UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460 OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES 

### January 25, 2010 MEMORANDUM Subject: Reproduction and Fertility Effects Study for Octhilinone (Acticide® OIT) in Support of the 2007 RED PC CODE: 099901 DP Barcode:337621 From: Thru: To: S.L. Malish, Ph.D. Toxicologist, Team 1

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### Risk Assessment and Science Support Branch (RASSa)- - . . Antimicrobials Division (AD)[7510P] 

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### Nader Elkassabany, Ph.D., Team Leader,"'7{**~** 

### Team One, RASSB/AD [7510P] Avivah Jakob, Chemical Review Manager, Team 36, AD [7510P] Sponsor: THOR GmbH Synonyms: Octhilinone: 2-n-Octyl-4-Isothiazolin-3-one, OIT; Acticide® OIT Background: This study was submitted in support ofthe 2007 RED . The executive summary is noted below . The DER is included in this package. EXECUTIVE SUMMARY: In a two-generation reproduction study (MRID 47815801) Acticide ® OIT (2-n-Octyl-4- isothiazolin-3-one) (95.9%) was administered to Wistar rats (24/sex/dose) in diet at dose levels of 0, 300,800, or 1500 ppm (equivalent to 0,13-15,43-51, or 96-120 mg/kg bw/day based on test article recovery). In the parental P and F I 

### generations, no treatment-related clinical signs were observed. No mortalities occurred in the P generation, but in the F I 

### parental generation, one animal in the 300 ppm group was found dead and another in the 800 ppm was killed in extreImis. These deaths were not considered to be treatment-related. In the first generation, body weights were statistically significantly decreased in dams treated with 1500 ppm test

1

### substance on days 7 and 14 oflactation. No treatment-related changes in body weight occurred in the second generation, although the body weights of males and females ofthe 1500 ppm group were lower during the complete treatment period. Females in the P generation showed decreased absolute spleen weight and increased relative adrenal weight at 1500 ppm. No organ weight changes were noted for the second generation. At the histopathological examination at 1500 ppm, minimal to moderate diffuse hyperplasia/hyperkeratosis ofthe forestomach was observed in nine males and ten females ofthe first generation and in eight males and six females ofthe second generation, all at the 1500 ppm dose level. These observations correlated with the macroscopic observations of irregular surface ofthe forestomach in four males in the first generation and in four males and one female in the second generation. The parental systemic LOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when corrected for mean value of test article recovery), based on decreased body weight, hyperplasia/hyperkeratosis of the forestomach, decreased spleen weight, and increased adrenal weight. The parental systemic NOAEL is 800 ppm (43-45 mg/kg bw/day in males, 48-51 mg/kg bw/day in females for both generations when corrected for the mean value of test article recovery). 

### The offspring ofboth generations exhibited decreased body weight gain and decreased spleen weight at the 1500 ppm dose level. The offspring LOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when corrected for mean value of test article recovery), based on decreased body weight gain and decreased spleen weight. The offspring NOAEL is 800 ppm (43-45 mg/kg bw/day in males, 48-51 mg/kg bw/day in females for both generations when corrected for the mean value of test article recovery). 

### Measured sperm count, motility, and morphology and estrous cycle regularity were unaffected by treatment in both generations. Reproduction parameters (mating performance, duration of gestation, fertility, number of implantation sites, number of live/dead pups at first litter check) and breeding parameters (postnatal loss, living pups on day 4 post partum, breeding loss, living pups on day 21 post partum, viability index, weaning index) were unaffected by treatment in the first and second generations. Therefore, the reproductive NOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when corrected for the mean value of test article recovery). 

### This study is ACCEPTABLE - GUIDELINE 

### and satisfies the guideline requirement for a two -generation reproductive study (OPPTS 870.3800; OEeD 416) in rat.

22-n -Octyl -4-isothiazolin -3 -one Reproduction and Fertility Effects (2006) 1Page 1 of 18 OPPTS 870 .3800 1 OECD 416 EPA Reviewer : Steven L. Malish, Ph.D. , Toxicologist Team 1, RASSBI Antimicrobial Division EPA Secondary Reviewer: Nader Elkassabany, Ph.D ., Team 2, RASSB/Antimicrobial Division DATA EVALUATION RECORD STUDY TYPE: Reproduction and Fertility Effects Study - OPPTS 870 .3800, OEeD 416 . PC CODE: 099901 DP BARCODE: 337621 TEST MATERIAL (PURITY): Acticide 

### ® OIT (2-n-Octyl-4-isothiazolin- 3-one) (95 .9%) SYNONYMS: Octhilinone CITATION: Beekhuijzen, M. E. W. (2006). Two -generation reproduction toxicity study with Acticide 

### ® OIT by dietary administration in Wi star rats. NOTOX B.V, Hambakenweterring 7, 5231, Hertogenbosch, The Netherlands . Laboratory Project Number 397823, November, 9, 2006. MRID 47815801. Unpublished. SPONSOR: THOR GmbH, Speyer, Germany EXECUTIVE SUMMARY: In a two-generation reproduction study (MRID 47815801) Acticide ® OIT (2 -n-Octyl-4- isothiazolin-3 -one) (95 .9%) was administered to Wistar rats (24/sex/dose) in diet at dose levels of 0, 300, 800, or 1500 ppm (equivalent to 0,13 - 15,43 -51, or 96 - 120 mg/kg bw/day based on test article recovery). In the parental P and FI generations, no treatment-related clinical signs were observed . No mortalities occurred in the P generation, but in the F I parental generation, one animal in the 300 ppm group was found dead and another in the 800 ppm was killed in extremis. These deaths were not considered to be treatment-related . In the fir st generation, body weights were statistically significantly decreased in dams treated with 1500 ppm test substance on days 7 and 14 of lactation. No treatment-related changes in body weight occurred in the second generation, although the body weights of males and females ofthe 1500 ppm group were lower during the complete treatment period. Females in the P generation showed decreased absolute spleen weight and increased relative adrenal weight at 1500 ppm. No organ weight changes were noted for the second generation. At the histopathological examination at 1500 ppm, minimal to moderate diffuse hyperplasia/hyperkeratosis ofthe forestomach was observed in nine males and ten females ofthe first generation and in eight males and six females ofthe second generation, all at the 1500 ppm dose level. These observations correlated with the macroscopic observations of irregular surface ofthe forestomach in four males in the first generation and in four males and one female in the second generation. The parental systemic LOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when2- n-Octyl-4-isothiazolin- 3 -one Reproduction and Fertility Effects (2006) 1 Page 2 of 18 OPPTS 870.38001 OECD 416 

### corrected for mean value of test article recovery), based on decreased body weight, hyperplasia/hyperkeratosis of the forestomach, decreased spleen weight, and increased adrenal weight. The parental systemic NOAEL is 800 ppm (43-45 mg/kg bw/day in males, 48-51 mg/kg bw/day in females for both generations when corrected for the mean value of test article recovery). The offspring ofboth generations exhibited decreased body weight gain and decreased spleen weight at the 1500 

### ppm dose level. The offspring LOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when corrected for mean value of test article recovery), based on decreased body weight gain and decreased spleen weight. The offspring NOAEL is 800 ppm (43-45 mg/kg bw/day in males, 48-51 mg/kg bw/day in females for both generations when corrected for the mean value of test article recovery). Measured sperm count, motility, and morphology and estrous cycle regularity were unaffected by treatment in both generations. Reproduction parameters (mating performance, duration of gestation, fertility, number of implantation sites, number of live/dead pups at first litter check) and breeding parameters (postnatal loss, living pups on day 4 post partum, breeding loss, living pups on day 21 

### post partum, viability index, weaning index) were unaffected by treatment in the first and second generations. Therefore, the reproductive NOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when corrected for the mean value of test article recovery). This study is ACCEPT ABLE - GUIDELINE and satisfies the guideline requirement for a two generation reproductive study (OPPTS 870.3800; 

### OECD 416) 

### in rat. COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements are provided.2- n-Octyl-4-isothiazolin-3 -one Reproduction and Fertility Effects (2006) 1Page 3 of 18 OPPTS 870.3800 1OECD 416 

### I. MATERIALS AND METHODS: A. MATERIALS: 1. Test material: Description: Lot/batch #: Purity: Compound stability: CAS # of TGAI : Structure: 

### 2-n-Octyl-4-isothiazolin-3-one (ACTICIDE® OIT) Amber solid to liquid, depending on ambient temperature K3946511 95.9% a.i. Stable 26530-20- 1



### . 0


# .

rf t.(S ,N ....(CH2l,cHs 2. Negative Control: Standard laboratory animal diet. 2a Vehicle: Test substance mixed in feed without use of vehicle; no positive control 3. Test animals: Species: Strain: Age at study initiation: Wt. at study initiation: Source: Housing:

Diet: Water: Environmental conditions: Acclimation period: Rat Wistar rats Crl: (WI) BR (P) 5-6 wks; (F I) 3 wks (P) Males: 174- 177 g; Females: 148 - 149 g (F 1) Males : 152 - 186 g; Females: 131 - 152g Charles River Deutschland, Sulzfeld, Gennany Pre-mating/after weaning: Housed 4 animals/sex/cage in Macrolon cages with sterilized sawdust as bedding material and paper as cage-emichment Mating : Females were caged 1: 1 with males in suspended stainless steel cages with wire mesh floors Post-mating: Males were individually housed in stainless steel cages with wire mesh floors Post-coitum: Females were individually house in Macrolon cages containing sterilized sawdust and paper Lactation: Offspring were kept with the dam until tennination Standard powder laboratory animal diet (Diet VRF- 1 from Altromin GmbH, Lage, Gennany) ad libitum Tap water ad libitum Temperature: 17.4-22.7° C (actual range) Humidity: 26 - 100% (actual range) Air changes: 151hr Photoperiod: 12 hrs darkl 12 hrs light 5 days prior to start of treatment 

### B. PROCEDURES AND STUDY DESIGN: 1. Mating procedure: One male and one female in the parental P generation from the same treatment group were housed together. Each morning following pairing, the trays under the cages were checked for ejected copulation plugs. The day on which the copUlation plug was2-n-Octyl-4-isothiazolin-3 -one Reproduction and Fertility Effects (2006) 1Page 4 of 18 OPPTS 870.38001 OECD 416 

### found was designated day 0 of gestation. Once mating occurred, the male and female were separated. 2. Study schedule: Parental animals were exposed to test substance in diet for at least 10 weeks prior to mating up to termination. F 1 

### parental animals were not mated until approximately 13 weeks after allocation from the F 1 

### litters. Culling ofF 1 

### and F2 

### offspring was adjusted on day 4 after birth to yield four males and four females per litter as nearly as possible. 

### 3. Animal assignment: Parental (P) animals were randomized into groups at least 5 days before study start, by computer-generated random algorithm according to body weight, with all animals within ±20% ofthe sex mean. Pups forming the F 1 

### generation were selected at random. 

# I TABLE 1. Animal assignment Test group Dose in diet a Animals/group (ppm) P Males P Females F J Males F J Females Control 0 24 24 24 24 Low (LDT) 300 24 24 24 24 Mid (MDT) 800 24 24 24 24 High (HDT) 1500 24 24 24 24 a Diets were administered from beginning ofthe study until sacrifice. 

### 4. Dose selection rationale: The dose levels were selected based on the results from a 14- day range finding study and a 90-day toxicity study . In the 14-day study, Wistar rats were exposed to 0, 2000, 6000, or 10000 ppm oftest material in the diet. Animals in the 6000 or 10000 ppm dose groups were found dead or killed in extremis on day 6 ofthe study. Animals at 2000 ppm showed a decreased body weight gain, increased food scatter, and irregular surface ofthe forestomach. In the 90-day study, Wistar rats were exposed to 0, 100, 300, or 1000 ppm oftest material in diet. The high dose for the two -generation reproduction study was chosen to be 1500 ppm based on slight toxicity observed at the 1000 ppm group and too severe toxicity at the 2000 ppm group. The mid dose was selected to be 800 ppm, and the low dose was selected to be 300 ppm. 5. Dosage preparation and analysis: Formulations were prepared bi-weekly by mixing appropriate amounts oftest substance with standard powder laboratory diet and were stored at room temperature. Prior to the start ofthe study, stability ofthe test substance in diet was evaluated. Homogeneity (top, middle, and bottom) was evaluated at weeks 8, 18,28, and 38. During the study, random samples oftreated food were analyzed at weeks 8,18,28, and 38 for accuracy of concentration. Results: Homogeneity analysis: Coefficients of variation for the diets of groups 2 and 4 were between 2.1% and 7.9% indicating homogeneity of mixture. Stability analysis: At least 6 weeks at room temperature for diets with a concentration of 100 and 3200 ppm . 

# I2- n-Octyl-4- isothiazolin-3 -one Reproduction and Fertility Effects (2006) 1 Page 5 of 18 OPPTS 870. 38001 OEeD 416 

### Concentration analysis: The test samples analyzed showed mean recoveries of 59%, 70%, and 80% at the 300, 800, and 1500 ppm diets, respectively. The analytical data indicated that the mixing procedure was adequate and that the variance between nominal and actual dosage to the study animals was acceptable. C. 

### OBSERVATIONS: 

### 1. Parental animals: Parental animals were checked for mortality/viability at least twice daily, and any animal exhibiting non-transient distress was sacrificed for humane reasons. Detailed clinical observations were made at least once daily and graded on fixed scales . Body weights were taken on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 7,14, and 21 post-coitum and on days 1,4, 7, 14, and 21 post-patium. Food consumption was measured weekly for males and females except during the mating period . Food consumption of mated females was measured on days 0, 7, 14, and 21 post-coitum and on days 1,4, 7 , 14, and 21 post-partum. Water consumption was not quantitatively measured. Vaginal smears were taken daily 3 weeks prior to pairing and throughout cohabitation in order to assess estrous cycle. Mating date, male pairing, confirmation of pregnancy, and delivery date were recorded. 2. Litter observations: According to the report, the following litter observations (X) were made (see Table 2). The day of vaginal opening or balanopreputial separation for several F 1 - 

### weanlings selected for mating and for all selected F2-pups was recorded. Ano-genital distance was measured on day 1 of lactation for all F2 pups. 

# I TABLE 2. FJ / F2 Litter Observations a Observation Day 1 Number of live pups X Pup weight X External alteration s X Number of dead pup s X Sex of each pup (M/F) X a Data obtained from page 25 in the study report. b Before standardization (culling). C After standardization (culling). Day 4 X X X Time of observation (lactation day) b Day 4 c Day 7 Day 14 Day 21 X X X X X X X X X X X X



### On day 4 postpartum, litters were standardized to a maximum of8 pupsllitter (4/sex/litter, as nearly as possible); excess pups were sacrificed and discarded. Dead pups were examined grossly for external and internal abnormalities, and a possible cause of death was determined for pups born or found dead. 3. Postmortem observations: a. Parental animals: All surviving parental males were sacrificed as soon as possible after successful mating. Maternal animals were sacrificed on day 21 post-partum or shortly 

# I2- n- Octyl -4- isothiazolin- 3 -one Reproduction and Fertility Effects (2006) 1Page 6 of 18 OPPTS 870.38001 OEeD 416 thereafter. For surviving males, sperm motility (all males) and morphology (all Pgeneration males and ten randomly selected samples from the F I -generation control and high-dose group males) were assessed from sperm samples taken from the proximal part of the vas deferens. Enumeration ofhomogenization-resistant spermatids and cauda epididymal sperm reserves were evaluated from one testis and one epididymis, respectively, for 10 randomly selected samples from the control and high-dose groups. The number of implantation sites in each uterus was assessed for all paired females . The uterus of nonpregnant females was stained using the Salweski technique to determine any very early post-implantation losses. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The following tissues (X) were prepared for microscopic examina tion and weighed (XX). Tissues that were only weighed are denoted by (XXX): 

### X Cervix 

### XX Spleen 

### X Coagulation gland 

### X Stomach 

### XX Epididymis (one) 

### XX Testis (one) 

### XX Ovarie s 

### XX Uterus 

### XX Prostate gland (weighed when fixed 

### X Vagina for at least 24 hours) 

### XX Seminal vesicles 

### X All gross lesions 

### XXX Adrenal glands XXX Liver 

### XXX B rain 

### XXX Pituitary (weighed when fixed for at least 24 hours) 

### XXX Kidneys XXX Thyroid b. 

### Offspring: F I offspring selected for the next generation were sacrificed via decapitation; all remaining pups were sacrificed using an oxygen/carbon dioxide procedure . The F I offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age or shortly thereafter. These animals were subjected to postmortem external examination ofthe cranium and macroscopic examination ofthoracic and abdominal tissues and organs . Gross lesions were preserved for future examination . The weights of brain, spleen, and thymus were taken for one randomly selected F I pup and one unselected F2 pup per sex per litter at day 21 post-partum or shortly thereafter. Offspring found dead before day 14 of lactation were sexed, externally examined, and stomach examined for presence of milk. 

### D. DATA ANALYSIS: 1. 

### Statistical analyses: If variables could be assumed to follow a normal distribution, the Dunnett-test (many-to -one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. The Steel-test (manyto -one rank test) was applied if the data could not be assumed to follow a normal distribution . The Fisher Exact test was applied to frequency data.2- n-Octyl -4-isothiazolin-3-one 

### 2. Indices: Reproduction and Fertility Effects (2006) 1Page 7 of 18 OPPTS 870.38001 OEeD 416 

### Reproductive indices: The following reproductive indices were calculated from breeding and parturition records of animals in the study: Percentage mating = 

### Number females mated/number females paired x 100 Fertility index = 

### Number ofpregnant females/number of females paired x 100 Conception rate 

### = Number ofpregnant females/number of females mated x 100 Gestation index 

### = Number of females bearing live pups/number ofpregnant females x 100 Duration of gestation = 

### Number of days between confirmation ofmating and the beginning of parturition Offspring viability indices: The following viability indices were calculated from lactation records of litters in the study: Percentage live males at first litter check 

### = Number of live male pups at first litter check/number of live pups at first litter check x 100 Percentage live females at first litter check = 

### Number of live female pups at first litter check/number of live pups at first litter check x 100 Percentage ofpostnatal loss days 0-4 post partum 

### = Number of dead pups on day 4 post partum/number of live pups at first litter check x 100 Percentage of breeding loss day 5 until weaning 

### = Number of dead pups between days 5 and 21 post partum/number of live pups on day 4 post partum x 100 Percentage of live males at weaning 

### = Number of live male pups on day 21 post partum/number oflive pups on day 21 post partum x 100 Percentage of live females at weaning = 

### Number of live female pups on day 21 post partum/number of live pups on day 21 post partum x 100 Viability index = 

### Number of live pups on day 4 post partum/number ofpups born alive x 100 Weaning index = 

### Number oflive pups on day 21 post partum/number oflive pups on day 4 post partum x 100 3. Historical control data: Provided in Appendix 7 ofthe study report. II. RESULTS: A. PARENTAL ANIMALS: 1. 

### Mortality and clinical signs: No mortality or treatment-related clinical signs were observed in the P parental generation. Incidental findings included alopecia, scabs, wounds, swelling2- n-Octyl-4-isothiazolin-3-one Reproduction and Fertil ity Effects (2006) 1 Page 8 of 18 OPPTS 870.38001 OEeD 416 

### ofthe genital region, and chromodacryorrheoa; these were not considered to be treatmentrelated. In the F 1 

### parental generation, one female in the 300 ppm treatment group was found dead on day 17 of lactation with no cause of death determined and no clinical signs oftoxicity before death. One female in the 800 ppm group was sacrificed on day 1 of lactation due to bad health. This female delivered sixteen dead pups and was observed to have uterine inflammation and retained fetuses upon necropsy. These deaths were not considered treatment-related. Incidental findings included alopecia of several body parts, hunched posture, chromodacryorrhea, broken or curled tail apex , scabs, swelling ofthe left flank or abdomen, and scales. A 300-ppm F 1 

### female showed an absent tail, and a 1500-ppm F 1 

### male showed a broken apex . None ofthese findings were considered treatment-related. 2. Body weight and food consumption: In the P parental generation, body weight was statistically significantly decreased in dams treated at 1500 ppm on days 7 and 14 of lactation. On several occasions, statistically significantly lower body weights and body weight gains were noted for males ofthe low- and high-dose groups. These changes were considered to be ofno toxicological significance because the data were within the historical control data range. Absolute and relative decreases in food consumption were observed in males and females treated at 1500 ppm from days 1 to 8 ofpre-mating. This was not considered to be toxicologically relevant but rather caused by decreased pal a tability of diet. F 1 

### parental generation body weights were lower during the entire treatment period in the group administered 1500 ppm, and this change was attributed to their lower starting body weight. A slight increase in body weight gain was noted in the males and females ofthe 800 and 1500 ppm group during pre-mating and not considered an adverse effect oftreatment. Females administered 800 ppm test material demonstrated decreased body weight gain during post coitum and lactation period; since no dose-response relationship was observed, this decreased body weight gain was not considered to be a treatment-related effect. Reported body weight and selected food consumption results are summarized in Table 4. I TABLE 4. Mean (±SD) Body weight and food consumption - pre- mating, post-coitum, and lactation a Observations/study week Dose group Control LDT MDT HDT P Generation males - Pre-mating Mean body weight (g) Week 1 177±9.3 177±7.1 174 ± 6.9 174±7.7 Week 2 231 ± 15.3 229 ± 11.1 227 ± 10 .9 219 ± 10.0** Week 3 291 ± 18.9 284 ± 13 .0 284 ± 15 .9 276 ± 12.9** Week 4 330 ± 22.6 318±15.9 322 ± 22 .0 316 ± 19 .7* Week 5 365 ± 29.2 347± 19 .9* 355 ± 26 .0 347± 22 .9 Week 6 396 ± 34.0 37 5 ± 23 .0 385 ± 31.7 373 ± 26 .4 Week 7 420 ± 36.6 397 ± 24 . 8* 407 ± 34.4 395 ± 29.4* Week 8 440 ± 39.7 411 ± 26.6* 426 ± 37 .3 413±31.4* Week 9 462 ± 42 . 3 430 ± 27 .5** 446 ± 42 .0 433 ± 33 .9*2- n- Octyl -4-isothiazolin-3 - one Reproduction and Fertility Effects (2006) 1Page 9 of 18 OPPTS 870.38001 OECD 416 TABLE 4. Mean (±SD) Body weight and food consumption - pre-mating, post-coitum, and lactation a Observations/study week Dose! roup Control LDT MDT HDT Week 10 475 ± 44.7 444 ± 30* 460 ± 44.3 449 ± 38 .2 Mean weight gain (g) Weeks 1- 10 (mean of means) 298 267* 286 275 Mean food consumption (g/animal/day) Weeks 1- 10 (mean ofmeans) 27 26 28 27 P Generation females - pre-mating Mean body weight (g) Week 1 149 ± 6.2 149 ± 8.0 148 ± 8 .4 148 ± 6.7 Week 2 172 ± 12 .0 176±9.2 172 ± 13.5 171±8 .3 Week 3 193 ± 12.7 194 ± 12.6 191 ± 16.3 190 ± 11.9 Week 4 211 ± 15.6 214 ± 15.9 207 ± 18.7 205 ± 13.3 WeekS 224 ± 17.4 230 ± 17.5 223 ± 19.7 219 ± 14.0 Week 6 236 ± 18.2 242± 16 . 1 235 ± 22 .2 231±16.4 Week 7 248 ± 18.2 249 ± 16 .3 244 ± 22.8 241±17.6 Week 8 255 ± 18.4 258 ± 14.3 24 8 ± 22.9 246 ± 18.8 Week 9 270 ± 20.5 272 ± 18.4 266 ± 23.8 263 ± 20.0 Week 10 278 ± 24 .5 277 ± 16 .9 274 ± 27.7 269 ± 19 Mean weight gain (g)

Weeks 1- 10 (mean of means) 129 128 125 121 Mean food consumption (g/animal/day) Week 1- 10 (mean of means) 19 20 20 19 FI Generation males - Pre-mating Mean body weight (g) Week 1 186 ± 25.9 189 ± 17. 9 182 ± 14.5 152 ± 19.0** Week 2 245 ± 27 .3 247 ± 18.2 23 9 ± 17.5 200 ± 22.1 ** Week 3 298 ± 29.0 298 ± 18.4 296 ± 23 .0 252 ± 22 .9** Week 4 348 ± 29 .5 350 ± 20.2 346 ± 26.8 304 ± 23.3** WeekS 388 ± 30.5 386 ± 22 .2 379 ± 31.1 337 ± 23.5** Week 6 419 ± 32 .3 413 ± 24.1 408 ± 37 .0 366 ± 23 .9** Week 7 441 ± 32.9 435 ± 26.7 428 ± 38.2 390 ± 26.0** Week 8 454 ± 33 . 8 447 ± 27.7 440 ± 40.6 402 ± 25.8** Week 9 480 ± 37 .1 469 ± 28.3 465 ± 43 . 6 425 ± 28.5** Week 10 498 ± 38 .5 488 ± 30.2 482 ± 46.3 442 ± 29.6** Mean weight gain (g) 312 299 300 290 Weeks 1- 10 (mean ofmeans) Mean food consumption (g/animal/day) 30 31 31 30 Weeks 1- 10 (mean of means) FI Generation females - pre-mating Mean body weight (g) Week 1 146 ± 17 .0 152 ± 10 .2 149±11.1 131 ± 13.9** Week 2 17 6±16 . 1 183 ± 11.5 179±14.3 161 ± 13 .8** Week 3 196 ± 14.3 202 ± 12. 7 201 ± 15.7 183 ± 15.9* Week 4 214 ± 15 . 8 222 ± 15 .3 2 16 ± 18 .3 201 ± 16.3* WeekS 231 ± 15 . 1 237 ± 15 .5 232 ± 20.4 217 ± 15.5* Week 6 241 ± 16.4 248 ± 15.8 242 ± 21.9 227 ± 16 .5* Week 7 246 ± 15.5 252± 17 .0 249 ± 22 .3 235 ± 16.42-n-Octyl-4-isothiazolin-3-one Reproduction and Fertility Effects (2006) / Page 10 of 18 OPPTS 870.38001 OECD 416 TABLE 4. Mean (±SD) Body weight and food consumption - pre-mating, post-coitum, and lactation a Observations/study week Dose roup Control LDT MDT Week 8 253 ± 14.2 261 ± 17.1 255 ± 25.4 Week 9 259 ± 15 .7 267 ± 17.8 264 ± 23.4 Week 10 265 ± 18.9 274 ±20 274 ±27.1 Mean weight gain (g) 119 122 125 Weeks 1-10 (mean ofmeans) Mean food consumption (g/animal/day) 20 21 2 1 Weeks 1-10 (mean ofmeans) P Generation females - post co itum and lactation Mean body weight (g)

Post coitum Day 0 275 ± 19 . 8 278 ± 16.7 275 ± 24 .7 Day 7 301 ± 24.8 305 ± 17.5 302 ± 25.5 Day 14 333 ± 26.5 336 ± 20.0 3 3 1 ± 26.0 Day21 436 ± 35.4 435 ± 34.6 424 ± 35.1 Lactation 319 ± 29.4 322 ± 21.3 3 12±28 .5 Day 1 Day 4 326 ± 28 . 8 328±21.0 3 1 8±26.7 Day 7 333 ± 25.6 336 ± 18.1 327 ± 33.2 Day 14 347 ± 25 .5 351 ± 18.5 3 3 3 ± 27.0 Day 21 330 ± 23.7 332 ± 18.8 324 ± 26.7 F 1 Generation females - post coitum and lactation Mean body weight (g)

Post coitum Day 0 276 ± 24 . 3 284 ± 20 .9 278 ± 26.6 Day 7 304 ± 25.4 310 ± 21.4 306 ± 29 .0 Day 14 338 ± 27 .6 341 ± 24.6 3 3 5 ± 31.3 Day 21 436 ± 34 .6 435 ± 24.8 423 ± 38 . 3 Lactation Day 1 320 ± 25.4 322 ± 23.0 3 1 8±29.4 Day 4 329 ± 28.4 333 ± 24.0 326 ± 31.0 Day 7 338 ± 29 .7 339±25.1 3 3 3 ± 33.3 Day 14 348 ± 33 .5 345 ± 23.8 3 3 6 ± 30.6 Day 21 331 ± 32.0 322 ± 19.1 3 16 ± 24.4 a Data obtained from pages 57 -60 , 65, 66, 340-343, 348, and 349 in the study report. * Statistically different from control, p<0.05 (Dunnett's test based on pooled variance). ** Statistically different from control, p<O.OI (Dunnett'S test based on pooled variance) . HDT 242 ± 16 . 8 252 ± 17 .5 260 ± 17 .4 129 21
267 ± 20.1 295 ± 19.8 323 ± 21.5 416 ± 30.2 304 ± 20 .5 309 ± 21.6 314±21.7* 323 ± 24 .9**

318±25.0 261 ± 18 .8 285 ± 20.0* 3 16 ± 20.5* 399 ± 29.6** 299± 19.5* 304 ± 20.4** 312 ± 19.8** 315 ± 18.2** 302± 17 . 1**



### 3. Test substance intake: Based on food consumption, body weight, and test material recovery in diet, the doses expressed as mean daily mg test substance/kg body weight during the 10- week pre-mating period are presented in Table 5. The values for the P and 

### Fl generation are similar and considered to be representative ofthe test substance intake for the entire study.2- n-Octyl-4-isothiazolin-3-one Reproduction and Fertility Effects (2006) I Page 11 of 18 OPPTS 870.38001 OEeD 416 TABLE 5. Mean test substance intake during premating (mg/kg body weight/day) corrected for mean value of recovery a Male Female LDT MDT HDT LDT MDT HDT (177 ppm (560 ppm (1275 ppm (177 ppm (560 ppm (1275 ppm recovered) recovered) recovered) recovered) recovered) recovered) P 13 43 96 15 48 107 F1 14 45 111 15 51 120 a Data obtained from p ages 33 and 38 in the study report . 

### 4. Reproductive function: a. Estrous cycle length and periodicity: Results from the evaluation ofvaginal smears indicated no treatment-related effects. b. Sperm measures: Results from the evaluation of sperm parameters revealed no treatment-related effects on sperm count, motility, or morphology. 5. Reproductive performance: In both generations, mating performance, duration of gestation, fertility parameters, number of implantation sites, and number of dead and living pups at first litter check were similar for the control and treated groups. Breeding parameters were unaffected by treatment up to 1500 ppm. In the P generation, eight males and eight females were suspected of infertility across dosing groups (2 males and 2 females in the control group, 5 males and 5 females in 300 ppm group, 1 male and 1 female in the 800 ppm group), and four males and four females were suspected of infertility in the F 1 

### parental generation. These observations were not considered to be treatment-related . Postnatal loss between days 0-4 post partum, living pups on day 4 post partum, breeding loss between days 5 -21 post partum, living pups on day 21 post partum, viability index and weaning index were similar for the control and treated groups for both generations. Results for the parental animals are summarized from the report in Table 6. TABLE 6 . Repro ductive performance and fertilityparameters U Observation Dose group (ppm) Control 300 800 1500 P Gene ration Mean (± SD) precoital interval (days) 3.2 3 .0 2.8 2. 7 

### I MALES

Number mated 24 24 24 24 Number fertile 22 19 23 24 Suspected infertile 2 5 1 0 Intercurrent deaths 0 0 0 0 

### I FEMALES

Number mated 24 24 24 24 Number fertile 22 19 23 24 Suspected infertile 2 5 1 0 Intercurrent deaths 0 0 0 0 Mean (± SD) gestation interval (days) 2 l.3 ± 0.6 2 l.6 ± 0 .6 2 1.5 ± 0 .6 2 1.2 ± 0 .7 Number of litters 22 19 2 3 24 Percentage mating 100 100 100 100 

### I
I2- n- Octyl -4-isothiazoli n-3 -one Reproduction and Fertility Effects (2006) 1 Page 12 of 18 OPPTS 870.38001 OECD 416 TABLE 6. Reproductive performance and f ertility parameters a O bservation Dose group (ppm) Control 300 800 1500 Fertility index 95 . 8 83.3 95 . 8 100 Conception rate 95.8 83.3 95 . 8 100 Gestation index 95.7 95 .0 95 .7 100 F 1 Generation Mean (±SD) precoital interval (days) 4.0 3 . 1 2 .9 2.0 MALES Number mated 24 24 24 24 Number fertile 22 24 24 22 Suspected infertile 2 0 0 2 Intercurrent deaths 0 0 0 0 FEMALES Number mated 24 24 24 24 Number fertile 22 24 24 22 Suspected infertile 2 0 0 2 Intercurrent deaths 0 1 1 0 Mean (±SD) gestation interval (days) 21.5 ± 0.7 21.4 ± 0.7 21.3 ± 0 .6 21.2 ± 0.4 Number of litters 22 24 24 22 Percentage mating 100 100 100 100 Fertility index 91.7 100 100 91.7 Conception rate 91.7 100 100 91.7 Gestation index 100 100 95 . 8 100 a Data obtained from pages 35, 40,81,82,365,366 , 741-745 in the study report. 

### 6. Parental postmortem results: Organ weights: The report noted decreased absolute spleen weight and increased relative adrenals weight in females treated with 1500 ppm test material in the P generation. Additionally, there was a decrease in absolute and relative ovary weight at 1500 ppm. This change, however, was not considered related to treatment because there were no corresponding pathological or histopathological changes, and female fertility was not affected. The report noted that no toxicologically significant changes were present in organ weights and organ:body weight ratios in the FJ generation. Males ofthe 1500 ppm dose group showed statistically significantly decreased terminal body weights at necropsy , decreased absolute weights ofthe brain, liver, kidneys, spleen and epididymides, and increased relative weights ofthe brain, pituitary and kidneys. Females ofthe 1500 dose group showed statistically significantly decreased terminal body weights at necropsy, decreased absolute weights ofthe brain and uterus and decreased absolute and relative weights of the thyroids and ovaries. These changes were most likely associated with decreased body weights in this group across this generation (including decreased terminal body weights) . In addition, no microscopic correlate was observed for the changes in the reproductive organs (epididymides, uterus and ovaries) . Therefore, the changes were not considered a sign oftoxicity.2-n-Octyl-4-isothiazolin-3-one Reproduction and Fertility Effects (2006) 1Page 13 of 18 OPPTS 870.38001 OEeD 416 

### Selected absolute and relative (to body weight) weight values for ovaries, spleen, and adrenals of females in the P generation are presented in Table 7 below. TABLE 7. Selected organ weights for P-generation females a Organ (weight meas urement) Dose group (ppm) Control 300 800 Ovaries (absolute, grams) 0.183 ± 0 .033 0.183 ± 0 .022 0.172 ± 0.028 Spleen (absolute, grams) 0.813 ± 0 . 13 7 0 .783 ± 0 . 148 0 .722 ± 0.094 Ovaries (relative, %) 0.056 ± 0 .009 0 .056 ± 0.007 0.054 ± 0.009 Adrenals (relative, %) 0 .027 ± 0.003 0.026 ± 0.003 0 .027 ± 0 .003 a Data extracted from pages 77 and 78 ofthe study report . * Statistically different from control, p<0.05 (Dunnett's test based on pooled variance) . ** Statistically different from control, p<O.O 1 (Dunnett's test based on pooled variance). 

### b . Pathology: 1500 0 . 139 ± 0 .022** 0.693 ± 0. 161 * 0 .045 ± 0.006 * * 0.030 ± 0.003**

1. 

### Macroscopic examination: In the P parental generation, irregular surface ofthe forestomach was observed in four males administered 1500 ppm test diet. This was the only observation considered related to test-substance administration. In the F 1 

### generation, four males and one female ofthe 1500 ppm dose group also exhibited an irregular surface ofthe forestomach. Other findings in either generation were not considered treatment-related and were incidental. 2. Microscopic examination: The report noted the following observations in the P parental generation. Diffuse hyperplasia/hyperkeratosis was observed in the forestomach ofnine males and ten females treated with 1500 ppm test substance. The severity was minimal to moderate and correlated with the gross observations in the stomach. Testicular tubular atrophy was observed in one mid-dose male. These observations were not considered to be treatment-related findings . In the F 1 

### parental generation, diffuse hyperplasia/hyperkeratosis of minimal to moderate severity was observed in the forestomach of eight males and six females in the 1500 ppm group , and correlated with the macroscopic observations of irregular surface ofthe forestomach. No other treatment-related findings were noted. Incidental findings included fetalloss/uterine inflammation, mammary adenoma in one control female , and hemopoietic foci in the spleen of one control female . B. OFFSPRING: 1. 

### Viability and clinical signs: The following findings were reported: For F 1 

### pups, no treatment-related clinical signs oftoxicity were observed at any dose. Incidental findings that were not considered to be treatment-related were little or no milk,2-n-Octyl-4- isothiazolin-3 -one Reproduction and Fertility Effects (2006) 1 Page 14 of 18 OPPTS 870.38001 OECD 416 small , pale, weak or cold appearance, exopthalmus, scabs, and reduced size ofthe left eye. One pup in the 300 ppm group had no tail. One pup in the 800 ppm group had a wounded right ear that was later reduced in size, and two pups in the 1500 ppm group showed scabs on the tail apex, absence of nails, and a broken andlor curled tail apex . These observations were considered to be within normal biological variation. 

### F2 pups did not exhibit any treatment-related clinical signs oftoxicity at any dose. Symptoms that were not treatment-related included weak or cold appearance , no milk, alopecia, thickened area ofthe shoulder, eye discharge and opacity, missing tail , and reduced testes size. Additionally, one pup ofthe 300 ppm showed an absent tail and one pup ofthe 800 ppm group showed an absent right eye. These findings were considered incidental and were not considered to be caused by treatment. Mean litter size and viability (survival) results from pups during lactation are summarized from the report in Table 8. TABLE 8. Litter parameters for F J and F2 generations a Observation Dose group (ppm) Control 300 800 1500 FJ Generation Mean implantation sites 15 . 1 12.7 14 .0 15.4 Number born live 326 266 313 335 Number born dead 3 2 7 0 Sex ratio Day 0 (% ) 53M / 47F 5 3M 1 47F 53M 147F 56M / 44F # Deaths 3 (0 .9) 6 (2.3) 3 (1.0) 5 (l.5) Days 0- 4 (%) # Deaths 2 (Ll) 0(0) 2 (1.1) 2 (Ll) Days 4-21 (%) Mean litter size Day 0 14.8 14 .0 13 .6 14 .0 Day 4 b 14. 7 l3.7 13 .5 13.8 Day 4 c 8 .0 7.9 7 .7 7. 9 Day 7 NR NR NR NR Day 14 NR NR NR NR Day 21 8 .0 7.9 7 .6 7 . 8 Birth index a 90.9 87 .9 95 .0 90.8 Live birth index e 99.0 99.3 97 . 8 100 Viability index f 99 . 1 97.7 99.0 98 .5 Lactation index g 98 .9 100 98.9 98.9 F2 Generation Mean implantation sites 14.8 16.4 15 .5 14 .9 Number born live 322 350 321 298 Number born dead 2 2 19 2 Sex ratio Day 0 (%) 48M / 52F 46M I 54F 50M I 50F 48M / 52F # Deaths 9 (2.8) 6 (1.7) 1 (0 .3) * 1 (0 . 3)* Days 0- 4 (% ) # Deaths 2 (0.9) 10 (4 .2)** 0(0) 1 (0 .5) Days 4-21 (%) Mean litter size Day 0 14.6 14 .6 13.4 13 .62-n-Octyl -4-isothiazolin -3 -one Reproduction and Fertility Effects (2006) 1Page 15 of 18 OPPTS 870.38001 OECD 416 TABLE 8. Litter parameters for FJ and F2 generations a Observation Dose group (ppm) Control 300 800 1500 Day 4 b 14.2 14.3 13.3 13.5 Day 4 c 9.8 10 9.5 9.6 Day 7 NR NR NR NR Day 14 NR NR NR NR Day 21 9.7 9.5 9.5 9 .5 Birth index d 91.5 89.6 90 .7 84.0 Live birth index e 99.4 99.4 94.4 99.3 Viability index f 97.2 98.3 99.7* 99.7* Lactation index g 98 .6 95.8 100 99.5 a Data obtained from pages 82, 189, 366, and 475 in the study report. bBefore standardization (culling); calculated by adding the number ofpups post-cull on day 4 plus the number of culled pups then dividing by the number of litters. C After standardization (culling). d Birth index = (total number of pups born [live and dead])/number of implantation scars. e Live birth index = (# pups born alive/ # pups born) x 100. f Viability index = (# pups alive on PND 4, pre-cull/ # pups born alive) x 100. g Lactation index = (# pups alive on PND 211 # pups alive on PND 4, post-cull) x 100. NR=Not reported * Statistically different from control, p<0.05 Fisher's exact test. ** Statistically different from control, p<O.Ol Fisher'S exact test. 

### 2. Body weight: Offspring body weights were statistically significantly lower in both male and female 

### FJ pups in the 1500 ppm group from day 14 oflactation onwards. In the 

### F2 pups, body weights were statistically significantly lower in the 1500 ppm group from day 21 of lactation onwards. Theses changes were attributed to pups beginning to switch to feed and therefore increasing compound intake. Selected mean pup body weight data are presented in Table 9.2- n-Octyl-4-isothiazolin-3 - one Reproduction and Fertility Effects (2006) 1 Page 16 of 18 OPPTS 870.3800 1OEeD 416 TABLE 9. Mean (±SD) Litter and pup weights (g) a Dose group (ppm) Lactati on 0 300 800 1500 day FJ Litters

1 6.4 ± 0.4 6 .6 ± 0.5 6.6 ± 0 .5 6.4 ± 0. 7 4 b NR NR NR NR 4 c 9.4 ± 0 .8 10.0 ± l.2 9 . 8 ± l.0 9.4± 1.4 7 15 . 1 ± 1.4 16.1 ± l.6 15. 7 ± l.7 14 .6 ± 2 .1 14 33 .9 ± 2.1 35 .5 ± 2 . 1 33 . 8 ± 3.l 30 . 8 ± 2.9** 21 57.6 ± 3.5 59. 7 ± 3.6 54.8 ± 5.1 47 .l ± 4.4** F J Pups - male 1 6.5 ± 0.4 6 .7 ± 0.5 6 . 8 ± 0 .5 6 .6 ± 0. 7 4 b NR NR NR NR 4 c 9 .6 ± 0.8 10 .2 ± l.2 1O . 1±1.l 9.7 ± 1.5 7 15.5 ± 1.4 16.4 ± 1. 7 16.2 ± 1.6 15.0±2.2 21 59 .0 ± 3 .6 61.2 ± 3.9 56.4 ± 5.3 48.1 ± 4. 7 ** FJ Pups - female 1 6.2 ± 0.4 6.4 ± 0 .6 6.4 ± 0 .5 6.2 ± 0.6 4 b NR NR NR NR 4 c 9.1±0.9 9.7 ± 1.3 9.6 ± 1.0 9.1 ± 1.3 7 14.8 ± 1.6 15 . 8 ± 1.8 15.3 ± 1.9 14.1 ± 2 . 1 21 56 . 1 ± 4.0 58 .2 ± 3 .7 53. 3± 5.1 4 5. 9 ± 4.3** a Data obtained from pages 83 and 367 in the study report. b Before standardization (culling) c After standardization (culling) NR = Not reported 0 6 .3 ±0 .6

NR 9 .3 ± l.0 14 .3 ± 1.6 29.9 ± 2.9 48.0 ± 5.6 6 .5 ± 0.6 NR 9.5 ± l.0 14 . 6 ± 1.6 48.8 ± 5.8

6 .2 ±0.6

NR 9.1 ± 1.0 14.0 ± 1.6 47.1 ± 5 .7 300 800 Fz Litters 6.5 ± 0.5 6.5 ± 0.4 NR NR 9 .7 ± l.0 9.8 ± 0.9 15.5 ± 1.5* 15.3±1.3 32.2 ± 2.4* 31.1 ± 2.4 52 .2 ± 4.1 * 48.6 ± 4 .9 Fz Pups - male 6.7 ± 0.5 6.7 ± 0.5 NR NR 10.0 ± 1.0 10.0 ± 0 .9 16.0 ± 1.5** 15 .7± 1.5 53.6 ± 4.4** 49.6 ± 5 . 8 Fz Pups - female 6.4 ± 0 .5 6.4 ± 0.4 NR NR 9.4 ± 1.0 9.6± 0.9 15 .0 ± 1.5 15.0 ± 1.4 50.9 ± 3 .9* 47.9 ± 4. 7

* Statistically different from control, p<0 .05 (Dunnett-test based on pooled variance) . ** Stati stically different from control, p<O.Ol (Dunnett-test based on pooled variance). 1500 6.4 ± 0 .6

NR 9 .3 ± l.2 14.2 ± l.6 28.1 ± 2.9 4l.7 ± 4.5**

6.5 ± 0.6

NR 9.6 ± 1.2 14 .6 ± l. 7 42.5 ± 4.9**

6 .2 ± 0 .6

NR 9 . 1 ± 1.2 13 .9±1.7 4l.0 ± 4.4 * *

3. Sexual maturation (F 11: 

### Sexual maturation was not affected by treatment, and no effects were seen in ano -genital distance or vaginal opening. The occurrence of balanopreputial separation (prepuce opening) was slightly delayed in males treated at 1500 ppm. This was considered to be caused by a slight delay in development due to lower body weights and was not considered to be treatment-related. 4. Offspring postmortem results: a. Organ weights: 

### Absolute and relative spleen weights (male and male pups) for F 1

### pups were significantly decreased at the high dose; this effect was considered treatmentrelated . Female pups at the 800-ppm dose level also exhibited decreased absolute and2-n-Octyl-4-isothiazolin-3 -one Reproduction and Fertility Effects (2006) 1Page 1 7 of 18 OPPTS 870.38001 OECD 416 

### relative spleen weight; however, this effect was not considered treatment-related since the values were within the historical control range; the magnitude ofthe change was slight; changes were not observed across generations; and changes were not consistent with males . Significantly decreased absolute and relative thymus weight for 1SOO-ppm females was not considered toxicologically significant since this effect was not observed in the second generation. TABLE 10. Selected organ weights for Frgeneration (males and female pups) a Organ (weight Dose group (ppm) measurement) Control 300 800 1500 MALES Spleen (absolute, grams) 0.340 ± 0.0529 0.362 ± 0.069 1 0.317 ± 0.0599 0 .244 ± 0.0481 ** Spleen (relative , %) 0 .59 ± 0.076 0 .60 ± 0.087 0 .55 ± 0 .077 0 .51 ± 0.078** FEMALES Spleen (absolute, grams) 0.349 ± 0.0487 0.355 ± 0.0608 0 . 304 ± 0.0413** 0.246 ± 0.0480** Spleen (relative, %) 0.61 ± 0.072 0.60 ± 0.084 0 .56 ± 0.070* 0.54 ± 0 .084** Thymus (absolute, grams) 0.264 ± 0 .0572 0.231 ± 0.0376* 0.236 ± 0 .0415 0 . 183 ± 0 .0292** Thymus (relative, %) 0.46 ± 0.081 0 .39 ± 0 .053 ** 0.44 ± 0.060 a Data extracted from pages 86 - 87 of the study report . * Statistically different from control, p<0.05 (Dunnett's test based on pooled variance) . ** Statistically different from control, p<O.OI(Dunnett's test based on pooled variance). 0.40 ± 0.060**



### Absolute and relative spleen weights (male and male pups) for 

### F2PUPS were significantly decreased at the high dose; this effect was considered treatment-related. All other changes in organ weights were not considered treatment-related . TABLE 11. Selected organ weights for Frgeneration male pups a Organ (weight Dose group (ppm) measurement) Control 300 800 Spleen (ab solute, grams) 0.276 ± 0 .0892 0.269 ± 0.0356 0.240 ± 0 .059 7 Spleen (relative, %) 0.55 ± 0 . 126 0.51 ± 0 .090 0.49 ± 0. 074 * a Data extracted from pages 371 -372 ofthe study report . 

## * Statistically different from control, p<0 .05 (Dunnett' s test based on pooled variance) . 

## ** Statistically different from control , p<O.O 1(Dunnett' s test based on pooled variance). b. 

### Pathology: 1500 0.194 ± 0 .0390**

0.47 ± 0.078**

1. 

### Macroscouic examination: No significant findings were noted in 

### Flor 

### F2 pups during macroscopic examination. Incidental findings that were not found to be dose-2-n-Octyl-4-isothiazolin-3-one Reproduction and Fertility Effects (2006) 1Page 18 of 18 OPPTS 870.38001 OECD 416 

### related or were within normal biological variation included small appearance, no milk, pelvic dilation ofthe kidneys, absent right eye, alopecia, reduced size testes and/or epididymides, constricted spleen and cannibalism. 2) Microscopic examination: Microscopic examination data were not presented for the offspring. III. DISCUSSION AND CONCLUSIONS: A. CONCLUSIONS: The investigators concluded that the parental systemic LOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when corrected for mean value oftest article recovery), based on decreased body weight, hyperplasia/hyperkeratosis ofthe forestomach, decreased spleen weight, and increased adrenal weight. The parental systemic NOAEL is 800 ppm (43-45 mg/kg bw/day in males, 48-51 mg/kg bw/day in females for both generations when corrected for mean value oftest article recovery). The offspring LOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when corrected for mean value oftest article recovery), based on decreased body weight gain and decreased spleen weight. The offspring NOAEL is 800 ppm (43-45 mg/kg bw/day in males, 48-51 mg/kg bw/day in females for both generations when corrected for mean value oftest article recovery). Reproduction parameters (mating performance, duration of gestation, fertility, number of implantation sites, number of live/dead pups at first litter check) and breeding parameters (postnatal loss, living pups on day 4 post partum, breeding loss, living pups on day 21 post partum, viability index, weaning index) were unaffected by treatment in the first and second generations. Therefore, the reproductive NOAEL is 1500 ppm (96-111 mg/kg bw/day in males, 107-120 mg/kg bw/day in females for both generations when corrected for mean value oftest article recovery). Adverse findings which did not exhibit a dose-related effect or did not have histopathological correlations with macroscopic findings were not considered to be caused by treatment. B. STUDY DEFI CIENCIES: No deficiencies in the study were noted . 

### C. STUDY CLASSIFICATION: This study is ACCEPTABLE - GUIDELINE and satisfies the guideline requirement for a two-generation reproductive study (OPPTS 870.3800; OEeD 416) in the rat.