V.C.P., Senior Pathologist, to determine if the changes were artifactual. It was determined that there was no increase of retinal folds in the fetuses of test article-treated animals compared to the fetuses of the control group animals. The findings were deemed to be artifact upon microscopic examination. Heads from the remaining fetuses remained attached to the carcasses and 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 by a method similar to that described by Dawson (1926). External, visceral and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no 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).

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as a percentage of 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

References:
Stuckhardt, J.L.; Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Carcinogenesis and Mutagenesis 1984, 4, 181-188.

Woo, D.C.; Hoar, R.M. Apparent hydronephrosis as a normal aspect of renal development in late gestation of rats: the effect of methyl salicylate. Teratology 1972, 6, 191-196.

Wilson, J.G. Embryological Considerations in Teratology. In Teratology: Principles and Techniques; Wilson, J.G. and Warkany, J., Eds.; The University of
Chicago Press: Chicago, IL, 1965; pp 251-277.

Dawson, A.B. A note on the staining of the skeleton of cleared specimens with Alizarin Red S. Stain Technology 1926, 1, 123-124.
Statistics:
All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests for a minimum significance level of 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) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor et al., 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (1964) 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 (1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, the Mann-Whitney U-test (Kruskal-Wallis, 1952) 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 particular 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.

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

1. Group Mean Litter Basis:

Postimplantation Loss/Litter = [No. Dead Fetuses, Resorptions (Early/Late)]/Group/[No. Gravid Females/Group]

2. Proportional Litter Basis:

Summation Per Group (%) = [Sum (Postimplantation Loss/Litter (%))]/[No. Litters/Group]

Where:

Postimplantation Loss/Litter (%) = [(No. Dead Fetuses, Resorptions (Early/Late)/Litter)/(No. Implantation Sites/Litter)] x 100



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.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Decreased defecation (4/25 females, beginning on Gest. D. 7), Mean body weight loss during gestation days 6-9 (not stat. sig.), • Reduced mean food consumption during gestation days 6-9, 9-12 and 12-21 (generally stat. sig.) and Dark red areas in stomach.

Details on maternal toxic effects:
MATERNAL CLINICAL OBSERVATIONS AND SURVIVAL
Female no. 35589 in the 10 mg/kg/day group died on gestation day 19, likely as a result of an intubation error. There were no significant clinical observations made for this animal during the days prior to death. At the necropsy, foamy contents in the trachea and dark red discoloration and white areas on the lungs were noted. In the absence of any deaths in the 30 mg/kg/day group, as well as necropsy findings suggestive of an intubation error, the death of female no. 35589 was not considered test article-related.

Female no. 35681 in the 30 mg/kg/day group aborted on gestation day 25. This female had decreased defecation during gestation days 7-22 and 24, moderate to severe brown matting at the base of the tail, in the urogenital area and the hindlimbs during gestation days 23-25, and small feces and soft stool on gestation day 24. Wet brown material was noted in the cage pan of this animal on gestation days 23 and 24. No other significant clinical observations were made for this animal during the days preceding abortion. In addition, a substantial body weight loss (22%) and large reduction in food consumption were noted for this animal following the start of dosing. At necropsy, multiple stomach findings were noted, including dark red, irregularly shaped areas and white areas on the cardiac portion of the stomach lining. Since abortions in this strain are not uncommon, occurring in at least one female in 12 of the 42 control groups in the WIL historical control data, the single abortion in the 30 mg/kg/day group was not attributed to test article administration.

All other females survived to the scheduled necropsy on gestation day 29. Test article-related findings at the daily observations were limited to decreased defecation, which occurred in 3/24 surviving females in the 30 mg/kg/day group, beginning on gestation day 7. Other findings at the daily and 1-hour post-dosing observations occurred similarly in the control group, were noted in a limited number of animals and/or did not occur in a dose-related manner.

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
A test article-related mean body weight loss was noted in the 30 mg/kg/day group during gestation days 6-9; the difference from the control group was not statistically significant. Mean body weight gains in this group were similar to that in the control group throughout the remainder of gestation (days 9-12, 12-21 and 21-29) and when the entire treatment period (gestation days 6-29) was evaluated. Mean body weight gains in the 3 and 10 mg/kg/day groups were unaffected by test article administration. Mean body weights, net body weights, net body weight gains and gravid uterine weights in the 3, 10 and 30 mg/kg/day groups were similar to those in the control group; none of the differences from the control group were statistically significant.

MATERNAL FOOD CONSUMPTION
Test article-related reductions in mean food consumption (g/animal/day and g/kg/day) occurred in the 30 mg/kg/day group during gestation days 6-9, 9-12 and 12-21; the differences from the control group were statistically significant (p<0.05) for gestation days 6-9 and 9-12. When the entire treatment period (gestation days 6-29) was evaluated, food consumption in the 30 mg/kg/day group was slightly reduced; the difference was statistically significant (p<0.05) when evaluated as g/kg/day and was attributed to the test article.

Except for a slight, statistically significant (p<0.05) reduction in food consumption (g/kg/day) in the 10 mg/kg/day group during gestation days 19-20, food consumption (g/animal/day and g/kg/day) in the 3 and 10 mg/kg/day groups was similar to that in the control group throughout the treatment period. A similar reduction in food consumption during gestation days 19-20 was not observed in the 30 mg/kg/day group; therefore, this isolated slight decrease in the 10 mg/kg/day group was considered to be incidental in nature and unrelated to test article administration.

MATERNAL NECROPSY DATA
Female no. 35589 in the 10 mg/kg/day group died on gestation day 19, likely as a result of an intubation error. This female had foamy contents in the trachea, dark red discoloration and white areas on the lungs and four early resorptions in utero. In the absence of any deaths in the 30 mg/kg/day group, as well as necropsy findings suggestive of an intubation error, the death of female no. 35589 was not considered test article-related.

Female no. 35681 in the 30 mg/kg/day group aborted one late resorption with no apparent malformations on gestation day 25. Gross internal observations for this female included dark red and white areas on the stomach, four late resorptions with no apparent malformations, one mummified late resorption and two former implantation sites in utero and one late resorption with no apparent malformations retained in the vagina.

At the scheduled necropsy on gestation day 29, test article-related effects consisting of dark red areas in the stomachs were noted for 5/24 surviving females in the 30 mg/kg/day group. Accessory spleens, dark red areas in the lungs and cystic oviducts occurred in both the control and test article-treated groups at similar incidences and were not attributed to test article administration. A major blood vessel variation in which the left carotid artery arose from the brachiocephalic trunk was noted for female no. 35603 in the control group. Female no. 35590 in the 3 mg/kg/day group had a missing accessory lobe of the lung and female no. 35577 in the 10 mg/kg/day group had tan discoloration of the lungs. These findings (major blood vessel variation, missing accessory lobe of the lung and tan discoloration of the lungs) did not occur in the 30 mg/kg/day group and therefore were not considered to be test article-related. All other females were internally normal. Four, four, six and four females in the control, 3, 10 and 30 mg/kg/day groups, respectively, were nongravid. No other internal findings were noted.

GESTATION DAY 29 LAPAROHYSTERECTOMY DATA
There were no test article-related effects on intrauterine parameters (viable fetuses, post-implantation loss, fetal weights and fetal sex ratio) at any dose level. All parameters were comparable to the control group and/or within the range of values in the WIL historical control data. A statistically significant (p<0.05) increased mean litter proportion of late resorptions (3.8% per litter) occurred in the 30 mg/kg/day group. This was not considered to be related to test article administration, because it was within the WIL historical control data range (0.0-6.2% per litter), occurred in the absence of other fetal effects and was due primarily to female no. 35638, which had three late resorptions and four viable fetuses. An increased mean litter proportion of post-implantation loss (10.7% per litter) and decreased mean litter proportion of viable fetuses (89.3% per litter) in the 30 mg/kg/day group were not considered test article-related since the differences from the control group were not statistically significant and both values were within the WIL historical control data range (0.6-23.1% per litter for post-implantation loss and 76.9-99.4% per litter for viable fetuses). Mean fetal weight and fetal sex ratio in the 30 mg/kg/day group were unaffected by test article administration. All intrauterine parameters (viable fetuses, post-implantation loss, fetal weights and fetal sex ratios) in the 3 and 10 mg/kg/day groups, as well as mean numbers of corpora lutea and implantation sites in all test article-treated groups, were similar to those in the control group.
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL MORPHOLOGICAL DATA
The numbers of fetuses (litters) available for morphological evaluation were 137(21), 136(21), 119(17) and 114(20) in the control, 3, 10 and 30 mg/kg/day groups, respectively. Malformations were observed in 1(1), 4(3), 7(6) and 0(0) fetuses (litters) in these same respective dose groups.

EXTERNAL MALFORMATIONS AND VARIATIONS
No test article-related external malformations or developmental variations were noted at any dose level. External malformations were observed in two and one fetuses in the 3 and 10 mg/kg/day groups, respectively. Fetus no. 35614-08 in the 3 mg/kg/day group had vertebral agenesis. Skeletally, this malformation consisted of all vertebrae posterior to sacral vertebra no. 3 being absent, and misshapen and/or fused sacral vertebra nos. 1 through 3. Fetus no. 35648-01 in the 3 mg/kg/day group had spina bifida in which the vertebral column was open in the sacral region. This malformation consisted skeletally of the dorsal aspect of lumbar arch no. 6 through sacral arch no. 4 (bilateral) being reflected more laterally than is normal. Fetus no. 35605-05 in the 10 mg/kg/day group had carpal flexure (bilateral), which had no apparent skeletal origin. Since these malformations occurred in single fetuses and were not observed for fetuses at higher dose levels of the test article, these findings were not considered to be test article-related.

No external developmental variations were found in any fetuses in this study.

VISCERAL MALFORMATIONS AND VARIATIONS
No test article-related visceral malformations or developmental variations were noted at any dose level. Soft-tissue malformations were noted in one, two and two fetuses in the control, 3 and 10 mg/kg/day groups, respectively. Fetus nos. 35628-04 and 35578-04 in the control and 10 mg/kg/day groups, respectively, had lobular agenesis in which the right accessory lobe of the lungs was absent. Fetus no. 35654-07 in the 10 mg/kg/day group had an enlarged atrium (bilateral). These soft-tissue malformations (lobular agenesis of the lung and enlarged atrium) were not attributed to the test article, since they did not occur in a dose-related manner. Fetus no. 35648-04 in the 3 mg/kg/day group had a malpositioned right adrenal gland (more posterior than normal) and a malpositioned posterior vena cava that coursed to the left side of the body immediately following the right atrium. Fetus no. 35614-08 in the 3 mg/kg/day group also had a malpositioned right adrenal gland (more posterior than normal) and a malpositioned inferior vena cava that coursed transversely across the right kidney and rejoined in the normal position. These two soft-tissue malformations (malpositioned adrenal gland and malpositioned vena cava) were not considered test article-related since they did not occur in fetuses in the high-dose group.

Soft-tissue developmental variations, including major blood vessel variations (left carotid arising from the brachiocephalic trunk or right carotid and right subclavian arteries arising independently from the aortic arch, with no brachiocephalic trunk), accessory spleen(s), small or absent gallbladders, duplicate spleens, hemorrhagic irides and retrocaval ureters, occurred similarly in the control and test article-treated groups and/or were within the WIL historical control data range.

SKELETAL MALFORMATIONS AND VARIATIONS
No test article-related skeletal malformations or developmental variations were noted at any dose level. Skeletal malformations were observed for three and four fetuses (litters) in the 3 and 10 mg/kg/day groups, respectively. Fetus nos. 35614-08, 35648-01 and 35660-04 in the 3 mg/kg/day group and nos. 35581-01 and 35653-05 in the 10 mg/kg/day group had vertebral anomalies with associated rib anomalies. Generally, these skeletal malformations consisted of missing, extra, fused and/or malpositioned cervical centra, lumbar centra and/or ribs and/or costal cartilage with malpositioned points of fusion with the sternum. Since these malformations did not occur in fetuses in the 30 mg/kg/day group and the mean litter proportions of this malformation in the 3 and 10 mg/kg/day groups (2.9% and 2.0% per litter, respectively) did not exceed the maximum value in the WIL historical control data (7.3% per litter), this malformation was not considered to be test article-related. Fetus no. 35581-03 in the 10 mg/kg/day group had a rib anomaly, in which left rib nos. 9 and 10 were fused. Fetus no. 35677-01 in the 10 mg/kg/day group had multiple fused sternebrae. These malformations were not considered test article-related since they occurred in single fetuses and were not noted in fetuses in the 30 mg/kg/day group.

Skeletal developmental variations occurred at similar frequencies in the control and test article-treated groups and/or occurred in mean litter proportions that were within the WIL historical control data range.

SUMMARY OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
No test article-related fetal external, visceral and skeletal malformations or developmental variations were noted at any dose level. Fetal external, visceral and skeletal malformations were observed in 1(1), 4(3), 7(6) and 0(0) fetuses (litters) in the control, 3, 10 and 30 mg/kg/day groups. A statistically significant (p<0.05) increased mean litter proportion of total malformations occurred in the 10 mg/kg/day group; this was not attributed to the test article since no malformations occurred in the 30 mg/kg/day group. All malformations in this study were considered to be spontaneous in origin. Fetal developmental variations in the test article-treated groups were observed at similar frequencies in the control group and/or the WIL historical control data or occurred infrequently, and were not attributed to the test article.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Highest dose tested
Abnormalities:
no effects observed
Developmental effects observed:
no

No additional information available.

Conclusions:
Based on the results of this study, a dose level of 10 mg/kg/day was considered to be the no-observed-adverse-effect-level (NOAEL) for maternal toxicity and a dose level of 30 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for developmental toxicity of 2-methyl-4-isothiazolin-3-one when administered orally by gavage to rabbits.
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 6-28 of gestation, to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity.

STUDY DESIGN

The test article, 2-methyl-4-isothiazolin-3-one, in the vehicle, deionized water, was administered orally by gavage to three groups of 25 artificially inseminated female New Zealand White rabbits once daily from gestation days 6 through 28. Dosage levels were 3, 10 and 30 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen at 5 mL/kg. 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 29, a laparohysterectomy was performed on all surviving females. The uteri, placentae and ovaries 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. Stomachs were retained for possible histopathologic examination. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

RESULTS

No test article-related maternal effects were noted in the 3 and 10 mg/kg/day groups. No test article-related developmental effects, fetal malformations or developmental variations were noted at any dose level.

Test article-related maternal effects were noted in the 30 mg/kg/day group and included:

• Decreased defecation (4/25 females, beginning on gestation day 7)

• Mean body weight loss during gestation days 6-9 (not statistically significant)

• Reduced mean food consumption during gestation days 6-9, 9-12 and 12-21 (generally statistically significant)

• Dark red areas in the stomach (6/25 females)

CONCLUSIONS

Based on the results of this study, a dose level of 10 mg/kg/day was considered to be the no-observed-adverse-effect-level (NOAEL) for maternal toxicity and a dose level of 30 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for developmental toxicity of 2-methyl-4-isothiazolin-3-one when administered orally by gavage to rabbits.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
chronic
Species:
rabbit
Quality of whole database:
Klimisch 1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Description of key information

No information available

Justification for classification or non-classification

Based on the results of the 2 -generation study (oral via drinking water) in rats and developmental toxicity studies in rats and rabbits (oral via gavage), this test material will not be classified for reproductive effects.