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 .J.~. r·l\..a~ I /~ 5 J/fJ 

Risk Assessment and Science Support Branch (RASSa)- - . . 

Antimicrobials Division (AD)[7510P] /J / _~ 

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 of the 

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 of the first generation and in eight males and six females of the second 

generation, all at the 1500 ppm dose level. These observations correlated with the 

macroscopic observations of irregular surface of the 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 of both 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; OEeD 416) in rat. 

2 
2-n-Octyl-4-isothiazolin-3-one 

Reproduction and Fertility Effects (2006) 1 Page 1 of 18 

OPPTS 870.38001 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 first 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 of the 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 of the forestomach was observed in nine males and 

ten females of the first generation and in eight males and six females of the second generation, all 

at the 1500 ppm dose level. These observations correlated with the macroscopic observations of 

irregular surface of the 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 
2-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 of both 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 twogeneration 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) 1 Page 3 of 18 

OPPTS 870.38001 OECD 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 

(F1) 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 was 
2-n-Octyl-4-isothiazolin-3-one 

Reproduction and Fertility Effects (2006) 1 Page 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 of F 1 and F 2 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% of the sex mean. Pups forming the F 1 generation were selected at 

random. 

I TABLE 1. Animal assignment 

Test group Dose in diet a Animals/grou p 

(ppm) P Males P Females FJ Males FJ 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 of the 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 of the study. 

Animals at 2000 ppm showed a decreased body weight gain, increased food scatter, and 

irregular surface of the 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 of test substance with standard powder laboratory diet and were stored 

at room temperature. Prior to the start of the 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 of treated 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. 

I 
2-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 F1-

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 alterations X 

Number of dead pups 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 

I 
2-n-Octyl-4-isothiazolin-3-one 

Reproduction and Fertility Effects (2006) 1 Page 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 of homogenization-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 examination 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 Ovaries 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 Brain 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 of the cranium and macroscopic examination of thoracic 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) 1 Page 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 of pregnant females/number of females paired x 100 

Conception rate = Number of pregnant females/number of females mated x 100 

Gestation index = Number of females bearing live pups/number of pregnant females x 100 

Duration of gestation = Number of days between confirmation of mating 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 of postnatal 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 of pups 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 of the 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, swelling 
2-n-Octyl-4-isothiazolin-3-one 

Reproduction and Fertility Effects (2006) 1 Page 8 of 18 

OPPTS 870.38001 OEeD 416 

of the 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 of toxicity 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 of the 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 of these 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 of the low- and high-dose groups. These changes were 

considered to be of no 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 of pre-mating. This was not 

considered to be toxicologically relevant but rather caused by decreased palatability 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 of the 800 

and 1500 ppm group during pre-mating and not considered an adverse effect of treatment. 

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 ± 34.0 375 ± 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) 1 Page 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 of means) 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 ± 14.3 248 ± 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 ± 18.2 239 ± 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) 

Weeks 312 299 300 290 1-10 (mean of means) 

Mean food consumption (g/animal/day) 

Weeks 30 31 31 30 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 2 176±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 ± 15.3 216 ± 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 ± 16.4 
2-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) 

Weeks 119 122 125 1-10 (mean of means) 

Mean food consumption (g/animal/day) 

Weeks 20 21 21 1-10 (mean of means) 

P Generation females - post coitum 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 ± 20.0 331 ± 26.0 

Day21 436 ± 35.4 435 ± 34.6 424 ± 35.1 

Lactation 319 ± 29.4 322 ± 21.3 312±28.5 

Day 1 

Day 4 326 ± 28.8 328±21.0 318±26.7 

Day 7 333 ± 25.6 336 ± 18.1 327 ± 33.2 

Day 14 347 ± 25.5 351 ± 18.5 333 ± 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 ± 24.6 335 ± 31.3 

Day 21 436 ± 34.6 435 ± 24.8 423 ± 38.3 

Lactation 

Day 1 320 ± 25.4 322 ± 23.0 318±29.4 

Day 4 329 ± 28.4 333 ± 24.0 326 ± 31.0 

Day 7 338 ± 29.7 339±25.1 333 ± 33.3 

Day 14 348 ± 33.5 345 ± 23.8 336 ± 30.6 

Day 21 331 ± 32.0 322 ± 19.1 316 ± 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* 

316 ± 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 of the test substance intake for the entire study. 
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Reproduction and Fertility Effects (2006) I Page 11 of 18 

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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 pages 33 and 38 in the study report. 

4. Reproductive function: 

a. Estrous cycle length and periodicity: Results from the evaluation of vaginal 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. Reproductive performance and fertilityparameters U 

Observation Dose group (ppm) 

Control 300 800 1500 

P Generation 

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 21.5 ± 0.6 21.2 ± 0.7 

Number of litters 22 19 23 24 

Percentage mating 100 100 100 100 

I 

I 
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TABLE 6. Reproductive performance and fertility parametersa 

Observation 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) pre coital 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 of the brain, liver, kidneys, spleen and epididymides, and increased relative 

weights of the brain, pituitary and kidneys. Females of the 1500 dose group showed 

statistically significantly decreased terminal body weights at necropsy, decreased absolute 

weights of the 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 of toxicity. 
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Reproduction and Fertility Effects (2006) 1 Page 13 of 18 

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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 measurement) 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 ± 0.137 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 of the 

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 of the 1500 ppm dose group also exhibited an 

irregular surface of the 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 of nine 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 of the 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 of toxicity were observed at any dose. 

Incidental findings that were not considered to be treatment-related were little or no milk, 
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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 of the 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 of toxicity at any dose. Symptoms 

that were not treatment-related included weak or cold appearance, no milk, alopecia, 

thickened area of the shoulder, eye discharge and opacity, missing tail, and reduced testes 

size. Additionally, one pup of the 300 ppm showed an absent tail and one pup of the 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 FJ 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 53M 147F 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 .6 
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Reproduction and Fertility Effects (2006) 1 Page 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 of pups 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. 
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Reproduction and Fertility Effects (2006) 1 Page 16 of 18 

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TABLE 9. Mean (±SD) Litter and pup weights (g) a 

Dose group (ppm) 

Lactation 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** 

FJ 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 45.9 ± 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). 

** Statistically 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 and 
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Reproduction and Fertility Effects (2006) 1 Page 17 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 of the 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 (absolute, grams) 0.276 ± 0.0892 0.269 ± 0.0356 0.240 ± 0.0597 

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 F lor F2 pups 

during macroscopic examination. Incidental findings that were not found to be dose-
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Reproduction and Fertility Effects (2006) 1 Page 18 of 18 

OPPTS 870.38001 OECD 416 

related or were within normal biological variation included small appearance, no 

milk, pelvic dilation of the 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 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 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 DEFICIENCIES: 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.