2-methyl-2H-isothiazol-3-one

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

other: clear light yellow liquid

Details on test material:

The test article, 2-methyl-4-isothiazolin-3-one (supplied as a 50% solution in water known as KordekTM 5732; Industrial Microbiocide) (Lot No. 8001 5123), was received from Rohrn and Haas Company, Spring House, Pennsylvania. GLP analysis of the test material revealed 51.4% active ingredient.

All though not stated in the report, the remainder of the test substance composition was water.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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.

Administration / exposure

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)

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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

Effect levels (P0)

1

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)

2

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)

3

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)

4

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)

5

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)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

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

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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	Females^a	Females - Gestation^b	Females - Lactation^c
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	Females^a	Females - Gestation^b	Females - Lactation^c
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).

Applicant's summary and conclusion

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.