UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D. C. 20460 OFFICE OF PREVENTION, PESTICID ES AND TOXIC SUBSTANCES HED DOC. NO. 0050286 DATE: November 20, 2001 MEMORANDUM SUBJECT: LINURON  Report of the Hazard identification Assessment Review Committee FROM: Robert F. Fricke, Ph. D. Reregistration Branch 2 Health Effects Division (7509C)

THROUGH: Jess Rowland, Co Chair and Elizabeth Doyle, Co Chair Hazard Identification Assessment Review Committee Health Effects Division (7509C)

TO: Carmen Rodia Chemical Review Manager Special Review and Registration Division (7508C)

CC: Tom Meyers (SRRD, 7508C) Carol Christensen (HED, RRB2, 7509C)

PC CODE: 035506 On September 13 and 27, 2001 the Hazard Identification assessment Review Committee (HIARC) reviewed the toxicology data base of linuron and selected endpoints/ doses for various exposure assessments. The HIARC also evaluated the potential for increased susceptibility of infants and children from exposure to linuron as required by the Food Quality Protection Act (FQPA) of 1996. The conclusions drawn at these meetings are presented in this report. 2 Committee Members in Attendance Members in attendance: Ayaad Assaad, William Burnam, Jonathan Chen, Paula Deschamp Pamela Hurley, John Liccione, David Nixon, Jess Rowland (Co Chair), and Brenda Tarplee Members in absentia:. Elizabeth Doyle (Co Chair) and Elizabeth Mendez Also in attendance: Susan Makris, Carol Christensen, Ken Doctor, and Pauline Wagner Data evaluation / presentation: Robert F. Fricke Reregistration Branch 2 3 N H Cl Cl O N CH 3 O CH 3 Linuron (035506) 1 INTRODUCTION On September 13 and 27, 2001 the Health Effects Division's (HED) Hazard Identification Assessment Review Committee (HIARC) reviewed the recommendations of the toxicology reviewer for Linuron with regard to the acute and chronic Reference Doses (RfD), the toxicological endpoints for short (1 30 days) and intermediate (1 6 months) term dermal occupational exposure, and the endpoints for short, intermediate and long term (greater than 6 months) inhalation occupational exposure. The potential for increased susceptibility of infants and children from exposure to Linuron was also evaluated. The conclusions drawn at this meeting are presented in this report.

2 HAZARD IDENTIFICATION 2.1 Acute Reference Dose (RfD)  Females 13+

Study Selected: Prenatal Oral Developmental/ Rat OPPTS 870.3700 MRID No.: 00018167 Executive Summary: In a developmental toxicity study (MRID 00018167), 27 presumed pregnant Crl: CD rats per group were administered 0, 50, 125, or 625 ppm of linuron (97% a. i.; Lot No. INZ 326 118) in the diet on gestation days (GD) 6 15, inclusive. Average doses to the treated dams were 5.0, 12.1, and 50 mg/ kg/ day, respectively. The day evidence of mating was found was designated GD 1. Maternal body weights and food consumption were recorded on GD 6, 10, 16, and 21. On GD 21, all surviving dams were sacrificed and all fetuses were weighed and examined for external malformations/ variations. Crown rump length was measured on each fetus. Approximately one half of the fetuses in each litter were fixed in Bouin's solution for visceral examination and the remaining one half were processed for skeletal examination.

All animals survived to scheduled termination without the appearance of any treatmentrelated clinical signs of toxicity. Gross necropsy was unremarkable. No treatment related clinical signs of toxicity were observed. Body weight gains and food consumption by the low and mid dose groups were similar to the controls throughout the study. Body weights of the high dose group were significantly (p # 0.05) less than the control group on GD 10, 16, and 21. Food consumption by the high dose group was significantly (p # 0.05) less than that of the controls for the intervals of GD 6 10 and 10 16. 4 Therefore, the maternal toxicity LOAEL is 625 ppm (50 mg/ kg/ day) based on reduced body weight gains and food consumption. The maternal toxicity NOAEL is 125 ppm (12 mg/ kg/ day).

No dose or treatment related effects were observed on fetal sex ratios, numbers of corpora lutea/ dam, implantations/ dam, live or dead fetuses/ dam, fetal body weights, or crown rump length. In the control, low, mid, and high dose groups post implantation loss was 5.8, 3.5, 4.4, and 14.0%, respectively, and the number of resorptions per litter with resorption was 1.6, 1.6, 1.2, and 2.1, respectively.

No treatment related external or visceral malformations/ variations were noted. In the highdose group bipartite thoracic vetebral centra was observed in 7 fetuses from 7 litters and unopposed sternebrae were observed in 3 fetuses from 3 litters. These anomalies were not found in the control group and were considered indicative of developmental delays.

Therefore, the developmental toxicity LOAEL is 625 ppm (50 mg/ kg/ day) based on increases in post implantation loss and in litter/ fetal resorptions. The developmental toxicity NOAEL is 125 ppm (12 mg/ kg/ day).

This study is classified as Acceptable/ Guideline and does satisfy the guidelines for a developmental toxicity study [OPPTS 870.3700 (83 3a)] in rats.

AcuteR D mg kg day UF mg kg day f = = 12 100 012 // () .//

Dose and Endpoint for Establishing Acute RfD: NOAEL = 12 mg/ kg/ day, based on increases in post implantation loss and litter/ fetal resorptions at the LOAEL of 625 ppm (50 mg/ kg/ day).

Uncertainty Factor( s): 100x (10x intraspecies variability, 10x interspecies extrapolation).

Comments about Study/ Endpoint/ Uncertainty Factor( s): The developmental effects are presumed to occur following a single exposure of females of child bearing age and, therefore, are appropriate for this risk assessment.

2.2 Acute Reference Dose (RfD)  General Population An appropriate end point attributable to a single dosesment for this population subgroup was not available in the database. 5 2.3 Chronic Reference Dose (RfD)

Study Selected: Chronic Toxicity (1 Year)/ Dog 870.4100 (§ 83 1b)

MRID No.: 40952601 Executive Summary: In a one year chronic toxicity study, linuron (96.2% a. i., Batch No. 6,569) was administered to groups of 4 male and 4 female beagle dogs in the diet at concentrations of 0, 10, 25, 125, or 625 ppm (MRID 40952601). Time weighted average doses for the treated groups were 0.29, 0.79, 4.2, and19 mg/ kg/ day, respectively, for males and 0.30, 0.77, 3.5, and 16 mg/ kg/ day, respectively, for females.

No treatment related clinical signs of toxicity or mortalities were observed at any dose level. Body weights, body weight gains, and food consumption were not affected by treatment. No treatment related ophthalmological lesions or changes in urinalysis parameters were noted and gross necropsy was unremarkable.

RBC counts, hemoglobin, and hematocrit were slightly (n. s.) decreased throughout the study in high dose males and females as compared with those of the controls. WBC and platelet counts were significantly (p # 0.05) increased in high dose females at 3, 6, and 9 months and platelet counts were increased (p # 0.05) in high dose males at 3 months. Met and sulfhemoglobin levels were significantly (p # 0.05) increased in the 625 ppm males and females at all time points as compared with those of the controls. In addition, for the 125 ppm groups methemoglobin levels were increased (p # 0.05) in males and females at 3 and 6 months while sulfhemoglobin levels were (p # 0.05) increased at 9 months in males and at 3, 9, and 12 months in females. Increased hematopoiesis was observed in the bone marrow from 3/ 4 high dose males and 4/ 4 high dose females, compared with none of the control males and only 1/ 4 control females.

Cholesterol levels were increased in the high dose groups at all time points as compared with control levels with statistical significance (p # 0.05) attained at 3, 6, 9, and 12 months for males and 3 months for females.

Absolute liver weights were slightly (n. s.) increased in the 625 ppm males and relative liver weights were significantly (p # 0.05) increased in the 125 and 625 ppm males. No effects on liver weights were noted in females.

Increases in the incidence and/ or severity of brown pigment (hemosiderin) deposition in the liver were observed microscopically in high dose males and females.

The LOAEL for linuron in male and female beagle dogs was established at 125 ppm (4.17 mg/ kg/ day in males and 3.5 mg/ kg/ day in females) based on abnormal hematology findings (increased met and sulfhemoglobin levels). The NOAEL was established at 25 ppm (0.79 mg/ kg/ day, males and 0.77 mg/ kg/ day, females). 6 This study is classified as Acceptable/ Guideline and satisfies the guideline requirements for a chronic toxicity study [OPPTS 870.4100 (83 1b)] in dogs.

Dose and Endpoint for Establishing Chronic RfD: NOAEL = 0.77 mg/ kg/ day, based on increased met and sulfhemoglobin levels at the LOAEL (4.17 mg/ kg/ day in males and 3.5 mg/ kg/ day in females).

Uncertainty Factor( s): 100x (10x intraspecies variability, 10x interspecies extrapolation).

Comments about Study/ Endpoint/ Uncertainty Factor( s): The route of administration and duration of exposure are appropriate for establishing a chronic RfD.

ChronicR D mg kg day UF mg kg day f = = 077 100 0 0077 .// () .//

2.4 Occupational Exposure 2.4.1 Oral Exposure <IP_ADDRESS> Short Term (1 30 days) Incidental Oral Exposure Study Selected: Reproduction and Fertility Effects/ Rat 870.3800 (§ 83 4)

MRID No.: 41463401, 41864701 Executive Summary: In a two generation reproduction study, linuron (96.2% a. i.) was administered to groups of 30 male and 30 female Crl: CDBR rats in the diet at concentrations of 0, 12.5, 100, or 625 ppm (MRID Nos: 41463401, 41864701). One litter was produced by each generation. Average premating doses for the treated F0 groups were 0.74, 5.8, and 36 mg/ kg/ day, respectively, for males and 0.92, 7.33, and 45.1 mg/ kg/ day, respectively, for females. Average premating doses for the treated F1 groups were 0.948, 7.77, and 54.0 mg/ kg/ day, respectively, for males and 1.12, 9.24, and 63.0 mg/ kg/ day, respectively, for females. F0 and F1 parental animals were administered test or control diet for 72 or 75 days, respectively, prior to mating and throughout mating, gestation, and lactation, and until necropsy.

No treatment related clinical signs of toxicity or mortalities were observed in the adult animals of either generation.

Body weights, body weight gains, and food consumption were significantly (p # 0.05) less than those of the controls beginning on day 7 for the high dose F0 animals (19%, males; 14%, females) and throughout premating for the mid (6%, males; 8%, females) 7 and high (24%, males; 25%, females) dose F1 animals. At the high dose, overall weight gains in F0 males and females were decreased 59% and 55%, respectively, and for highdose F1 males and females, 76% and 75%, respectively. Mean daily food consumption levels for the high dose groups during the premating interval were 80 85% of the control levels. Absolute body weights of the mid dose F0 males were occasionally significantly (p # 0.05) less than the controls with premating weight gains and food consumption 88% and 94% (both, p # 0.05), respectively, of the control levels. For the mid dose F1 males and females, final premating body weights, body weight gains, and food consumption were 92 94% of the control levels. For the mid and high dose dams of both generations, lower body weights during gestation and lactation were considered a continuation of premating effects.

No treatment related lesions were noted at necropsy of the F0 males or females. In highdose F1 males, gross lesions of the testes included reduced in size (9/ 30), abnormally large (3/ 30), soft (5/ 30), small epididymides (8/ 30), and unspecified deformities of the epididymides (5/ 30); these were noted as significant incidences, but control rates were not given in the original DER. Microscopically, increased incidences (p # 0.05) of testicular and epididymal lesions were found in high dose F1 males as compared with the controls: atrophy (14/ 30), fibrosis (8/ 30), and hyperplasia (7/ 30) in the testes and arteritis (6/ 30), inflammation (5/ 30), and oligospermia (12/ 30) in the epididymides. Only one incidence each of atrophy and oligospermia were observed in control animals. Absolute testes weights of the high dose F1 males were significantly (p # 0.05; 80% of control) less than the controls. In high dose F1 females, gross findings (n. s.) included cystic ovaries (4/ 30), dilatation (3/ 30), and fluid filled uterine horns (2/ 30), none of which were observed in control animals.

In addition, lesions of the eye were noted in high dose F1 males and females and were reviewed in MRID 41864701. A significant (p # 0.05) increase was seen in the number of high dose F1 males with any type of corneal or conjunctival change (14/ 30 vs 4/ 30 controls). The lesions included corneal degeneration/ basophilia and conjunctival inflammation/ basophilia. Degeneration of the lens was observed in 3/ 30 high dose males and 3/ 29 high dose females compared with only 1/ 30 control male. However, a clear treatment related effect in females was not considered to be definitive.

The systemic toxicity LOAEL was established at 100 ppm (average premating doses 5.8 9.24 mg/ kg/ day) based on reduced body weight gains in males and females during both generations. The systemic toxicity NOAEL was established at 12.5 ppm (average premating doses 0.74 1.12 mg/ kg/ day).

No treatment related adverse effects were found on the reproductive performance of either generation. For the control, low, mid, and high dose group, pup viability for lactation days 0 4 was 99.4, 98.0, 99.8, and 91.7% (p # 0.05), respectively, for the F1 generation and 96.8, 92.7, 99.5, and 76.2% (p # 0.05), respectively, for the F2 generation. The mean number of pups per litter in the high dose F2 group was significantly (p # 0.05) less than the control throughout lactation. The number of litters 8 with pups showing clinical signs was significantly (p # 0.05) increased in both generations. Body weights of the mid and high dose F1 male and female pups and of the high dose F2 male and female pups were significantly (p # 0.05) less than those of the controls throughout lactation. These data were not considered for setting the reproductive toxicity NOAEL in the original DER.

The reproductive toxicity NOAEL was greater than or equal to 625 ppm (average premating doses 36 63.0 mg/ kg/ day) and the reproductive toxicity LOAEL was not established.

This study is classified as Acceptable/ Guideline and satisfies the guideline requirements for a reproductive toxicity study [OPPTS 870.3800 (83 4)] in rats.

Dose and Endpoint Selected: 5.8 mg/ kg (maternal NOAEL) based statistically and biologically significant decrease in premating body weights in F0 and F1 animals observed at 36 mg/ kg/ day.

Comments about Study/ Endpoint: The systemic toxicity (body weight decrease) is relevant for the populations (infants and children) and duration (1  30 days) of concern. The NOAEL of 5.8 mg/ kg/ day used for this exposure period is based on the decrease in body weight seen during days 1 through 30 at 36 mg/ kg/ day and is used only for this risk assessment. This NOAEL/ LOAEL differs from the overall NOAEL/ LOAEL (0.7/ 5.8) established for the study. In an open literature publication [McIntyre, B. S. et al., Toxicol Appl Pharmacol 167( 2): 87 99 (2000)] the effects observed (retention of areolae/ nipples in male rats) at 12.5 mg/ kg/ day were not considered to be biologically significant for risk assessment since the findings were not replicated in any other studies. .

2.4.2 Intermediate Term (1 to 6 Months) Incidental Oral Exposure Study Selected: One year chronic study  Dog 870.4100 (§ 83 1b)

MRID No.: 40952601 Executive Summary: See Chronic RfD (2.3)

Dose and Endpoint Selected: 0.77 mg/ kg/ day, based on increased met and sulfhemoglobin concentrations at 3.5 mg/ kg/ day.

Comments about Study/ Endpoint: The effects seen in this study after 3 and 6 months treatment were within the time frame for intermediate term exposure.

2.5 Dermal Absorption 9 Study Selected: Dermal penetration 870.7600 (§ 85 2)

MRID No.: 163837 Executive Summary: In this dermal absorption study, four groups of 20 rats (one female group and three male groups) were dosed with 14 C (2.35 : Ci/ mg) linuron at 0.12, 1.00, or 7.4 mg/ 2 in 2 (2.82, 23.5, or 17.4 : Ci, respectively).

Based on the results of this study, a dermal absorption factor of 16% was observed for an exposure of 8 to 10 hr (2%/ hr).

Percentage (%) Dermal Absorption: 16% for 8 to 10 hour exposure (2% per hour)

2.6 Dermal Exposure 2.6.1 Short Term (1 30 days) Dermal Exposure Study Selected: Reproduction and Fertility Effects/ Rat 870.3800 (§ 83 4)

MRID No.: 41463401, 41864701 Executive Summary: See short term oral exposure (<IP_ADDRESS>)

Dose and Endpoint Selected: Parental NOAEL of 5.8 mg/ kg based statistically and biologically significant decrease in premating body weights in F0 and F1 animals observed at 36 mg/ kg/ day.

Comments about Study/ Endpoint: No repeat dose dermal toxicity study is available. Therefore, an oral NOAEL of 5.8 mg/ kg/ day was selected for this exposure period. The NOAEL used for this exposure period is based on the decrease in body weight seen during days 1 through 30 at 36 mg/ kg/ day and is used only for this risk assessment. This NOAEL/ LOAEL differs from the overall NOAEL/ LOAEL (0.7/ 5.8) established for the study. A dermal absorption factor of 16% should be used for route to route extrapolation.

2.6.2 Intermediate (1 to 6 Months) Term Dermal Exposure Study Selected: One year chronic study  Dog 870.4100 (§ 83 1b)

MRID No.: 40952601 Executive Summary: See Chronic RfD (2.3)

Dose and Endpoint Selected: 0.77 mg/ kg/ day, based on increased met and sulfhemoglobin concentrations after 3 and 6 months treatment at 3.5 mg/ kg/ day. 10 Comments about Study/ Endpoint: No repeat dose dermal toxicity study is available. The systemic toxicity (increased met and sulfhemoglobin concentrations) is relevant for this duration (seen after 3 and 6 months). A dermal absorption factor of 16% should be used for route to route extrapolation.

2.6.3 Long term (greater than 6 months) Dermal Exposure Study Selected: One year chronic study  Dog 870.4100 (§ 83 1b)

MRID No.: 40952601 Executive Summary: See Chronic RfD (2.3)

Dose and Endpoint Selected: 0.77 mg/ kg/ day; based on increased met and sulfhemoglobin concentrations after 9 and 12 months treatment at 3.5 mg/ kg/ day. A dermal absorption factor of 16% should be used for route to route extrapolation.

Comments about Study/ Endpoint: This dose and endpoint were selected to establish the chronic RfD. A dermal absorption factor of 16% should be used for route to route extrapolation.

2.7 Inhalation Exposure 2.7.1 Short Term (1 30 days) Inhalation Exposure Study Selected: Reproduction and Fertility Effects/ Rat 870.3800 (§ 83 4)

MRID No.: 41463401, 41864701 Executive Summary: See short term oral exposure (<IP_ADDRESS>)

Dose and Endpoint Selected: Parental NOAEL of 5.8 mg/ kg based statistically and biologically significant decrease in premating body weights in F0 and F1 animals observed at 36 mg/ kg/ day.

Comments about Study/ Endpoint: In the absence of an inhalation study, an oral study was selected. The NOAEL used for this exposure period is based on the decrease in body weight seen during days 1 through 30 at 36 mg/ kg/ day and is used only for this risk assessment. This NOAEL/ LOAEL differs from the overall NOAEL/ LOAEL (0.7/ 5.8) established for the study.

2.7.2 Intermediate Term (1 to 6 Months) Incidental Inhalation Exposure Study Selected: One year chronic study  Dog 870.4100 (§ 83 1b) 11 MRID No.: See Chronic RfD (2.3)

Executive Summary: See Chronic RfD (2.3)

Dose and Endpoint Selected 0.77 mg/ kg/ day, based on increased met and sulfhemoglobin concentrations after 3 and 6 months treatment at 3.5 mg/ kg/ day.

Comments about Study/ Endpoint: In the absence of an inhalation study, an oral study was selected.

2.7.3 Long term (greater than 6 months) Inhalation Exposure Study Selected: One year chronic study  Dog 870.4100 (§ 83 1b)

MRID No.: See Chronic RfD (2.3)

Executive Summary: See Chronic RfD (2.3)

Dose and Endpoint Selected: 0.77 mg/ kg/ day, based on increased sulfhemoglobin concentrations after 9 and 12 months treatment at 3.5 mg/ kg/ day.

Comments about Study/ Endpoint: See Chronic RfD (2.3)

2.8 Margins of Exposure for Occupational Risk Assessments Margin of exposure of 100 is adequate for occupational dermal and inhalation exposure risk assessments. The MOEs for residential use will be determined by the FQPA Safety Factor Committee.

3 CLASSIFICATION OF CARCINOGENIC POTENTIAL 3.1 Combined Chronic Toxicity/ Carcinogenicity Study in Rats MRID No.: 00029680, 00029679 Executive Summary: In a chronic toxicity/ oncogenicity study (MRID 00029680 and MRID 00029679), INZ 326 (Linuron; 96.9 97.2% a. i.; Lot No. 20427, Batch No. 90, E 6110 29B) was administered in the diet to groups of 70 ChR CD® rats/ sex/ dose at concentrations of 0, 50, 125, and 625 ppm (0, 2.09, 5.11, and 27.1 mg/ kg/ day for males and 0, 3.13, 7.75, and 48.3 mg/ kg/ day for females) for up to 2 years. Additional groups of 10 rats/ sex/ dose were administered the same diets for 12 months for interim evaluation. All clinical pathology data were reanalyzed (MRID 00164117) due to inappropriate statistical methods used in the original study report. 12 Linuron had no effect on mortality at any dose, and there were no treatment related clinical signs reported. Absolute body weights of the high dose male group were decreased to 88 91% of controls during weeks 1 19, with decreased body weight gain during weeks 0 13 and 52 104 resulting in body weight gain for the entire study being 89% of controls. Absolute body weights of the high dose female group were decreased throughout the study, with the magnitude of the decrease generally increasing throughout the course of the study. The body weight gain of this group for the entire study was 57% of controls. Body weight gain by mid dose males was decreased to 64% of controls during the week 52 76 interval, with body weight loss during the week 76 104 interval being increased to 148% of controls. Body weight gain by the mid dose female group was decreased to 75% of controls during the week 52 76 interval. There were no toxicologically significant treatment related effects on food consumption; however, food efficiency values of high dose females were decreased to 74 88% of controls during weeks 1 4 and to 58% of controls for the entire study period.

Histopathology observations consistent with hemolysis were observed at increased incidences in high and mid dose males and females from the main study and interim sacrifice groups, including hemosiderin in Kupffer cells and increased hemosiderosis in bone marrow, spleen, and/ or mesenteric lymph nodes, and transient decreases in the erythrocyte count, hemoglobin concentration, and hematocrit of the high dose female group were noted at 6 and/ or 12 months.

Male rats of the main study group had significantly increased incidences of mineralization/ calculi in the renal pelvis, transitional cell hyperplasia in the renal pelvis, and subacute perivasculitis and/ or vasculitis in the epididymides at the mid and high dose treatment levels. Female rats of the main study group had significantly increased incidences of calculi in renal tubules at the mid and high dose treatment levels and significantly increased incidences of hepatic sinusoidal ectasia and collecting duct ectasia in the kidney at the high dose treatment level. The high dose female group also had a non statistically significantly increased incidence of transitional cell hyperplasia in the renal pelvis compared to controls. Hepatocellular megalocytosis/ syncytium formation with fibroplasia radiating between hepatic cords, and occasional increased hepatocellular intracytoplasmic basophilia was only observed in the main study high dose female group (15/ 68 animals examined at that site) and may have been related to induction of hepatocellular protein synthesis. The significance of this finding is unknown but considered adverse.

The lowest observed adverse effect level (LOAEL) for Linuron in ChR CD® rats is 125 ppm (5.11 mg/ kg/ day for males and 7.75 mg/ kg/ day for females), based on decreased body weight gains in both sexes, microscopic observations consistent with hemolysis (hemosiderin in Kupffer cells and increased hemosiderosis in bone marrow, spleen, and/ or mesenteric lymph nodes), and increased incidences of microscopic changes in the epididymides (perivasculitis/ vasculitis) and renal pelvis (transitional cell hyperplasia and mineralization/ calculi) of males and kidneys (calculi in renal tubules) of females. The corresponding no observed adverse effect level is 50 ppm (2.09 mg/ kg/ day for males and 3.13 mg/ kg/ day for females). 13 There was a treatment related increase in the incidence of testicular interstitial adenomas at the 125 and 625 ppm treatment levels (5.7, 27.5, and 53.6% for control, mid, and high dose males, respectively; p< 0.01). Common neoplasms, included pituitary adenomas of the pars anterior in both male and female rats and mammary fibroadenomas in female rats. Decreased incidences of both these tumor types were noted in the high dose female group. Dosing was considered adequate based on the decreases in body weight and body weight gain of high dose females.

This chronic toxicity/ oncogenicity study in the rat is Acceptable/ Guideline and does satisfy the guideline requirement for a chronic toxicity/ oncogenicity oral study [OPPTS 870.4300 (§ 83 5)] in the rat; however, it must be noted that results from concentration and stability analyses indicate potential variation between nominal and actual diet concentrations which make the exact doses to the animals questionable.

Discussion of Tumor Data: In a chronic toxicity/ oncogenicity study, INZ 326 (linuron; 96.9 97.2% a. i.) was administered in the diet to groups of 70 ChR CD' rats/ sex/ dose at concentrations of 0, 50, 125, and 625 ppm (0, 2.09, 5.11, and 27.1 mg/ kg/ day for males and 0, 3.13, 7.75, and 48.3 mg/ kg/ day for females) for up to 2 years.

There was a treatment related increase in the incidence of testicular interstitial adenomas at the 125 and 625 ppm treatment levels (5.7, 27.5, and 53.6% for control, mid, and high dose males, respectively; p< 0.01). Common neoplasms, included pituitary adenomas of the pars anterior in both male and female rats and mammary fibroadenomas in female rats. Decreased incidences of both these tumor types were noted in the high dose female group.

Adequacy of Dose Levels: Dosing was considered adequate based on the decreases in body weight and body weight gain of high dose females.

3.2 Carcinogenicity Study in Mice MRID No. 00124195 Executive Summary: In an oncogenicity study (MRID 00124195), INZ 326 (97.0% a. i., Haskell Laboratory identification no. 10720) was administered to groups of 80 male and 80 female Charles River CD® 1 mice in the diet at concentrations of 0, 50, 150, or 1500 ppm. The test diets were given for 24 months. The concentrations of 50, 150, and 1500 ppm resulted in mean daily compound intakes for males of 8, 23, and 261 mg/ kg/ day; and for females of 12, 35, and 455 mg/ kg/ day, respectively, calculated from food intake and body weight measurements.

No significant treatment related effects were seen in clinical signs or survival. Body weights were consistently and significantly lower in males and females at 1500 ppm than in the control groups throughout the study. At 52 weeks, the group mean body weights and weight 14 gains of high dose males were 8% and 15% lower than the controls, respectively, and highdose females were decreased by 11% and 21%. At 104 weeks, the body weights and weight gains of high dose males were 10% and 20% less than the controls, and in high dose females were 8% and 14% less than the controls, respectively. The overall food intake for high dose males was decreased by about 14% and by 10% for high dose females compared to the controls. Food efficiency for the 2 year study was not significantly affected in treated animals.

Increases of 9 18% in group mean erythrocyte cell volume and mean cell hemoglobin were seen in males and females after 6 months of treatment at 1500 ppm. Erythrocyte counts were decreased by 9% and 14% in high dose males and females, respectively, compared to the controls at 6 months. These hematology values returned to near control levels later in the study. A significant increased incidence of hemosiderin deposits in the spleens of both sexes at 1500 ppm is suggestive of a compensated hemolytic anemia in high dose animals. Methemoglobin levels were increased in high dose females by 117% compared to the control group, and were increased in high dose males by 613% compared to the 50 ppm group (the male control value was not available). Differential white cell counts were within normal parameters for both sexes at all doses.

The absolute and relative (to body) liver weights were increased by 20% and 24%, respectively in high dose males and by 65% in high dose females compared to the controls. Microscopic evidence of liver toxicity at 1500 ppm included increased incidences of focal hepatocellular cytoplasmic alteration, focal centrilobular peliosis, and centrilobular hepatocytomegaly in both sexes, and increased incidence of hepatocellular cytoplasmic vacuolation in females.

The LOAEL for INZ 326 in mice is 1500 ppm in the diet for males (261 mg/ kg/ day) and females (455 mg/ kg/ day), based on microscopic liver changes, methemoglobinemia, and decreased body weight and weight gain in both sexes. The NOAEL was 150 ppm for males (23 mg/ kg/ day) and females (35 mg/ kg/ day).

Treatment of up to 104 weeks with 1500 ppm INZ 326 resulted in a significant increase in the incidence of hepatocellular adenomas (control, 6%; 1500 ppm, 25%, p < 0.05) in female Charles River CD® 1 mice under the conditions of this study. Dosing was considered adequate based on the liver changes, methemoglobinemia, and decreased body weights.

This oncogenicity study in the mouse is Acceptable (Guideline) and does satisfy the guideline requirement for an oncogenicity study [OPPTS 870.4200 (§ 83 2)] in mice. There were a number of deficiencies in this study, but none that would alter the conclusions reached through the available data.

Discussion of Tumor Data: Treatment of up to 104 weeks with 1500 ppm INZ 326 resulted in a significant increase in the incidence of hepatocellular adenomas (control, 6%; 1500 ppm, 25%, p < 0.05) in female Charles River CD® 1 mice under the conditions of this study. 15 Adequacy of the Dose Levels: Tested: Dosing was considered adequate based on the decreases in body weights, body weight gains and other systemic effects (microscopic liver changes, methemoglobinemia) in high dose males (261 mg/ kg/ day) and females (455 mg/ kg/ day).

3.3 Special Oncogenicity Study  Aged Male Rats MRID No.: 45506501 Executive Summary: A special study (MRID 45506501) was conducted to determine whether linuron (94.5% a. i.) administered in the diet to aged male Crl: CD( SD) BR rats would induce testicular interstitial cell (Leydig cell) hyperplasia and adenomas. Two groups of 25 rats each were given 625 ppm linuron beginning at 12 or 18 months of age and continuing for 6 and 12 months; terminal sacrifice was carried out at 24 months of age. Another group of 25 rats was given basal diet and served as controls. Compound intake for both treated groups was 22 23 mg/ kg/ day for the treatment interval.

Treatment related clinical signs of toxicity were limited to swollen testes observed in 2, 4, and 5 rats in the control, 6, and 12 month groups. A total of 6, 4, and 4 rats, respectively, were found dead or were sacrificed in extremis prior to scheduled termination; further details were not included.

Body weights of both treated groups were significantly reduced as compared with the control group within two weeks after the initiation of feeding the test article. Final body weights for the control, 6, and 12 month groups were 848.5, 670.1, and 668.5 g, respectively (additional data not given in original DER). Weight gains during the treatment intervals were significantly (p # 0.05) less than the controls with a weight loss for animals treated for 6 months ( 107.8 g vs 45.6 g for control) and a weight gain 2.1% of the control level by the animals treated for 12 months. Food consumption and food efficiency values were slightly reduced for both treated groups as compared with the controls throughout the treatment intervals, but statistical significance was not attained.

At necropsy discoloration of the testes was observed in 2/ 25, 4/ 25, and 6/ 25 rats in the control, 6 and 12 month groups, respectively. However the observation did not correlate with either interstitial cell adenoma or hyperplasia.

For rats sacrificed at study termination, a dose related increases in the incidence of testicular adenoma were observed. The incidences in the control, 6, and 12 month groups were 0/ 19, 2/ 21, and 6/ 21 (p # 0.05), respectively. In addition, testicular hyperplasia was observed in 6/ 19, 7/ 21, and 14/ 21 (p # 0.05) rats, respectively. No increased incidences of hyperplasia or adenoma in the adrenal cortex, adrenal medulla, or the pituitary gland were observed in the treated groups as compared with the control group. 16 In conclusion, linuron induced hyperplasia and adenomas of the testes in aged rats. In addition, life time feeding was not necessary to induce oncogenic responses in this tissue, in fact linuron may be more potent as an oncogen than previously estimated from the two year study.

This study is classified as Acceptable/ Nonguideline as a special feeding study in aged rats.

Discussion of Tumor Data: A special study was conducted to determine whether linuron (94.5% a. i.) administered in the diet to aged male Crl: CD( SD) BR rats would induce testicular interstitial cell (Leydig cell) hyperplasia and adenomas. Two groups of 25 rats each were given 625 ppm linuron beginning at 12 or 18 months of age and continuing until sacrifice at 24 months of age; another group of 25 rats was given basal diet and served as controls. Compound intake for both treated groups was 22 23 mg/ kg/ day for the treatment interval.

For rats sacrificed at study termination, a dose related increased incidence of testicular adenoma was observed. The incidence rates in the control, 6, and 12 month groups were 0/ 19, 2/ 21, and 6/ 21 (p # 0.05), respectively. In addition, testicular hyperplasia was observed in 6/ 19, 7/ 21, and 14/ 21 (p # 0.05) rats, respectively. No increased incidences of hyperplasia or adenoma in the adrenal cortex, adrenal medulla, or the pituitary gland were observed in the treated groups as compared with the control group.

In conclusion, linuron induced hyperplasia and adenomas of the testes in aged rats. In addition, life time feeding was not necessary to induce oncogenic responses in this tissue, in fact, linuron may be a more potent oncogen than previously estimated from the 2 year study.

Adequacy of Dose Levels: Dose levels were the same as the previously conducted oncogenicity study in the rat.

3.4 Classification of Carcinogenic Potential Linuron was placed in special review for carcinogenic effects in 1982. Linuron was later classified as a group C carcinogen with a Q1* of 2 x 10 5 on the basis of a dose related increase in interstitial cell hyperplasia and adenomas in a two year rat feeding study (00029680) and hepatocellular tumors that appeared in low dose male and high dose female mice in a two year feeding study (00124195). Subsequent review by the HED peer review committee and the Science Advisory Panel resulted in the removal of the Q1* for quantification of risk. At present, Linuron is classified as a Group C carcinogen requiring no quantification of human cancer risk [Federal Register 54( 17): 4072].

4 MUTAGENICITY 4.1 Gene Mutations 4.1.1 Salmonella typhimurium/ Escherichia coli reverse gene mutation assay MRID No.: 00131738 17 Executive Summary: In a reverse gene mutation assay in bacteria, S. typhimurium strains TA98, TA100, TA1535, and TA1537 were exposed to Linuron (95 97%) in dimethylsulfoxide (DMSO) at concentrations of 0.5, 0.75, 1.0, 2.5, and 5.0 : g/ plate in the absence of mammalian metabolic activation (S9 mix) and 1, 5, 10, 50, and 100 : g/ plate in the presence of S 9 mix. Duplicate plates were utilized for each test concentration, and two independent assays were performed. The S9 fraction was obtained from Aroclor 1254 induced Charles River CD rat liver.

Linuron was cytotoxic at $ 5 : g/ plate without S 9 mix; and was cytotoxic at $ 50 : g/ plate with S 9 mix. No treatment related increase in mutant frequency was noted in any strain tested with or without exogenous metabolic activation. The solvent (DMSO) and positive control (N methyl N' nitro N nitrosoguanidine, 2 nitrofluorene, 9 aminoacridine, and 2

aminoacridene) values were appropriate in the respective strains. There was no evidence of induced mutant colonies over background with or without S9 activation.

This study is classified as Acceptable/ Guideline. It satisfies the requirements for FIFRA Test Guideline [OPPTS 870.5100 (§ 84 2)] for in vitro mutagenicity (bacterial reverse gene mutation) data.

4.1.2 Chinese Hamster Ovary (CHO)/ HGPRT cell forward gene mutation assay MRID No.: 00137152 Executive Summary: In a mammalian cell gene mutation assay in vitro, triplicate (in the absence of activation) or duplicate (in the presence of activation) cultures of Chinese hamster ovary (CHO) CHO K1 BH4 cells were exposed to Linuron (Lot No. 1N2 326 141, 94.5% a. i.) in F12 medium at concentrations of 0.05, 0.25, 0.35, 0.40, 0.45, and 0.50 mM in the absence of mammalian metabolic activation (S9 mix), and at 0.25, 0.50, 0.75, 0.90, and 1.0 mM in the presence of Charles River S9 mix. The S9 fraction was obtained from Aroclor 1254 induced 8 to 9 week old male Charles River CD rats.

Linuron was tested up to concentrations limited by cytotoxicity. Cytotoxicity was observed at 0.45 and 0.5 mM under nonactivated conditions and at 0.75 mM and above with 0.5 mg S9 protein/ mL and at 1.0mM and above with 1.0 mg S9 protein/ mL. (Percentage cell survival were not provided in the DER). There was no increase in mutant frequency in cells treated with linuron in either the presence or absence of metabolic activation. The positive (ethyl methane sulfonate (EMS) without S9 mix and dimethylbenzanthracene with S9 mix) and solvent (DMSO) controls responded appropriately. No evidence of an increased mutant frequency was observed in the presence or absence of metabolic activation. 18 This study is classified as Acceptable/ Guideline. It satisfies the requirements for FIFRA Test Guideline [OPPTS 870.5300 (§ 84 2)] for in vitro mutagenicity (mammalian forward gene mutation) data.

4.2 Chromosome Aberrations 4.2.1 In vivo bone marrow cytogenetic assay MRID No.: 00137153 Executive Summary: In a mammalian cell cytogenetics chromosomal aberration assay in bone marrow cells of Sprague Dawley rats, 5 rats per sex per harvest time were administered Linuron (94.5%, lot number not given) by single gavage at doses of 0, 100, 300, or 1000 mg/ kg. Bone marrow cells were harvested 6, 12, 24, or 48 hours after test compound administration and 48 hours after the positive control dose. The vehicle was corn oil (20 mL/ kg) and the positive control was a single 40 mg/ kg dose of cyclophosphamide.

One high dose rat in the 24 hour group was found dead and 8 of 10 high dose rats in the 48 hour group died prior to sacrifice on day 2. Low and mid dose animals exhibited slight depression, ataxia, and/ or prostration. Treated animals also had decreased body weights compared to controls. There was no significant increase in the frequency of aberrations in bone marrow cells of treated animals compared to controls at any sampling time. Values in treated animals ranged from 0.3 0.8% aberrant cells/ group; the positive control group had 19.6% aberrant cells, indicating that this control responded appropriately. There was no change in mitotic index of dosed groups compared to controls. There is no evidence that Linuron induced chromosomal aberrations in bone marrow cells of rats over background levels.

This study is classified as Acceptable/ Guideline. It satisfies the requirements for FIFRA Test Guideline OPPTS [870.5385 (§ 84 2)] for in vivo cytogenetic mutagenicity data.

4.3 Other Mutagenic Mechanisms 4.3.1 Unscheduled DNA synthesis (UDS) in WI 38 human fibroblasts assay MRID No.: 00132583 Executive Summary: In an unscheduled DNA synthesis assay, primary rat hepatocyte cultures were exposed to Linuron (94.5% a. i. in dimethylsulfoxide; Lot No. T80311 81) in Williams' Medium E (WME) at concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1.0, 10, and 50.0 mM (trial 1) or 0.01, 0.1, 1.0, 10, and 50.0 mM (trial 2) for 18 hours.

Linuron was cytotoxic in one of 2 cultures at 1.0 mM in trial 1; no other cytotoxicity data 19 were provided. The mean net nuclear grain counts were calculated from 25 randomly selected nuclei per slide. There was no increase in mean net nuclear grain counts in treated cells compared to the solvent control, indicating no induction of UDS activity. The solvent (DMSO) and positive control (1.0 mM dimethylbenzanthracene) values were appropriate. There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver grain counts] was induced.

This study is classified as Acceptable/ Guideline. It satisfies the requirements for FIFRA Test Guideline [OPPTS 870.5550 (§ 84 2)] for other genotoxic mutagenicity data.

4.4 Conclusions: Linuron was not mutagenic in bacteria or in cultured mammalian cells. There was also no indication of a clastogenic effect up to toxic doses in vivo.

The submitted test battery satisfies the Pre 1991 mutagenicity initial testing battery guidelines. No further testing is required at this time.

5 FQPA CONSIDERATIONS 5.1 Adequacy of the Data Base The toxicology data base is complete but is inadequate for an FQPA assessment. The required developmental toxicity studies in the rat and rabbit and reproduction studies in the rat meet guideline requirements.

5.2 Neurotoxicity Data No acute or subchronic neurotoxicity studies on Linuron are available. Evaluation of subchronic, chronic and reproduction toxicity, did not indicate any treatment related effects on the central or peripheral nervous system of mice, rats, or rabbits. No changes in clinical signs, brain weights, gross necropsy results or histopathological results suggested any part of the nervous system as a target organ. 20 5.3 Developmental Toxicity 5.3.1 Prenatal Developmental Study  Rat MRID No: 00018167 Executive Summary: In a developmental toxicity study, 27 presumed pregnant Crl: CD rats per group were administered 0, 50, 125, or 625 ppm of linuron (97% a. i.; Lot No. INZ 326 118) in the diet on gestation days (GD) 6 15, inclusive. Average doses to the treated dams were 5.0, 12, and 50 mg/ kg/ day, respectively. The day evidence of mating was found was designated GD 1. Maternal body weights and food consumption were recorded on GD 6, 10, 16, and 21. On GD 21, all surviving dams were sacrificed and all fetuses were weighed and examined for external malformations/ variations. Crown rump length was measured on each fetus. Approximately one half of the fetuses in each litter were fixed in Bouin's solution for visceral examination and the remaining one half were processed for skeletal examination.

All animals survived to scheduled termination without the appearance of any treatmentrelated clinical signs of toxicity. Gross necropsy was unremarkable. No treatmentrelated clinical signs of toxicity were observed. Body weight gains and food consumption by the low and mid dose groups were similar to the controls throughout the study. Body weights of the high dose group were significantly (p # 0.05) less than the control group on GD 10, 16, and 21. Food consumption by the high dose group was significantly (p # 0.05) less than that of the controls for the intervals of GD 6 10 and 10 16.

The maternal toxicity LOAEL is 625 ppm (50 mg/ kg/ day) based on reduced body weight gain and food consumption. The maternal toxicity NOAEL is 125 ppm (12 mg/ kg/ day).

No dose or treatment related effects were observed on fetal sex ratios, numbers of corpora lutea/ dam, implantations/ dam, live or dead fetuses/ dam, fetal body weights, or crown rump length. In the control, low, mid, and high dose groups post implantation loss was 5.8, 3.5, 4.4, and 14.0%, respectively, and the number of resorptions per litter with resorption was 1.6, 1.6, 1.2, and 2.1, respectively.

No treatment related external or visceral malformations/ variations were noted. In the high dose group bipartite thoracic vetebral centra was observed in 7 fetuses from 7 litters and unapposed sternebrae were observed in 3 fetuses from 3 litters. These anomalies were not found in the control group and were considered indicative of developmental delays.

The developmental toxicity LOAEL is 625 ppm (50 mg/ kg/ day) based on increases in post implantation loss and in litter/ fetal resorptions. The developmental toxicity NOAEL is 125 ppm (12 mg/ kg/ day). 21 This study is classified as Acceptable/ Guideline and does satisfy the guidelines for a developmental toxicity study [OPPTS 870.3700 (83 3a)] in rats. Deficiencies included no information on dietary formulation preparation or analyses, the treatment period should have been GD 7 16, body weight on GD 0 were not collected, fetal anomalies were not classified as malformations or variations, and fetuses were not individually identified.

5.3.2 Developmental Toxicity Study in the Rabbit MRID Nos: 00153867 and 40437201 Executive Summary: In a developmental toxicity study, 25 presumed pregnant New Zealand white rabbits per group were administered 0, 5, 25, or 100 mg/ kg/ day of linuron (96.2% a. i.) by gavage on gestation days (GD) 7 19, inclusive. Doses were chosen based on the results of a range finding study. The vehicle was 0.5% hydroxypropylmethylcellulose On GD 29, all surviving does were sacrificed and the livers weighed. All fetuses were weighed and examined for external and visceral malformations/ variations including free hand sectioning of the brain. All fetuses were eviscerated and processed for skeletal examination.

No dose or treatment related clinical signs of toxicity, maternal deaths, or necropsy findings were observed in any group. Absolute body weights and food consumption for the low and mid dose groups and body weight gain by the low dose group were not affected by treatment.

Body weight of the high dose group was significantly (p # 0.05) less than that of the controls on GD 19. Body weight gains during GD 13 16 were slightly less for the middose group (50%, n. s.) and significantly less for the high dose group ( 0.01 g vs 0.08 g for the controls; p # 0.05) as compared with the controls. Similarly, during GD 16 20 body weight gains were slightly less for the mid dose group and significantly (p # 0.01) less for the high dose group (0.00 g for mid dose and 0.12 g for high dose vs 0.02 g for the controls) as compared with the controls. Both mid and high dose groups had significantly (p # 0.05) greater body weight gains as compared with the controls during GD 20 24. Food consumption by the high dose group was significantly (p # 0.05) less than that of the controls on GD 13 16 and 16 20.

In the high dose group, absolute and relative liver weights were increased to 129% (p # 0.01) and 135% (n. s.), respectively of controls.

The maternal toxicity LOAEL was established at 25 mg/ kg/ day based on reduced body weight gain. The maternal toxicity NOAEL was established at 5 mg/ kg/ day.

The number of fetuses( litters) available for evaluation in the control, low, mid, and high dose groups was 135( 20), 135( 20), 121( 17), and 79( 13), respectively. 22 Five high dose does aborted between days 20 25 compared with one control doe on GD 22. The mean number and percentage of resorptions and number of dead fetuses were similar between the treated and control groups. In the high dose group, slight (n. s.) decreases in the mean number of live fetuses/ litter (6.1 vs 6.8 for controls) and mean fetal body weight (41.99 g vs 45.8 g for controls) were observed.

No treatment related external or visceral fetal malformations/ variations were noted. In the control, low, mid, and high dose groups, skull alterations (irregularly shaped fontanelle, hole in parietals, parietals contain intraparietals, and unossified) were observed in 1( 1), 9( 5), 5( 3), and 19( 6) fetuses( litters), respectively. The litter incidence for the high dose group was significantly (p # 0.05) greater than that of the control group. It should be noted that 6 fetuses from 5 low dose litters also had a variety of external malformations of the head and body.

The developmental toxicity LOAEL was established at 100 mg/ kg/ day based on alterations of the bones of the skull. The developmental toxicity NOAEL was established at 25 mg/ kg/ day.

This study is classified as Acceptable/ Guideline and does satisfy the guidelines for a developmental toxicity study [OPPTS 870.3700 (83 3b)] in rabbits.

5.4 Reproductive Toxicity 5.4.1 Three Generation Reproduction  Rat MRID No.: 00146071 & 00155168 Executive Summary: In a three generation reproduction study, Linuron (94.5% a. i.) was administered to groups of 20 male and 20 female Crl: CD® rats in the diet at concentrations of 0, 25, 125, or 625 ppm. Two litters were produced by the F0 and F1 generations and one litter was produced by the F2 generation. Average premating doses were 0, 2, 9, and 44 mg/ kg/ day, respectively, for F0 males; 0, 2, 10, and 50 mg/ kg/ day, respectively, for F0 females; 0, 2, 9, and 50 mg/ kg/ day, respectively, for F1 males; 0, 2, 11, and 59 mg/ kg/ day, respectively, for F1 females; 0, 2, 9, and 48 mg/ kg/ day, respectively, for F2 males; and 0, 2, 11, and 67 mg/ kg/ day, respectively, for F2 females. F1 and F2 adults were chosen from the F1b and F2b litters, respectively. F0, F1, and F2 male and female parental animals were administered test or control diet for at least 90 days prior to mating, throughout mating, gestation, and lactation, and until necropsy. At weaning 10 F2b pups/ sex/ group were subjected to gross necropsy with microscopic examination of selected tissues. Following the reproductive toxicity evaluations, F1 and F2 adults were maintained on their respective diets, for up to a total of 22 months on study, for hematological evaluations (MRID 00155168).

Premature deaths of several adults in each generation were considered incidental to 23 treatment. No treatment related clinical signs of toxicity were observed in males or females during premating in any generation. Necropsy findings were not reported for adults. No effects on body weights or body weight gains were seen in the low dose groups of any generation; food consumption and food efficiency were not affected by treatment.

Body weights of the high dose parental animals were significantly (p # 0.05) less than those of the controls beginning on day 7 for the F0 adults and throughout premating for the F1 and F2 adults. Compared with their control levels, body weights for the high dose males and females were 83 90% and 88 93%, respectively, for the F0 adults, 77 81% and 74 87%, respectively, for the F1 adults, and 72 79% and 74 81%, respectively, for the F2 adults. Premating weight gains for the high dose males and females were significantly (p # 0.05) less than those of the controls in all generations. Body weights of the mid dose males were less than those of the controls during each generation, but statistical significance was reached only occasionally. Body weights of the mid dose females from all generations were 88 94% of the control levels with statistical significance (p # 0.05) attained at most time points. Lower body weights of the mid and/ or high dose dams after weaning of their litters were considered a continuation of the premating effects on body weights.

Hematology results for the F1 rats were inconclusive. No treatment related hematological effects were noted in F2 males after continuous feeding for 20 months. However, for F2 females a mild anemia was observed in the mid and high dose groups at 20 and 22 months. In mid and high dose females, RBC counts were decreased to 90 91% of the controls, hemoglobin was decreased to 89 93% of controls, and the percent of reticuloytes was increased to 147 213% of the control levels. Although statistical significance was not attained for all endpoints at both sampling intervals, the changes in red cell parameters are considered to be biologically significant.

Treatment related lesions observed in the liver of high dose F2b weanlings are considered systemic toxicity. In the control, low, mid, and high dose groups, the incidence (average severity) of hepatocellular atrophy was 1/ 10 (2.0), 0/ 10 (0), 2/ 10 (1.5), and 8/ 10 (2.1), respectively, for males and 2/ 10 (3.0), 1/ 10 (3.0), 2/ 10 (2.0), and 10/ 10 (2.5), respectively, for females. The incidence and severity of decreased cytoplasmic vesiculation was the same as that of hepatocellular atrophy for all groups except the high dose males in which 10/ 10 were affected with an average severity score of 2.0.

The LOAEL for systemic toxicity was established at 125 ppm (average premating dose 9 mg/ kg/ day, males and 10 mg/ kg/ day, females) based on reduced body weights of males and females and anemia in females. The systemic toxicity NOAEL is 25 ppm (premating dose 2 mg/ kg/ day in males and females).

Fertility, pup survival, and pup body weights were not affected in the low or mid dose 24 groups in any generation. In the high dose groups, fertility was decreased with each successive litter and generation. The fertility indices for production of the F1a, F1b, F2a, F2b, and F3 litters were 100, 89.5, 63.2, 61.1, and 52.6%, respectively. Mean live litter size at birth and pup viability during lactation days 0 4 were significantly (p # 0.05) or slightly reduced for all litters produced by the high dose groups. Both of these parameters generally declined with each successive litter and generation. Mean live litter sizes were 6.2 9.3 pups for the high dose groups compared with 11.7 13.3 pups for the control groups. Viability indices for lactation days 0 4 were 58.8 92.0% for the highdose litters compared with 92.1 100% for the control litters.

The reproductive toxicity LOAEL was established at 625 ppm (premating dose 44 mg/ kg/ day in males and 48 mg/ kg/ day in females) based on reduced fertility. The reproductive toxicity NOAEL is 125 ppm (average premating dose 9 mg/ kg/ day in males and 10 mg/ kg/ day in females).

Body weights of the high dose pups from all generations were consistently reduced throughout lactation as compared to those of the controls with statistical significance (p # 0.05) attained at most time points. Body weights of the high dose pups from both litters of the F1 and F2 generations were approximately 82 94% of the control levels one day after birth and declined to approximately 66 80% of the control levels at weaning. In contrast body weights of the high dose F3 pups were 84 89% of the controls throughout lactation. Differences in absolute and/ or relative organ weights in high dose F2b weanlings were considered to be due to lower final body weights.

The offspring toxicity LOAEL was established at 625 ppm (premating dose 44 mg/ kg/ day, males and 48 mg/ kg/ day, females) based on decreased pup survival, and lower pup body weights. The offspring toxicity NOAEL is 125 ppm (average premating dose 9 mg/ kg/ day in males and 10 mg/ kg/ day in females).

This study is classified as Acceptable/ Guideline and satisfies the requirements for a reproduction study (870.3800 [83 4]) in rats.

5.4.2 Two Generation Reproduction  Rat MRID No: 41463401 Executive Summary: In a two generation reproduction study, linuron (96.2% a. i.) was administered to groups of 30 male and 30 female Crl: CDBR rats in the diet at concentrations of 0, 12.5, 100, or 625 ppm. One litter was produced by each generation. Average premating doses for the treated F0 groups were 0.74, 5.8, and 36 mg/ kg/ day, respectively, for males and 0.92, 7.3, and 45.1 mg/ kg/ day, respectively, for females. Average premating doses for the treated F1 groups were 0.95, 7.77, and 54.0 mg/ kg/ day, respectively, for males and 1.12, 9.24, and 63.0 mg/ kg/ day, respectively, for females. F0 and F1 parental animals were administered test or control diet for 72 or 75 days, respectively, prior to mating and throughout mating, gestation, and lactation, and until 25 necropsy.

No treatment related clinical signs of toxicity or mortalities were observed in the adult animals of either generation.

Body weights, body weight gains, and food consumption were significantly (p # 0.05) less than those of the controls beginning on day 7 for the high dose F0 animals and throughout premating for the mid and high dose F1 animals. At the end of premating, body weights of the high dose F0 males and females were 81% and 86%, respectively, of the controls with overall weight gains 59% and 55%, respectively, of the control values. For the high dose F1 males and females final premating body weights were 76% and 75%, respectively, with weight gains 77% and 77%, respectively, of the control levels. Mean daily food consumption levels for the high dose groups during the premating interval were 80 85% of the control levels. Absolute body weights of the mid dose F0 males were occasionally significantly (p # 0.05) less than the controls with premating weight gains and food consumption 88% and 94% (both, p # 0.05), respectively, of the control levels. For the mid dose F1 males and females, final premating body weights, body weight gains, and food consumption were 92 94% of the control levels. For the mid and high dose dams of both generations, lower body weights during gestation and lactation were considered a continuation of premating effects.

No treatment related lesions were noted at necropsy of the F0 males or females. In highdose F1 males, gross lesions of the testes included reduced in size (9/ 30), abnormally large (3/ 30), soft (5/ 30), small epididymides (8/ 30), and unspecified deformities of the epididymides (5/ 30); these were noted as significant incidences, but control rates were not given in the original DER. Microscopically, increased incidences (p # 0.05) of testicular and epididymal lesions were found in high dose F1 males as compared with the controls: atrophy (14/ 30), fibrosis (8/ 30), and hyperplasia (7/ 30) in the testes and arteritis (6/ 30), inflammation (5/ 30), and oligospermia (12/ 30) in the epididymides. Only one incidence each of atrophy and oligospermia were observed in control animals. Absolute testes weights of the high dose F1 males were significantly (p # 0.05; 80% of control) less than the controls. In high dose F1 females, gross findings (n. s.) included cystic ovaries (4/ 30), dilatation (3/ 30), and fluid filled uterine horns (2/ 30), none of which were observed in control animals.

In addition, lesions of the eye were noted in high dose F1 males and females and were reviewed in MRID 41864701. A significant (p # 0.05) increase was seen in the number of high dose F1 males with any type of corneal or conjunctival change (14/ 30 vs 4/ 30 controls). The lesions included corneal degeneration/ basophilia and conjunctival inflammation/ basophilia. Degeneration of the lens was observed in 3/ 30 high dose males and 3/ 29 high dose females compared with only 1/ 30 control male. However, a clear treatment related effect in females was not considered to be definitive.

The systemic toxicity LOAEL was established at 100 ppm (average premating doses 5.8 9.24 mg/ kg/ day) based on reduced body weight gains in males and females 26 during both generations. The systemic toxicity NOAEL was established at 12.5 ppm (average premating doses 0.74 1.12 mg/ kg/ day).

No treatment related adverse effects were found on the reproductive performance of either generation. For the control, low, mid, and high dose group, pup viability for lactation days 0 4 was 99.4, 98.0, 99.8, and 91.7% (p # 0.05), respectively, for the F1 generation and 96.8, 92.7, 99.5, and 76.2% (p # 0.05), respectively, for the F2 generation. The mean number of pups per litter in the high dose F2 group was significantly (p # 0.05) less than the control throughout lactation. The number of litters with pups showing clinical signs was significantly (p # 0.05) increased in both generations.

The reproductive toxicity NOAEL was greater than or equal to 625 ppm (average premating doses 36 63.0 mg/ kg/ day) and the reproductive toxicity LOAEL was not established.

Body weights of the mid and high dose F1 male and female pups and of the high dose F2 male and female pups were significantly (p # 0.05) less than those of the controls throughout lactation LOAEL for offspring toxicity was established at 100 ppm (average premating doses 5.8 9.24 mg/ kg/ day) based on reduced F1 pup body weights. The offspring toxicity NOAEL was established at 12.5 ppm (average premating doses 0.74 1.12 mg/ kg/ day).

This study is classified as Acceptable/ Guideline and satisfies the guideline requirements for a reproductive toxicity study [OPPTS 870.3800 (83 4)] in rats.

5.5 Special Studies 5.5.1 Biochemical and Histopathological Effects in Rats MRID No: 00164093 Executive Summary: A special study was conducted to determine the biochemical and histopathological effects under a variety of conditions of linuron (94.5% a. i.) administration to Crl: CD® BR rats. The rats utilized for various parts of this study were young adult males approximately 22 days old (young), retired breeder males approximately 11 months old (old), and male and female F1b and F2b rats from a multigeneration study (MRID 41463401) maintained on diets of 0, 12.5, 100, or 625 ppm (0, 0.75, 4.1, and 22 mg/ kg/ day, respectively, for males and 0, 1.1, 6.1, and 37 mg/ kg/ day, respectively, for females). Additionally, Leydig cell enzyme analyses were conducted in vitro.

The activities of five testicular steroidogenic enzymes were assayed in vitro using horse 27 testicular microsomal preparations in the presence of 0, 0.5, 5, 50, 500, or 5000 : M linuron or linuron metabolites. Enzyme activities measured included aromatase, 17 20 desmolase (desmolase), 3 $ hydroxysteriod dehydrogenase/ isomerase (isomerase), 17 hydroxylase (hydroxylase), and 17 ketosteriod reductase (reductase). Linuron concentrations of 500 and 5000 : M resulted in significantly decreased aromatase and desmolase and increased reductase activities. At 50 : M the activities of aromatase, desmolase, isomerase, and hydroxylase were decreased by 10 20% and reductase was increased by 20% as compared with the controls. Effects of linuron metabolites on enzyme activities were highly variable and generally only seen at 5000 : M.

The testosterone metabolic clearance rate was measured in young male rats. Groups of 5 animals were administered 0 or 200 mg/ kg/ day for eight days, castrated, and then infused with testosterone at 3 or 6 : g/ hour. Blood samples were taken every 30 minutes for 180 minutes after the start of infusion. No differences between the treated and control groups were noted at the lower infusion rate. At the higher infusion rate, the mean plateau concentrations (60 180 minutes) in the control and treated rats were 769± 344 and 605± 67.4 : g/ dL, respectively, resulting in calculated metabolic clearance rates of 780 and 992 mL/ h, respectively. Due to the large variability between individual animals it was concluded that linuron does not affect testosterone clearance in young, castrated rats.

Two trials evaluated the response of Leydig cells to luteinizing hormone (LH). In trial 1, the groups consisted of five young or five old males treated with 0 or 200 mg/ kg/ day for three days, and five 0 ppm and five 625 ppm F2b males which were approximately 11 months old. In trial 2, the groups consisted of five young or five old males treated with 0 or 200 mg/ kg/ day for seven days, and five each of the 0, 12.5, 100, and 625 ppm F2b males which were approximately 19 months old. Leydig cells were isolated from the testes and incubated with up to 1000 ng LH/ tube. Results from trial 1 showed no differences in the response between the treated and control young rats. However in old rats, Leydig cells from the treated animals were less responsive than the controls both in maximum response and potency. In contrast, Leydig cells from the chronically treated F2b males were significantly more responsive to LH stimulation as compared with their controls. In trial 2, Leydig cells from treated young and old rats had decreased maximum responses and potency as compared with controls, with old rats more affected than young rats. From the chronically exposed rats, Ledig cells were moderately responsive from the control and low dose groups, minimally responsive from the mid dose group, but significantly greater in response from the high dose group. Taken together, the two trials were reasonably consistent with a preliminary conclusion that there are dose and timerelated effects of linuron upon the sensitivity of rat Leydig cells to stimulation by LH.

F1b and F2b rats were sacrificed at two years of age for histopathological evaluation of selected tissues. The number of high dose males of both generations with small or discolored testes was increased as compared with the controls (stated in text of DER; incidence rates not readable). No other gross observations were noted. Microscopically, mid and high dose males had increased incidences of interstitial cell adenomas and hyperplasia. Combining data from both generations, adenomas were observed in 1/ 19, 28 0/ 25, 6/ 25, and 2/ 16 animals and hyperplasia was observed in 2/ 19, 0/ 25, 7/ 25, and 3/ 19 animals from the control, low, mid, and high dose groups, respectively. The lower incidences in the high dose group were probably due to fewer animals available for examination. In females the combined incidence rate for cervical endometrial hyperplasia was 0/ 28, 6/ 30, 9/ 29, and 13/ 29, respectively, and of cervical cystic hyperkeratosis was 0/ 28, 1/ 30, 1/ 29, and 7/ 29, respectively. These lesions in both males and females are consistent with findings in a 2 year chronic study.

In conclusion, the biochemical and histopathological data presented in this report suggest that linuron may affect testosterone metabolism in horse testicular microsomes for a range of concentrations which overlap the dose levels given rats chronically. However, the net effect of these enzyme changes and the relevance to the rat in vivo are uncertain. Evidence in young and old rats exposed repeatedly (3 7x) or for 11 or 19 months suggests that Leydig cell incubates are differentially altered in their sensitivity to LH. Microscopic lesions in the testes and cervix have been confirmed in other studies.

This study is classified as Acceptable/ Nonguideline as a special mechanistic study in rats.

5.5.2 Special Reproduction Study  Leydig Cell Tumorigenisis in Rat MRID No: 41630101 Executive Summary: A special mechanism study was conducted in male Crl: CD( SD) BR rats. Linuron (96.2% a. i) was administered at 200 mg/ kg/ day by gavage for 14 days to groups of 10 growing (32 33 days of age) and adult (93 days of age) rats. Additional groups of 10 rats each were used as negative control, pair fed control, and positive control (flutamide, 10 mg/ kg/ day). All rats were observed daily and body weights and food consumption were recorded. At termination, blood was collected for serum hormone analyses and the organs of the reproductive tract were weighed. In addition, blood was collected from the F0 and F1 males and organ weights were recorded from the F0 males from a multigeneration study (MRID 41463401). Premating doses for the multigeneration study were 0.74 0.95, 5.8 7.8, and 36 54 mg/ kg/ day. Finally, linuron and four of its metabolites were evaluated in vitro for their ability to compete for binding to the androgen receptor.

No treatment related clinical signs of toxicity were observed in the growing rats, the positive controls, or the F0 and F1 rats. Adult rats treated with the test article for 14 days had significantly (p # 0.05) increased incidences of discharge and/ or stains in the perioral, perinasal, and or periocular regions (9/ 10), eye discharge (5/ 10), and weak appearance (9/ 10) as compared with both the negative and pair fed control groups. These signs were not observed in the control groups with the exception of one pair fed animal with discharge and/ or stains.

Final body weights, body weight changes, and food consumption of the 14 day treated groups, of the pair fed control groups, and of the mid and high dose F0 and F1 groups 29 were significantly (p # 0.05) less than that of their concurrent negative control group values. Final body weight and weight change for the 14 day adult group were also significantly (p # 0.05) less than those of their pair fed control. Body weights and body weight changes were significantly (p # 0.05) reduced only in the adult positive control group as compared with their negative controls (food consumption not measured in positive controls).

For growing rats, absolute and relative accessory sex organ unit, prostate, ventral prostate, and seminal vesicle weights were significantly (p # 0.05) reduced as compared with both negative and pair fed control groups. Absolute and relative dorsal lateral prostate and levator ani muscle weights and absolute testes weights were significantly (p # 0.05) reduced and relative testes weights were significantly increased (p # 0.05) as compared with the negative controls. Significant (p # 0.05) differences in the pair fed control group as compared with the negative control group included decreased absolute accessory sex organ unit, ventral prostate, dorsal lateral prostate, seminal vesicles, and levator ani muscle weights and relative testes and levator ani muscle weights. In the positive control rats, absolute and relative testes weights were not affected, but all other absolute and relative organ weights were significantly (p # 0.05) less than their negative controls.

For adult rats, absolute and relative accessory sex organ unit, prostate, and ventral prostate weights were significantly (p # 0.05) reduced as compared with both negative and pair fed control groups. Absolute epididymides, seminal vesicle, coagulating gland, and levator ani muscle weights significantly (p # 0.05) reduced as compared with the negative controls. Significant (p # 0.05) differences in the pair fed control group as compared with the negative control group included decreased absolute accessory sex organ unit, coagulating gland, and levator ani muscle weights. In the positive control rats, testes weights were not affected, but all other absolute and relative organ weights were significantly (p # 0.05) or slightly (n. s.) less than their negative controls.

Significant differences (p # 0.05) in organ weights for the high dose F0 males as compared with the controls included decreased absolute epididymides, dorsal lateral prostate, and levator ani muscle weights and increased relative testes, epididymides, and ventral prostate weights. Organ weights were unaffected in the two lower dose groups.

Serum testosterone, estradiol, and luteinizing hormone levels in both growing and adult rats were similar to the control levels. However, F0 and F1 males had significantly (p # 0.05) increased levels of estradiol (155 and 115%, respectively) and luteinizing hormone (175 and 168%, respectively). In the positive control groups, testosterone, estradiol, and luteinizing hormone levels were increased (p # 0.05) 304, 123, and 304%, respectively, in growing rats and 915, 100 (n. s.), and 346%, respectively, in adult rats.

Linuron and three other compounds [1 (3,4 dichlorophenyl) 3 methoxyurea; 3,4 dichloroanaline; 3,4 dichlorophenylurea; and 1( 3,4 dichlorophenyl) 3 methylurea] appeared to compete with testosterone for binding to the androgen receptor in vitro, although the results were highly variable. IC50 values for linuron and flutamide were 30 approximately 18,000 ± 3,500 and 64,000± 11,000 nM, respectively. 3,4 dichlorophenylurea did not displace testosterone from the receptor, therefore an IC50 value could not be determined. The remaining metabolites had IC50 ranging from 110,000 to 260,000 nM.

In conclusion, linuron may be a weak androgen receptor antagonist based on decreased accessory sex organ weights for growing and adult rats, increased serum luteinizing hormone levels in F0 and F1 rats, and competitive androgen receptor binding in vitro. These data support the hypothesis that rats exposed to linuron could develop interstitial hyperplasia and subsequent adenomas (Leydig cell tumors) via a mechanism of sustained hypersecretion of luteinizing hormone induced by the antiandrogenic potential of linuron.

This study is classified as Acceptable/ Nonguideline as a special mechanistic study in rats.

5.5.3 Special Reproduction Study  Cross mating  Rat MRID No: 00159846 Executive Summary: A special study (MRID 00159846) was conducted to evaluate the effects of linuron (94.5% a. i.) on the reproduction and lactation performance of crossmated male and female Crl: CD®( SD) BR rats. The rats utilized for this study were the F2 adults from a multigeneration study and the current study was initiated within two weeks after weaning of the last F3a litter. High dose (625 ppm) and control animals were cross mated to produce F3b and F3c litters; different pairings were made for production of each litter. The data from the original F2 control group from the multigeneration study was used as control data for the current study, but the animals were not remated. The fertility index was calculated in the report as (no. litters delivered/ no. females mated) x 100. The reviewer calculated the following indices: male fertility index = (no. males impregnating females/ no. males exposed to females) x 100; female fertility index = (no. females conceiving/ no. females exposed to males) x 100; and fecundity index = (no. pregnancies/ no. copulations) x 100.

During production of both litters, the fertility index and the number of pups/ litter at birth and at weaning were reduced as compared with the control values. The fertility indices for control females crossed with treated males and for treated females crossed with control males were 11.8 and 47.4%, respectively, for the F3b litters and 41.2 and 42.1%, respectively, for the F3c litters as compared with 89.5% for the controls. The number of pups/ litter at birth was 4.0 9.2 for the cross mated groups and 13.1 for the controls. At weaning the number of pups/ litter was 4.0 4.3 for the F3b litters and the F3c litters from treated females crossed with control males compared with 8.1 pups/ litter for both the control group and the F3c litters from control females crossed with treated males.

In production of both litters, male and female fertility indices for the groups in which the males were treated were reduced (47 71% for males and 47 60% for females) compared with those for groups in which the females were treated (90 86% for males and 85 85% 31 for females). The fecundity index was reduced only for treated males crossed with control females during production of the F3b litters (25%) as compared with the other groups (56.2 63.6%).

Pup viability was decreased in litters from treated dams mated with control males as compared with litters from control dams mated with treated males. Pup viability for days 0 4 was 77.5% in litters from treated dams and 98.5% in litters from control dams while viability for days 1 4 was 88.6% and 98.7%, respectively. Litter survival was 75% from treated dams compared with 100% from control dams. Mean pup body weights from treated dams were also slightly or significantly (p # 0.05) less than those from the control dams throughout lactation.

The cross mating results suggest that linuron may cause paternally mediated effects based on decreased fertility and fecundity as well as maternally mediated effects based on decreased pup viability and litter survival.

This study is classified as Acceptable/ Nonguideline as a special cross mating study in rats.

5.6 Additional Information from Literature Sources 5.6.1 McIntyre BS, Barlow NJ, Wallace DG, Maness SC, Gaido KW, Foster PM, Effects of in utero exposure to linuron on androgen dependent reproductive development in the male Crl: CD( SD) BR rat. Toxicol Appl Pharmacol 167( 2): 87 99 (2000)

Pregnant rats were administered linuron by gavage at 0, 12.5, 25, or 50 mg/ kg/ day (n = 11/ group) from gestation day 12 to 21.

Anogenital distance of resulting offspring was unaffected, whereas male areola/ nipple retention per rat was increased at 12.5, 25, and 59 mg/ kg/ day (1.0, 1.6, and 3.7, respectively) compared to the control (< 1.0).

Hypoplastic testes in adult offspring were seen in 2/ 56 rats (2/ 10 litters), 8/ 69 rats (4/ 11 litters), and 5/ 44 rats (3/ 8 litters), while hypoplastic epididymides occurred in 1/ 56 rats (1/ 10 litters), 8/ 69 rats (4/ 11 litters), and 2/ 44 rats (1/ 8 litters) in the 12.5, 25, and 50 mg/ kg/ day dose groups, respectively.

Partial agenesis of the epididymides was observed in 3/ 44 rats (2/ 8 litters) only in the 50 mg/ kg/ day group. These data indicate that in utero exposure to linuron preferentially impairs testosterone mediated, rather than DHT mediated, reproductive development. This effect is distinctly different from the effects induced by flutamide, an AR antagonist that shares structural similarities with linuron.

5.6.2 Lambright C, Ostby J, Bobseine K, Wilson V, Hotchkiss AK, Mann PC, 32 Gray LE, Cellular and molecular mechanisms of action of linuron: an antiandrogenic herbicide that produces reproductive malformations in male rats. Toxicol Sci 56( 2): 389 99 (2000)

In vitro, linuron binds human AR (hAR), and and acts as an hAR antagonist. Linuron competed with an androgen for rat prostatic AR EC50 = 100 300 microM) and human AR (hAR) in a COS cell binding assay EC50 = 20 microM).

Linuron inhibited dihydrotestosterone (DHT) hAR induced gene expression in CV 1 and MDA MB 453 KB2 cells EC50 = 10 microM) at concentrations that were not cytotoxic.

Linuron (oral 100 mg/ kg/ d for 7 days) treatment reduced testosterone and DHT dependent tissue weights in the Hershberger assay using castrate immature testosterone propionate treated male rats and altered the expression of androgen regulated ventral prostate genes (oral 100 mg/ kg/ d for 4 days). [The Hershberger assay is an EDSTAC Tier 1 assay to provide in vivo screening data relevant to androgen agonism and antagonism for preliminary hazard identification.]

Histological effects of in utero exposure to linuron (100 mg/ kg/ d, day 14 18) or DBP (500 mg/ kg/ d, day 14 to postnatal day 3) on the testes and epididymides also are shown here.

These results support the hypothesis that linuron is an AR antagonist both in vivo and in vitro, but it remains to be determined if linuron alters sexual differentiation by additional mechanisms of action.

5.6.3 Gray LE, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby J, Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p, p' DDE, and ketoconazole) and toxic substances dibutyland diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health 15( 1 2): 94 118 (1999)

Linuron (100 mg/ kg/ day) treatment induced a level of external effects consistent with its low affinity for AR [reduced anogenital distance (AGD), retained nipples, and a low incidence of hypospadias].

Linuron treatment also induced malformed epididymides and testis atrophy.

The results suggest that Linuron may display several mechanisms of endocrine toxicity, one of which involves AR binding 5.6.4 Vinggaard, AM, Hnida, C, Breinholt, V and Larsen, JC, Screening of 33 Selected Pesticides for Inhibition of CYP19 Aromatase Activity In vitro, Toxicol in Vitro 14: 227 234 (2000)

Linuron was tested for its ability to affect CYP19 aromatase activity in human placental microsomes using the classical [ 3 H] 20 method. Linuron did not affect CYP19 aromatase activity in human placental microsomes. The positive control, 4 hydroxyandrostendione (1 : m), caused a 74% inhibition of CYP19 aromatase activity and a 94% inhibition of aromatase activity in JEG 3 human carcinoma cells.

5.6.5 Vinggaard, AM, Breinholt, V and Larsen, JC, Screening of selected pesticides for oestrogen receptor activation in vitro, Food Addit Contam 16 (12), 533 542 (1999)

Linuron, tested in the oestrogen receptor in vitro using an MCF7 cell proliferation assay and a Yeast Oestrogen screen, did not effect the proliferation response after 6 days of exposure.

5.6.6 Cook, J. C., Mullin, L. S., Frame, S. R., Biegel, L. B., Investigation of a Mechanism for Leydig Cell Tumorigenesis by Linuron in Rats, Toxicol Appl Pharmacol 119( 2): 195 204 (1993)

Serum testosterone, estradiol, and luteinizing hormone (LH) concentrations in sexually immature rats and testosterone and LH concentrations in sexually mature rats were significantly increased.

Linuron significantly decreased body weight and relative epididymides, accessory sex organ, prostate, and seminal vesicle weights in sexually immature rats and body weight and relative accessory sex organ and prostate weights in mature rats.

Serum estradiol and LH concentrations were significantly increased in sexually mature rats. Estradiol, LH, and testosterone concentrations were not affected in immature males.

Linuron significantly decreased body weights and increased serum LH and estradiol concentrations in both parental and F1rats.

Linuron was found to compete with testosterone for binding to the androgen receptor.

The authors conclude that linuron apparently induces Leydig tumors by disrupting the hypothalamic/ pituitary/ testicular axis which leads to a sustained hypersecretion of LH. 34 5.7 Determination of Susceptibility There is no qualitative/ quantitative evidence of increased susceptibility of rabbit developmental study; developmental effects were seen at a dose higher than that causing maternal toxicity. In the rat developmental study, increases in post implantation losses and increases in fetal resorptions/ litter were seen as a dose that caused decreases in maternal body weight and food consumption. The HIARC determined that the developmental effects are not indicative of qualitative evidence of susceptibility., since increases in resorptions were marginal and there was no change in the number of live fetuses to corroborate the increases in post implantation losses.

There was no quantitative evidence of susceptibility either in the 2 generation or the 3 generation reproduction studies. In the 2 generation study, reduced body weight gains of pups were seen at the same dose that caused decreases in parental body weights. In the 3 generation study, offspring effects (deceased pup survival and pup body weight) were seen a dose ( 44 mg/ kg/ day) higher than the dose that caused decreases in body weight gain in the parental animals (9 mg/ kg/ day).

However, when the reproductive effects were examined, testicular atrophy was seen at the same dose (625 ppm, 45 mg/ kg/ day) in both studies. In both studies, while the F0 males were not affected, testicular lesions and reduced fertility were seen in the F1 males. This effect in the F1 males is an indication of qualitative evidence of susceptibility.

5.8 Evidence that suggest study requiring a Developmental Neurotoxicity Study:

From special studies and open literature publications, Linuron was shown to be an endocrine disruptor. Key findings include:

(1) Linuron and some of its metabolites are androgen receptor antagonists [§ 5.5.2] (2) Rats treated with linuron had reduced anogenital distance, retention of nipples, and a low incidence of hypospadias. [§ 5.5.3]. (3) The responsiveness of Leydig cells to luteinizing hormone was decreased in both immature (22 days) and mature (11 months) male rats treated with linuron. Mature rats were less responsive that immature ones [§ 5.4.3] (4) F0 and F1 males had significantly increased levels of estradiol and and luteinizing hormone [§ 5.4.4]. (5) Linuron inhibits activities of steroidogenic enzymes [§ 5.4.3] (6) A dose dependent increase in areola/ nipple retention in male rats [§

Although there was no evidence for increased susceptibility in rats, based on the findings that linuron is an endocrine disruptor, increased testicular lesions and decreased fertility, the HIARC concluded that a development neurotoxicity study in the rat is required.

6 HAZARD CHARACTERIZATION 35 The toxicological database for linuron is considered adequate for hazard characterization. At the time of this review, there are no toxicological data gaps and no outstanding toxicological concerns.

Linuron has low acute toxicity, with toxicity categories of III for oral (LD50 2600 mg/ kg), dermal (LD50 > 2000 mg/ kg) and toxicity category IV for inhalation ( LC50 > 218 mg/ L/ hr). Primary eye and skin irritation studies were category III and IV, respectively; no dermal sensitization was observed in rabbits.

Chronic toxicity studies in the dog, rat and mouse showed altered hematological findings. Beagle dogs fed linuron at dietary concentration of 625 ppm, resulted in hemolytic anemia and secondary erythropogenic activity evidenced by slightly reduced hemoglobin, hematocrit, and erythrocyte counts accompanied by hemosiderin deposition in liver Kupffer cells and erythroid hyperplasia of bone marrow. ChR CD rats fed diets containing Linuron at 125 ppm (5.11 mg/ kg/ day in males and 7.75 mg/ kg/ day in females), microscopic observations consistent with hemolysis (hemosiderin in Kupffer cells and increased hemosiderosis in bone marrow, spleen, and/ or mesenteric lymph nodes). Rats also showed decreased body weight gains in both sexes increased incidences of microscopic changes in the epididymides (perivasculitis/ vasculitis) and renal pelvis (transitional cell hyperplasia and mineralization/ calculi) of males and kidneys (calculi in renal tubules) of females. Systemic toxicity observed in mice included increased methemoglobin formation and vacuolation and hemosiderosis of the spleen.

Oncogenicity studies in the rat and mouse did not show consistent tumor profiles between sexes and species. In rats there was a significant increase in the incidence of testicular interstitial cell adenomas in males, while no treatment related neoplasms were observed in females. In a mouse oncogenicity study a statistically significant increase in liver tumors was observed in females. Based on the results of these studies, linuron was classified as an unquantifiable Group C carcinogen (a possible human carcinogen for which there is limited animal evidence).

In a developmental toxicity study using rats, the highest dose level caused maternal toxic effects including decreased body weight gain and food consumption, as well as increased postimplantation loss and fetal resorptions. In a study using rabbits, linuron caused decreases in maternal body weight, food consumption and liver weight, as well as more abortions, fewer fetuses per litter, decreased fetal body weight, and an increased incidence of fetuses with skeletal skull variations.

In a 2 generation reproductive toxicity study using rats, linuron caused effects on the parents including decreased body weight gain and abnormalities in the eyes and testes. Rats exposed to linuron could develop cell tumors in testicular tissue. A 3 generation study using rats showed reduced body weights and fertility, decreased pup survival, and decreased weanling body, liver and kidney weights, as well as liver atrophy.

The chronic toxicity of linuron has been evaluated in two year feeding studies in both the rat and dog. Body weights were measured weekly during the entire study. In the rat study, after one 36 week of treatment, a significant decrease in body weight gain was observed at 600 ppm in males (59% of control) and females (53% of control). These decreases persisted throughout the entire study, with females showing a consistently lower body weight gains (68 to 76%) than males (82 to 93%). The decreases in body weight gains correlated to some degree with decreased food consumption.

Linuron was not mutagenic in bacteria or in cultured mammalian cells. There was also no indication of a clastogenic effect up to toxic doses in vivo. In oncogenicity studies in the rat and mouse tumors were observed, however, no sex and species differences were noted. In the mouse oncogenicity study, a significant increase in malignant lymphomas were observed in the spleen of females. In the combined chronic toxicity/ oncogenicity study in rats, testicular adenomas were observed in 16 and 58% of males at 600 and 1800 ppm, respectively, compared to 6% in both control and low dose groups. In female rats, the incidence of endometrial polyps was observed at 600 (15%) and 1800 (13%) ppm, compared to 4% for control and 8% at 200 ppm.

There is ample evidence from special studies submitted by the registrant as well as open literature studies which indicate that linuron is an endocrine disruptor. These findings include, in part: (1) competitive androgen receptor antagonist [§ 5.6.2]; but not an estrogen receptor antagonist [§ 5.6.5]; (2) competitive inhibition of the transcriptional activity of dihydrotestosterone (DHT) human androgen receptor (hAR) in vitro [§ 5.6.1, §5.6.3], decreased anogenital distance [§ 5.6.3] and/ or an increase in the retention of areolae/ nipples [§ 5.6.1, §5.6.3] in male offspring following in utero exposure to linuron; (3) inhibition of steroidogenic enzymes [§ 5.6.4], and (4) decreased responsiveness of Leydig cells to luteinizing hormone in both immature (22 days) and mature (11 months) male rats treated with linuron, mature rats were less responsive that immature ones [§ 5.5.1]; (5) F0 and F1 males had significantly increased levels of estradiol and luteinizing hormone [§ 5.5.2].

7 DATAGAPS Developmental neurotoxicity study Rat 28 day inhalation study  Rat. 37 8 ACUTE TOXICITY Acute Toxicity of Linuron, Technical Guideline No. Study Type MRID No. Results Toxicity Category 81 1 Acute Oral (Rat) 00027625 2600 mg/ kg III 81 2 Acute Dermal (Rabbit) 00027625 > 2000 mg/ kg III 81 3 Acute Inhalation (Rat) 00053769 > 218 mg/ L/ hr IV 81 4 Primary Eye Irritation 42849001 Slight conjunctival redness at 24 hrs; clear at 72 hrs III 81 5 Primary Skin Irritation 42849002 Not an irritant IV 81 6 Dermal Sensitization 00146868 Not a sensitizer N/ A 38 9 SUMMARY OF TOXICOLOGY ENDPOINT SELECTION The doses and toxicological endpoints selected for various exposure scenarios are summarized below.

EXPOSURE SCENARIO DOSE (mg/ kg/ day) ENDPOINT STUDY Acute Dietary (Females 13 50) NOAEL= 12 UF = 100 Increased post implantation loss and fetal/ litter resorptions at 50 mg/ kg/ day (LOAEL). Rat Prenatal Developmental Toxicity (MRID 00018167)

Acute RfD (Females 13 50 years old) = 0.12 mg/ kg Acute Dietary (General Population) No appropriate effects attributed to a single exposure was identified.

Chronic Dietary NOAEL = 0.77 UF = 100 Increased met and sulfhemoglobin levels at LOAEL (4.17 mg/ kg/ day, males; 3.5 mg/ kg/ day, females). Chronic Oral / Dog Chronic RfD = 0.0077 mg/ kg/ day Incidental Oral Short Term NOAEL= 5.8 Statistically and biologically significant decrease in premating body weights in F0 and F1 animals observed at 36 mg/ kg/ day (LOAEL). 2 Generation Reproduction Study/ Rat Incidental Oral Intermediate Term NOAEL = 0.77 Increased met and sulfhemoglobin concentrations after 3 and 6 months exposure4.17 mg/ kg/ day (males) and 3.5 mg/ kg/ day (females). Chronic Oral / Dog Dermal 1 Short Term NOAEL= 5.8 Statistically and biologically significant decrease in premating body weights in F0 and F1 animals observed at 36 mg/ kg/ day (LOAEL). 2 Generation Reproduction Study/ Rat Dermal 1 Intermediate Term NOAEL = 0.77 Increased met and sulfhemoglobin concentrations after 3 and 6 months exposure4.17 mg/ kg/ day (males) and 3.5 mg/ kg/ day (females). Chronic Oral / Dog Dermal 1 Long Term NOAEL = 0.77 Increased met and sulfhemoglobin concentrations after 9 and 12 months exposure4.17 mg/ kg/ day (males) and 3.5 mg/ kg/ day (females). Chronic Oral / Dog Inhalation 2 Short Term NOAEL= 5.8 Statistically and biologically significant decrease in premating body weights in F0 and F1 animals observed at 36 mg/ kg/ day (LOAEL). 2 Generation Reproduction Study/ Rat Inhalation 2 Intermediate Term NOAEL = 0.77 Increased met and sulfhemoglobin concentrations after 3 and 6 months exposure4.17 mg/ kg/ day (males) and 3.5 mg/ kg/ day (females). Chronic Oral / Dog Inhalation 2 Long Term NOAEL = 0.77 Increased met and sulfhemoglobin concentrations after 9 and 12 months exposure4.17 mg/ kg/ day (males) and 3.5 mg/ kg/ day (females). Chronic Oral / Dog 1 The dermal absorption factor of 16% should be applied to extrapolate from the oral route to the dermal route. 2 100% absorption rate (default value) should be used to extrapolate from the oral route to the inhalation route.