1 TXR NO. 0051033 DATE: August 12, 2002 MEMORANDUM SUBJECT: HEXAZINONE  3 rd Report of the Hazard Identification Assessment Review Committee.

FROM: David G. Anderson Toxicologist. 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: Carol Christensen, Risk Assessor Reregistration Branch 2 Health Effects Division (7509C)

PC Code: 107201 On Dec 4, 2001, the Health Effects Division (HED) Hazard Identification Assessment Review Committee (HIARC) reviewed the recommendations of the toxicology reviewer for HEXAZINONE with regard to the acute and chronic Reference Doses (RfDs) and the toxicological endpoint selection for use as appropriate in occupational/ residential exposure risk assessments. The potential for increased susceptibility of infants and children from exposure to HEXAZINONE was also evaluated as required by the Food Quality Protection Act (FQPA) of 1996.

On April 02, 2002, HIARC assessed the potential for increased susceptibility of infants and children from exposure as required by the Food Quality Protection Act (FQPA) of 1996 under the 2002 OPP 10X guidance document. At this meeting, the HIARC applied a database uncertainty factor to account for the lack of a developmental toxicity study in rabbits.

On July 30, 2002, HIARC reviewed the new rabbit developmental toxicity study submitted to the Agency on May 3, 2002. The conclusions drawn at the three meetings are presented in this report. 2 Committee Members in Attendance Members present were: Bill Burnam, Jonathan Chen, Pamela Hurley, John Liccione, Elizabeth Mendez, Jess Rowland, Brenda Tarplee.

Member( s) in absentia: Ayaad Assaad, David Nixon, Beth Doyle, Sue Makris, Steve Knizer.

Data evaluation prepared by: David G. Anderson, RRB2 Also in attendance were: Carol Christensen, Pauline Wagner Data Evaluation/ Report presentation David G. Anderson Toxicologist Report Concurrence Brenda Tarplee, Senior Scientist Science Information Management Branch 3 INTRODUCTION On December 4, 2001, the Health Effects Division (HED) Hazard Identification Assessment Review Committee (HIARC) reviewed the recommendations of the toxicology reviewer for HEXAZINONE with regard to the acute and chronic Reference Doses (RfDs) and the toxicological endpoint selection for use as appropriate in occupational/ residential exposure risk assessments. The potential for increased susceptibility of infants and children from exposure to HEXAZINONE was also evaluated as required by the Food Quality Protection Act (FQPA) of 1996.

On April 02, 2002, HIARC assessed the potential for increased susceptibility of infants and children from exposure as required by the Food Quality Protection Act (FQPA) of 1996 under the 2002 OPP 10X guidance document. At this meeting, the HIARC applied a database uncertainty factor to account for the lack of a developmental toxicity study in rabbits.

On July 30, 2002, HIARC reviewed the new rabbit developmental toxicity study submitted to the Agency on May 3, 2002. The conclusions drawn at the three meetings are presented in this report.

I FQPA HAZARD CONSIDERATIONS 1. Adequacy of the Toxicity Data base The HIARC considered that the toxicology database for hexazinone is complete, except for a needed confirmatory 28 day inhalation study.

2. Evidence of Neurotoxicity The HIARC concluded that there is not a concern for neurotoxicity resulting from exposure to hexazinone.

3. Developmental Toxicity Study Conclusions 3.1 Developmental toxicity in rats (1987)

Executive Summary: In a developmental toxicity study (MRID 40397501), hexazinone (99.26% a. i.; Lot# S30306A, Batch# 2/ 36) in 0.5% methylcellulose was administered orally via gavage to 25 Crl: CD ® BR female rats/ group at dose levels of 0, 40, 100, 400, or 900 mg/ kg on gestation day (GD) 7 through 16. The dosing volume was 10 mL/ kg (calculated by reviewers). All dams were sacrificed on GD 22 and their fetuses removed by cesarean and examined.

When compared to concurrent controls, no treatment related changes in clinical signs, gross pathology, pregnancy rate, live fetuses, resorptions, pre or post implantation loss, corpora lutea, or implantations were noted at any dose level tested. No treatment related findings were noted in the 100, or 40 mg/ kg groups. 4 In the 900 mg/ kg dams, one treatment related death occurred; alopecia and an enlarged stomach containing fluid and food were noted at necropsy. Decreased (p< 0.05) body weight gains were noted during GDs 15 17 ( 37%) and 17 22 ( 17%). In addition, body weight gains were decreased (p< 0.05) for the overall (GDs 7 17) treatment interval ( 30%). Gravid uterine weights were not reported; however, adjusted (for gravid uterine weight) terminal body weights were slightly decreased ( 6%; p 0.05). A significant (p 0.05) trend by linear combination of dose ranks from ANOVA was noted for GDs 9 11, 15 17, 7 17, and 17 22. Decreased (p 0.05) food consumption was observed throughout treatment (GDs 7 17; 9 16 22%) and post treatment (GDs 17 22; 9 9%). A significant (p 0.05) trend by linear combination of dose ranks from analysis of variance (ANOVA) was noted for GDs 7 9, 9 11, 11 13, 13 15, 15 17, 7 17, and 17 22. Also a significantly decreased food consumption was seen at 400 mg/ kg/ day (GD9 11; 9 1%) and (GD 7 17; 9 8%).

In addition, relative (to body) liver weights were increased (p 0.05) in the 400 (5.2%) and 900 (5.6%) mg/ kg dams compared to concurrent controls (4.9%). Decreased (p 0.05) terminal body weights were noted at 900 mg/ kg ( 6%). A significant (p 0.05) trend by linear combination of dose ranks from ANOVA was noted for relative liver weights and adjusted terminal body weights. In addition, a significant (p 0.05) trend by linear combination of dose ranks from ANOVA was noted for absolute liver weights. Despite this trend, the observed increases in absolute liver weights at 100, 400, and 900 mg/ kg ( 1 8%) were not statistically significant. Liver weight findings are considered equivocal.

The maternal LOAEL is 400 mg/ kg/ day based on decreased food consumption during dosing and nominal decreases in body weight gain from day 7 to day 17 and at all measured intervals between and at 900 mg/ kg/ day on mortality and decreased body weight gains and food consumption. The maternal NOAEL is 100 mg/ kg/ day.

In the 900 mg/ kg/ day group, male and female fetal weights were decreased ( 21%; p 0.05); a significant (p 0.05) trend by Jonckheere's test was noted for this parameter. At 400 mg/ kg/ day, the female fetal weights that were significantly decreased were not considered to be biologically significant. At necropsy, an increased (p 0.05) incidence of misaligned sternebra (1), a variation, was observed (fetal incidence: 0.0217; litter incidence: 0.30) relative to concurrent controls (fetal incidence: 0.0066; litter incidence: 0.09). In addition, an increased (p 0.05) incidence of misaligned sternebrae (2+), a variation, was noted (fetal incidence: 0.0217; litter incidence: 0.30) relative to concurrent controls (fetal incidence: 0.0033; litter incidence: 0.04). Furthermore, an increased incidence of kidneys with no papilla was observed (fetal incidence: 0.0347; litter incidence: 0.25) relative to concurrent controls (fetal incidence: 0.0062; litter incidence: 0.04). Although the incidence was not statistically significant, a dose related trend (p 0.05) was observed.

The developmental toxicity LOAEL is 900 mg/ kg/ day, based on decreased male and female fetal weight, increased incidence of kidneys with no papilla (malformation), and an increased incidence of misaligned sternebrae (variation). The developmental toxicity NOAEL is 400 mg/ kg/ day. The developmental toxicity study in the rat is classified acceptable/ guideline (OPPTS 870.3700; 5 §83 3 a) and satisfies the guideline requirement for a developmental toxicity study in the rat. An unacceptable developmental feeding study in the rat shows (MRID# 00114486) a maternal body weight decrement at 482 mg/ kg/ day with a NOAEL of 94.5 mg/ kg/ day with a developmental NOAEL of 482mg/ kg/ day. This study tends to add support the acceptable rat developmental (by gavage) toxicity study.

3.2 Developmental Toxicity in the rabbit (2002)

Executive Summary: In a developmental toxicity study (MRID 45677801) [DXP A3674] hexazinone (98.78 % a. i., batch/ lot# DXP A3694 329) was administered to 22 female [Hra:( NZW) SPF strain] rabbits/ group by gavage] at dose levels of 0, 20, 50, 125 or 175 mg/ kg bw/ day from days 7 through 28 of gestation.

Maternal toxicity was demonstrated by statistically significant findings at 125 mg/ kg/ day of a body weight gain decrement of 102 g between gd 7 9 and 57 g between gd 9 11, food consumption decrement of 18%, abortions, death and clinical signs such as diarrhea, stained cageboard and tail. Only 1 dam and 1 litter survived to termination at 175 mg/ kg/ day.

The maternal LOAEL is 125 mg/ kg bw/ day, based on body weight gain decrement, decreased food consumption, abortions, death and clinical signs including abnormal gait at 175 mg/ kg/ day. The maternal NOAEL is 50 mg/ kg bw/ day.

No dose related external, visceral or skeletal findings were noted. Since only 1 dam with 1 litter survived to termination at the 175 mg/ kg/ day dose, evaluation of embryo/ fetal effects at this dose are meaningless. Random malformations were seen in control and the highest dose group with no dose relationship. Mean male and female fetal weight was reduced by 5% and female fetal weight was reduced by 10% at 125 mg/ kg/ day. Although this weight reduction was not list as being statistically significant, the author stated that this slight weight reduction at 125 mg/ kg/ day was a developmental effect level and this reviewer agrees.

The developmental LOAEL is 125 mg/ kg bw/ day, based on mean male and female fetal weight decrement and female fetal weight decrement. The developmental NOAEL is 50 mg/ kg bw/ day.

The developmental toxicity study in the rabbit is classified acceptable (guideline) and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in the rabbit.

3.3 Developmental Toxicity in the rabbit (1980)

Executive Summary: In a developmental toxicity study (MRID 00028863), hexazinone (100% a. i.; Lot/ batch # E21216A) was administered orally via gavage in a dosing volume of 1 mL/ kg) to 17 female New Zealand White rabbits/ group at dose levels of 0, 20, 50, or 125 mg/ kg on GD 6 through 19. All does (except those that died or delivered prematurely) were sacrificed on GD 29, and their fetuses removed by cesarean and examined.

When compared to concurrent controls, no treatment related changes in mortality, clinical signs, body weights, gross pathology, fetal weights, sex ratios, pre implantation or post 6 implantation losses, or the number of corpora lutea, implantations, resorptions, live fetuses, or dead fetuses were observed.

At 125 mg/ kg, food consumption was decreased (p 0.05), relative to concurrent controls, at the beginning of treatment from GD 7 through 11 ( 61 89%). Decreases in food consumption, that were not statistically significant, continued throughout treatment (GDs 12 19; 9 2 37%). Diminished food consumption resulted in decreased (not statistically significant) body weight gains in the does ( 241.5 g) relative to concurrent controls ( 7.2 g) during GDs 6 11. However, weight gain in these animals recovered quickly and was higher than control animals during subsequent treatment intervals (GDs 11 15 and 15 19).

The maternal LOAEL is 125 mg/ kg/ day based on transient decreases in food consumption and body weight gains. The maternal NOAEL is 50 mg/ kg/ day.

At 125 mg/ kg/ day, the following skeletal variations were noted (data presented as fetal incidence vs. 0 controls): (i) lagging ossification in extremities (0.0882); (ii) malaligned thoracic vertebrae (0.0294); and (iii) flexed wrist( s) (0.0294). In addition, non ossified thumb, an anomaly, was noted at an increased incidence (0.0294) relative to concurrent controls (0). In the absence of historical control data, these findings are considered treatment related. In addition, it could not be determined how many of these nominally increased incidences were from different litters, which would have increased concern for developmental toxicity.

The developmental toxicity LOAEL is 125 mg/ kg/ day, based on possible skeletal abnormalities and total abnormalities. The developmental toxicity NOAEL is 50 mg/ kg/ day.

The developmental toxicity study in the rabbit is classified unacceptable/ upgradable, pending submission of acceptable purity, concentration, stability and litter data and historical control data. A letter dated 9/ 26/ 01 from the registrant provided no additional information about this rabbit developmental toxicity study other than that the doses were not analyzed and that a repeat rabbit developmental toxicity was currently being conducted.

4. Reproductive Toxicity Study Conclusions (1991)

4.1 Executive Summary: In a two generation reproduction study, hexazinone (98% a. i., Lot No. T02118994) was administered to groups of 30 male and 30 female Sprague Dawley rats in the diet at concentrations of 0, 200, 2000, or 5000 ppm (MRID 42066501). One litter was produced in the first generation and two litters were produced in the second generation. Test substance intake for the treated F0 groups was 11.8, 117, and 294 mg/ kg/ day, respectively, for males and 14.3, 143, and 383 mg/ kg/ day, respectively, for females. Test substance intake for the treated F1 groups was 15.3, 154, and 399 mg/ kg/ day, respectively, for males and 17.7, 180, and 484 mg/ kg/ day, respectively, for females. F0 and F1 parental animals were administered test or control diet for 73 or 105 days, respectively, prior to mating, throughout mating, gestation, and lactation, and until necropsy.

Deaths of several F0 and F1 parental animals were considered incidental to treatment. No treatment related clinical signs of toxicity were observed in the adult animals of either generation. 7 Gross necropsy was unremarkable and no microscopic lesions were observed in selected tissues from the reproductive tracts of male and female parental animals.

Body weights and body weight gains of the F0 males were not affected by treatment. Premating body weight gains by the mid and high dose F0 females were 76% and 62% (p # 0.05 for both), respectively, of the control level resulting in final premating body weights 93% and 87% (p # 0.05), respectively, of the controls. Body weights of the high dose F1 males and females were significantly reduced (p # 0.05) during the premating interval with overall weight gains 87% and 82%, respectively, of the control group amounts. Reductions in body weights and body weight gains during premating for the mid and high dose F0 and high dose F1 dams continued during gestation and lactation. Food consumption during premating was similar between the treated and control groups for males and females of both generations. However, during gestation significantly (p # 0.05) lower food consumption was noted for the high dose F1 dams during production of both litters and for the mid dose F1 dams during production of the second litter.

There was a statistically significant increase in absolute P0 testes weight that appeared to be dose related, but a nominally decrease absolute F1 adult testes weight in the 5000 ppm dose groups. The F1 testes weight change did not appear to dose related. The testes weight changes in males would appear to be incidental.

Therefore, the systemic toxicity LOAEL is 2000 ppm (117 154 mg/ kg/ day for males and 143 180 mg/ kg/ day for females) based on reduced body weight and body weight gains by F1 males and F0 and F1 females. The systemic toxicity NOAEL is 200 ppm (11.8 15.3 mg/ kg/ day for males and 14.3 17.7 mg/ kg/ day for females). No reproductive effects were seen in the study except for the weight effects on offspring.

Live birth and viability indices and litter survival were similar between the treated and control groups. The lactation index for the F2b high dose litters was 85.8% (p # 0.05) compared to 97.5% for the control group. Pup body weights were decreased throughout lactation in the mid and high dose groups of all litters as compared with the control groups with statistical significance (p # 0.05) attained at most time points. The lower pup body weights were more pronounced in females than in males. F1 and F2a female pup weights were statistically significantly decreased at birth, day 7 and 14 of lactation at $ 2000 ppm. There were no obvious reproductive effects other than the pup weight decrement.

Therefore, the offspring LOAEL is 2000 ppm (117 154 mg/ kg/ day for males and 143 180 mg/ kg/ day for females) based on reduced female pup body weights at birth and during lactation. The reproductive toxicity NOAEL was 200 ppm (11.8 15.3 mg/ kg/ day for males and 14.3 17.7 mg/ kg/ day for females).

This study is classified as Acceptable/ Guideline and satisfies the guideline requirements for a reproductive toxicity study [OPPTS 870.3800 (§ 83 4)] in rats. 8 5. Additional Information from Literature Sources The published literature found on hexazinone were the papers on the same studies submitted to the Agency [ Kennedy, GL and Kaplan, AM (1984) Chronic Toxicity, Reproductive, and Teratogenic Studies of Hexazinone. Fundamental and applied Toxicology 4, 960 971].

6. Pre and /or Postnatal Toxicity The HIARC concluded that there is not a concern for pre and postnatal toxicity resulting from exposure to hexazinone.

A. Determination of Susceptibility No quantitative or qualitative evidence of increased susceptibility was seen following in utero or pre/ post natal exposure to rats or rabbits. In the rat developmental toxicity study, developmental effects were seen at a higher dose than the dose that caused maternal toxicity. In the rabbit study, fetal weight decrement was seen at the same dose that caused maternal weight decrement. In the two generation reproduction study, offspring toxicity was seen at the same dose that caused parental toxicity.

B. Degree of Concern Analysis and Residual Uncertainties There is no evidence for increased susceptibility, therefore, no residual concerns .

C. Hazard based Special FQPA Safety Factors The special FQPA Safety Factor can be reduced to 1x due to lack of evidence for increased susceptibility and no residual concerns.

7. Recommendation for a Developmental Neurotoxicity Study A. Evidence that suggests requiring a Developmental neurotoxicity study:

None B. Evidence that do not support the need for a Developmental Neurotoxicity study No evidence of neurotoxicity or neuropathology was seen in the database. A 12 13% absolute female brain decrement was seen in the chronic dog study, but the brain weight decrement was associated with severe body weight decrement and may have been secondary to the malnutrition in these animals.

Hexazinone differs structurally from atrazine such that it is unlikely to bind to the same receptor and not likely to result in similar effects.

Based on the weight of evidence presented, the HIARC concluded that a developmental nuerotoxicity study is not required. 9 II. HAZARD IDENTIFICATION 1. Acute Reference Dose (RfD)( Population Subgroup: Females 13 50)

Study Selected: Developmental toxicity Study in Rats § 870.3700 MRID No.: 40397501 Executive Summary: In a developmental toxicity study (MRID 40397501), hexazinone (99.26% a. i.; Lot# S30306A, Batch# 2/ 36) in 0.5% methylcellulose was administered orally via gavage to 25 Crl: CD ® BR female rats/ group at dose levels of 0, 40, 100, 400, or 900 mg/ kg on gestation day (GD) 7 through 16. The dosing volume was 10 mL/ kg (calculated by reviewers). All dams were sacrificed on GD 22 and their fetuses removed by cesarean and examined.

When compared to concurrent controls, no treatment related changes in clinical signs, gross pathology, pregnancy rate, live fetuses, resorptions, pre or post implantation loss, corpora lutea, or implantations were noted at any dose level tested. No treatment related findings were noted in the 100, or 40 mg/ kg groups.

In the 900 mg/ kg dams, one treatment related death occurred; alopecia and an enlarged stomach containing fluid and food were noted at necropsy. Decreased (p< 0.05) body weight gains were noted during GDs 15 17 ( 37%) and 17 22 ( 17%). In addition, body weight gains were decreased (p< 0.05) for the overall (GDs 7 17) treatment interval ( 30%). Gravid uterine weights were not reported; however, adjusted (for gravid uterine weight) terminal body weights were slightly decreased ( 6%; p 0.05). A significant (p 0.05) trend by linear combination of dose ranks from ANOVA was noted for GDs 9 11, 15 17, 7 17, and 17 22. Decreased (p 0.05) food consumption was observed throughout treatment (GDs 7 17; 9 16 22%) and post treatment (GDs 17 22; 9 9%). A significant (p 0.05) trend by linear combination of dose ranks from ANOVA was noted for GDs 7 9, 9 11, 11 13, 13 15, 15 17, 7 17, and 17 22. Also a significantly decreased food consumption was seen at 400 mg/ kg/ day (GD9 11; 9 1%) and (GD 7 17; 9 8%).

In addition, relative (to body) liver weights were increased (p 0.05) in the 400 (5.2%) and 900 (5.6%) mg/ kg dams compared to concurrent controls (4.9%). Decreased (p 0.05) terminal body weights were noted at 900 mg/ kg ( 6%). A significant (p 0.05) trend by linear combination of dose ranks from ANOVA was noted for relative liver weights and adjusted terminal body weights. In addition, a significant (p 0.05) trend by linear combination of dose ranks from ANOVA was noted for absolute liver weights. Despite this trend, the observed increases in absolute liver weights at 100, 400, and 900 mg/ kg ( 1 8%) were not statistically significant. Liver weight findings are considered equivocal.

The maternal LOAEL is 400 mg/ kg/ day based on decreased food consumption during dosing and nominal decreases in body weight gain from day 7 to day 17 and at all measured intervals between and at 900 mg/ kg/ day on mortality and decreased body weight gains and food consumption. The maternal NOAEL is 100 mg/ kg/ day. 10 In the 900 mg/ kg/ day group, male and female fetal weights were decreased ( 21%; p 0.05); a significant (p 0.05) trend by Jonckheere's test was noted for this parameter. At 400 mg/ kg/ day, only female fetal weights were significantly decreased (2%) but were not considered to biologically significantly depressed. At 900 mg/ kg/ day, an increased (p 0.05) incidence of misaligned sternebrae (1), a variation, was observed (fetal incidence: 0.0217; litter incidence: 0.30) relative to concurrent controls (fetal incidence: 0.0066; litter incidence: 0.09). In addition, an increased (p 0.05) incidence of misaligned sternebrae (2+), a variation, was noted (fetal incidence: 0.0217; litter incidence: 0.30) relative to concurrent controls (fetal incidence: 0.0033; litter incidence: 0.04). Furthermore, an increased incidence of kidneys with no papilla was observed (fetal incidence: 0.0347; litter incidence: 0.25) relative to concurrent controls (fetal incidence: 0.0062; litter incidence: 0.04). Although the incidence was not statistically significant, a dose related trend (p 0.05) was observed.

The developmental toxicity LOAEL is 900 mg/ kg/ day, based on decreased male and female fetal weight and increased incidence of kidneys with no papilla (malformation), and an increased incidence of misaligned sternebrae (variation). The developmental toxicity NOAEL is 400 mg/ kg/ day.

The developmental toxicity study in the rat is classified acceptable/ guideline (OPPTS 870.3700; §83 3 a) and satisfies the guideline requirement for a developmental toxicity study in the rat.

Dose and Endpoint for Establishing RfD: Developmental NOAEL is 400 mg/ kg/ day. The LOAEL is 900 mg/ kg/ day based on increased kidneys with no papillae and misaligned sternebrae.

Uncertainty Factor (UF): 100 (10X intraspecies variation; 10X interspecies extrapolation)

Comments about Study/ Endpoint/ Uncertainty Factor: The malformations (kidneys with no papillae) are presumed to occur after a single and thus appropriate for this risk assessment. In the rabbit developmental study (MRID# 45677801) abortions occurred later in gestation (GD 18 27) and therefore was not considered to be appropriate for this exposure scenario. Also, the one death in the maternal animal at 125 mg/ kg/ day that occurred on gestation day 8 was discounted due to a lack of dose response and one death in control rabbits. At 175 mg/ kg/ day, maternal death occurred late in gestation.

2. Acute Reference Dose (RfD)( General Population)

An appropriate endpoint attributable to a single dose was not identified in the oral studies, including the rat and rabbit developmental studies. Acute RfD (Females 13 50) = 400 mg/ kg (NOAEL) = 4.0 mg/ kg 100 (UF) 11 3. Chronic Reference Dose (RfD)

Study Selected: One Year Chronic Dog Study Guideline #: 870.4100 MRID No.: 42162301 Executive Summary: In a one year chronic toxicity study, hexazinone (98% a. i., Lot No. T02118994) was administered to groups of 5 male and 5 female beagle dogs in the diet at concentrations of 0, 200, 1500, or 6000 ppm (MRID 42162301). Time weighted average doses for the treated groups were 5.00, 41.24, and 161.48 mg/ kg/ day, respectively, for males and 4.97, 37.57, and 166.99 mg/ kg/ day, respectively, for females.

All animals survived to scheduled necropsy. Treatment related clinical signs of toxicity included the observation of thinness in 1/ 5 mid dose males, 3/ 5 high dose males, and 1/ 5 high dose females. Body weights of the high dose groups were significantly (p # 0.05) less than those of the control throughout most of the study. Final body weights of the high dose males and females were 78% and 67%, respectively, of the control levels. Food consumption by the high dose groups was slightly (n. s.) less than that of the controls throughout the study with statistical significance (p # 0.05) attained for females at week 52. Overall food consumption (weeks 1 52) for high dose males and females was 85% (n. s.) and 74% (p # 0.05), respectively, of the control group levels. Body weights and food consumption for the low and mid dose groups were not affected by treatment.

No treatment related ophthalmological lesions, changes in urinalysis parameters, or gross necropsy findings were noted. A moderate macrocytic anemia was observed in the high dose groups as evidenced by slight or significant (p # 0.05) decreases in RBC counts, hemoglobin, and hematocrit and increases in MCV and MCH in one or both sexes throughout the study. Cholesterol levels were significantly (p # 0.05) decreased in the high dose groups beginning at week 13 for males (52 64% of controls) and at week 26 for females (45 51% of controls). Albumin levels were significantly (p # 0.05) decreased in the mid dose males (93% of controls) at week 13 only, and in the high dose males (74 78% of controls) and females (75 82% of controls) throughout the study. Beginning on week 13 or 26, the high dose groups had aspartate aminotransferase levels 140 203% (p # 0.05) of the control values and alanine aminotransferase levels 206 276% (p # 0.05) of the control values. Alkaline phosphatase levels were significantly (p # 0.05) increased in the mid dose males (259 409% of controls) and females (163 194% of controls; n. s.) beginning at week 26 and in the high dose males (346 1363% of controls) and females (307 559% of controls) beginning at week 13.

For the high dose animals, decreases in absolute testes weights in males and kidney, heart, and brain weights in females ( 12%) and increases in relative liver weights in males and females were considered due to lower final body weights of these animals as compared with controls.

Microscopic lesions in the liver of high dose animals included concentric membranous bodies in 4 males and 5 females, centrilobular single cell necrosis in 3 males and 3 females, hepatocellular pigment in 3 males and 3 females, and vacuolation in 3 males and 4 females. In addition 12 vacuolation was observed in one mid dose male and pigment and membranous bodies were each observed in one mid dose female. These lesions were not seen in control or low dose animals.

Therefore, the LOAEL for hexazinone in male and female beagle dogs is 1500 ppm (41.24 and 37.57 mg/ kg/ day, respectively) based on thinness in one male and hepatotoxicity as evidenced by changes in clinical chemistry parameters and microscopic lesions. The NOAEL is 200 ppm (5.00 and 4.97 mg/ kg/ day, respectively).

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 RfD: NOAEL of 5.0 mg/ kg/ day based body weight decrement, elevated clinical chemistry values (serum alkaline phosphatase, serum aspartate aminotransferase), other changes in clinical chemistry values, and histopathologic lesions of the liver at 38 mg/ kg/ day (LOAEL).

Uncertainty Factor( s): 100 (10X intraspecies variation; 10X interspecies extrapolation)

Comments about Study/ Endpoint/ Uncertainty Factor( s): Dose and endpoint identified following long term exposure in the most sensitive species.

4. Incidental Oral Exposure: Short Term (1 30 days) and Intermediate Term (1 Month 6 Months)

Toxicity endpoints for incidental oral exposure were not selected since there are no residential exposure expected or anticipated based on the current use pattern.

5. Dermal Absorption No dermal absorption study is available. For dermal absorption, the NOAEL from the 21 day dermal toxicity study at the limit dose of 1000 mg/ kg/ day was considered a lower bound for the LOAEL, which was compared with LOAEL of 125 mg/ kg/ day from the new developmental toxicity study in rabbits (MRID# 45677801). The ratio of these two numbers was used to estimate a dermal absorption factor of 12.5%.

Dermal Absorption Factor: 12.5% Chronic RfD = 5.0 mg/ kg/ day (NOAEL) = 0.05 mg/ kg/ day 100 (UF) 13 6. Short Term Dermal (1 Day  1 Month) Exposure Study Selected: None MRID No.: None Executive Summary: None Dose and Endpoint for Risk Assessment: Not applicable Comments about Study/ Endpoint: Quantification of dermal risk is not required since no hazard was identified via the dermal route (no systemic toxicity was seen at the Limit Dose (1000 mg/ kg/ day) in the 21 day dermal study in rabbits) and there are no concerns for pre/ post natal toxicity .

7. Intermediate Term Dermal (1 6 Months)

Study Selected: Chronic Feeding Study in Dogs § 870.4100 MRID No.: 42162301 Executive Summary: [See Section II. 3. on the Chronic Reference Dose (RfD).]

Dose/ Endpoint for Risk Assessment: NOAEL of 5.0 mg/ kg/ day is based on elevated clinical chemistry values (serum alkaline phosphatase, serum aspartate aminotransferase), other changes in clinical chemistry values, liver microscopic findings and body weight decrement and the clinical observation of thinnest in one males at 38 mg/ kg/ day.

Comments about Study/ Endpoint: The elevation in clinical chemistry parameters were seen at 3 and 6 months and thus appropriate for the exposure period. Since an oral NOAEL was selected, a 12.5% dermal absorption factor should be used for route to route extrapolation.

8. Long Term Dermal (longer than 6 Months) Exposure Study Selected: Chronic Feeding Study in Dogs § 870.4100 MRID No.: 42162301 Executive Summary: [See Section II. 3. on the Chronic Reference Dose (RfD).]

Dose/ Endpoint for Risk Assessment: NOAEL of 5.0 mg/ kg/ day is based on elevated clinical chemistry values (serum alkaline phosphatase, serum aspartate aminotransferase), other changes in clinical chemistry values, liver microscopic findings and body weight decrement and the clinical observation of thinnest in one males at 38 mg/ kg/ day.

Comments about Study/ Endpoint: The elevation in clinical chemistry parameters were seen at 3 , 14 6 and 12 months and thus appropriate for the exposure period. Since an oral NOAEL was selected, a 12.5% dermal absorption factor should be used for route to route extrapolation.

9. Short Term Inhalation Exposure (1 day to 1 Month)

Study Selected: Developmental Toxicity in the Rabbit § 870.3700 MRID No.: 45677801 Executive Summary: In a developmental toxicity study (MRID 45677801) [DXP A3674] hexazinone (98.78 % a. i., batch/ lot# DXP A3694 329) was administered to 22 female [Hra:( NZW) SPF strain] rabbits/ group by gavage] at dose levels of 0, 20, 50, 125 or 175 mg/ kg bw/ day from days 7 through 28 of gestation.

Maternal toxicity was demonstrated by statistically significant findings at 125 mg/ kg/ day of a body weight gain decrement of 102 g between gd 7 9 and 57 g between gd 9 11, food consumption decrement of 18%, abortions, death and clinical signs such as diarrhea, stained cageboard and tail. Only 1 dam and 1 litter survived to termination at 175 mg/ kg/ day.

The maternal LOAEL is 125 mg/ kg bw/ day, based on body weight gain decrement, decreased food consumption, abortions, death and clinical signs including abnormal gait at 175 mg/ kg/ day. The maternal NOAEL is 50 mg/ kg bw/ day.

No dose related external, visceral or skeletal findings were noted. Since only 1 dam with 1 litter survived to termination at the 175 mg/ kg/ day dose, evaluation of embryo/ fetal effects at this dose are meaningless. Random malformations were seen in control and the highest dose group with no dose relationship. Mean male and female fetal weight was reduced by 5% and female fetal weight was reduced by 10% at 125 mg/ kg/ day. Although this weight reduction was not listed as being statistically significant, the author stated that this slight weight reduction at 125 mg/ kg/ day was a developmental effect level and this reviewer agrees.

The developmental LOAEL is 125 mg/ kg bw/ day, based on mean male and female fetal weight decrement and female fetal weight decrement. The developmental NOAEL is 50 mg/ kg bw/ day.

The developmental toxicity study in the rabbit is classified acceptable (guideline) and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in the rabbit.

Dose/ Endpoint for Risk Assessment: The maternal NOAEL of 50 mg/ kg/ day based on based on maternal body weight and food consumption decrement and fetal weight decrement at 125 mg/ kg/ day (LOAEL).

Comments about Study/ Endpoint: In the absence of a inhalation study an oral NOAEL was selected. Absorption via the inhalation route is assumed to be equivalent to oral absorption. 15 9. Intermediate Term inhalation (1 Month to 6 Months) Exposure.

Study Selected: Chronic Feeding Study in Dogs § 870.4100 MRID No.: 42162301 Executive Summary: [See Section II 3. on the Chronic Reference Dose (RfD).]

Dose/ Endpoint for Risk Assessment: NOAEL of 5.0 mg/ kg/ day based on elevated clinical chemistry values (serum alkaline phosphatase, serum aspartate aminotransferase), other changes in clinical chemistry values, liver microscopic findings and body weight decrement and clinical observation of thinnest in one males at 38 mg/ kg/ day.

Comments about Study/ Endpoint: The affects seen in clinical chemistry values at 3, 6 and 12 months are appropriate for these exposure periods. Absorption via the inhalation route is presumed to be equivalent to oral absorption.

10. Long Term Inhalation (longer than 6 Months) Exposure.

Study Selected: Chronic Feeding Study in Dogs § 870.4100 MRID No.: 42162301 Executive Summary: [See Section II 3. on the Chronic Reference Dose (RfD).]

Dose/ Endpoint for Risk Assessment: NOAEL of 5.0 mg/ kg/ day based on elevated clinical chemistry values (serum alkaline phosphatase, serum aspartate aminotransferase), other changes in clinical chemistry values, liver microscopic findings and body weight decrement and clinical observation of thinnest in one males at 38 mg/ kg/ day.

Comments about Study/ Endpoint: The affects seen in clinical chemistry values at 3, 6 and 12 months are appropriate for these exposure periods. Absorption via the inhalation route is presumed to be equivalent to oral absorption.

11. Margins of Exposure for Occupational/ Residential Risk Assessment A margin of exposure of 100 is adequate for occupational dermal and inhalation exposure. There is no non occupational (residential) exposures identified at this time. 16 12. Recommendation for Aggregate Exposure Risk Assessments Aggregate exposure risk assessment is not required since there are no non occupational (residential) uses at the present time.

III. CLASSIFICATION OF CARCINOGENIC POTENTIAL 1. Combined Chronic Toxicity/ Carcinogenicity Study in Rats MRID No.: 00108638 Executive Summary: In this combined chronic/ oncogenicity study (MRID 00108638), hexazinone (94 96% a. i.; Lot/ Batch #: 6897 40 and 74.25) was administered in the diet to ChR CD rats (36/ sex/ group) for up to 25 months at nominal doses of 0, 0, 200, 1000, or 2500 ppm (equivalent to 0, 0, 10.2, 53.4, and 138.3 mg/ kg/ day in males and 0, 0, 12.5, 67.5, and 178.6 mg/ kg/ day in females). No treatment related differences were observed in mortality, clinical signs, food consumption, hematology, clinical chemistry, organ weights, and gross or microscopic pathology. No adverse effects were observed in the 200 ppm animals. There were several signs of general toxicity, but a target organ could not be clearly identified at any dose.

Terminal body weights were decreased 8% in1000 ppm and 20%, p 0.05, in the 2500 ppm females. A decrease in overall body weight gain (Days 0 728; calculated by the reviewers) was also observed in the 1000 ( 10%) and 2500 ppm ( 25%) females. Nominal decreases in body weight and body weight gain ( 3 to 5%) occurred in males at 1000 ppm during the study, which may have been biologically significant at the end of the study ( 12% body weight and 14% for body weight gain).

Decreases (p values not calculated) in total food efficiency were observed in females at 1000 10 and 2500 ppm ( 25%) and in males at 1000 ppm during the study with overall decrement in food efficiency in 1000 ppm males ( 10%). In males at 2500 ppm, food efficiency was depressed for the first 6 months of the study ( 25%), but from 6 months to the end of the study, it was increased 139%.

The reviewer noted problems interpreting the body weights and food efficiency in males at the top dose level, which were not consistent with the mid dose level. For the first 6 months of the study in males, a body weight decrement due to probable toxicity was seen at 1000 and 2500 ppm. After 6 months food efficiency in males at the 1000 ppm remained less than controls ( 56%) while food efficiency at 2500 ppm in males was higher than controls (+ 139%)( Table 4). By the end of the study, male body weight at 1000 ppm was 12% and body weight gain 14%, where as body weight and body weight gain in males at 2500 ppm was +3% for both weight and gain. The reason for this recovery in male body weight decrement at 2500 ppm is unknown, but it appears to be real. [Since absolute and relative liver weights were decreased in males at 2500 ppm, liver enzyme induction allowing the recovery seems unproven.] The body weight decrement at 1000 and 2500 ppm with recovery in body weight at 2500 ppm indicates the an adequate dose level to test for carcinogenicity in males was approached, but probably not attained. Other treatment groups were similar to the average of concurrent controls. 17 Dosing was considered adequate based on decreased body weights, body weight gains, and food efficiency in the 2500 and 1000 ppm females and food efficiency and body weights in the 2500 ppm males for the first 6 months. Body weight ( 12%) and body weight gain ( 14%) in males at 1000 ppm were decreased at the end of the study, in addition overall food efficiency ( 14%) was decreased by the end of the study.

Thus female body weight and body weight gain was decreased sufficiently to adequately test for carcinogenicity. Male body weight and body weight gain at1000 ppm appeared to be adequate to test for carcinogenicity by the end of the study, but the lack of dose response in male body weight and body weight gain at 2500 ppm (showing recovery after 6 months such that body weight and body weight gain were higher than control values) may indicate problems with the interpretation of the body weights and body weight gains at 1000 ppm.

In the 2500 ppm males, creatinine was increased (NS) in the urine at months 18 and 24 and bilirubin was detected at month 18 and 24. The Sponsor reported that the urine was more alkaline in the 2500 ppm treatment groups (data not reported). Also at 2500 ppm, decreased (p 0.05) absolute and relative liver and kidney organ weights were observed in the males and increased (p 0.05) relative (to body) stomach and kidney organ weights were observed in the females. However, histopathological data did not corroborate these findings.

The LOAEL is 1000 ppm for males and females (equivalent to 53.3 for males and 67.5 mg/ kg/ day for females) based on decreased body weight and food efficiency in males and females. In males at 2500 ppm the body weight decrement and food efficiency decrement occurred only for the first 6 months of the study. The NOAEL is 200 ppm for males and females (10.2 for males and 12.5 mg/ kg/ day for females).

Neoplasm data were evaluated by two pathologists. An increase incidence of thyroid C cell adenomas was observed in the 2500 ppm males, but when analyzed by the Fisher's exact test (p> 0.10) or life table methods (p> 0.20) no significant differences were observed between controls and treated groups. Using life table analyses a significant (p< 0.05) dose response trend was observed in thyroid C cell tumors in only one of the two pathology reports. Female rats did not show any potential for C cell thyroid adenomas. The incidence, malignancy and latency of tumors were comparable among control and treated rats of both sexes.

Under the conditions of this study, carcinogenic potential of hexazinone is considered negative.

The submitted study is classified as acceptable for guideline 870.4300 combined chronic/ carcinogenicity study in rats.

Discussion of Tumor Data: Neoplasm data were evaluated by two pathologists. An increase incidence of thyroid C cell adenomas was observed in the 2500 ppm males, but when analyzed by the Fisher's exact test (p> 0.10) or life table methods (p> 0.20) no significant differences were observed between controls and treated groups. Using life table analyses a significant (p< 0.05) dose response trend was observed in thyroid C cell tumors in only one of the two pathology reports. Female rats did not show any potential for C cell thyroid adenomas. The incidence, malignancy and latency of tumors were comparable among control and treated rats of both sexes. 18 The study in rats does not support a carcinogenic potential for hexazinone.

Adequacy of the Dose Levels Tested: The dose levels in female rats was adequate to test for the carcinogenic potential hexazinone. There was an adequate body weight decrement and body weight gain decrement at the two top dose levels. The dose levels in male rats approached an adequate dose level to test for carcinogenic potential of hexazinone. There may have been an adequate body weight and body weight gain at the mid dose level at the end of the study to test for carcinogenic potential of hexazinone, however body weights of males at the top dose level behaved atypically. At the top dose level, males showed a adequate body weight decrement only for the first 6 months of the study and by the end of the study, body weights were higher than the average of the two controls. The reason for this reversible body weight decrement is unknown, but the top dose level in males was one half the top dose level of 5000 ppm in the 90 day subchronic study in males where liver toxicity was seen. These body weight decrements in males and females are supported by a corresponding decrements in food efficiency.

2. Carcinogenicity Study in Mice MRID No. 41359301,42509301 and 4320290 § 870.4200 Executive Summary: In this mouse oncogenicity study (MRIDs 00079203, 41359301, 42509301 and 43202901), hexazinone ( 95% a. i.; Lot/ Batch #: H 11, 265 and 265 2) was administered in the diet to CD 1 mice (80/ sex/ group) for up to 104 weeks at nominal doses of 0, 200, 2500 or 10,000 ppm (equivalent to 28, 366 and 1635 mg/ kg/ day in males and 0, 34, 450 and 1915 mg/ kg/ day in females).

No treatment related differences were observed in mortality, food consumption, food efficiency or hematology.

Hepatotoxicity was evident at the terminal sacrifice. Macroscopic liver nodule/ mass (% treated vs % controls; n = 28 55) was observed in males at 2500 (39% vs 7%) and 10,000 ppm (33%). Increased incidences (% treated vs 0% controls; n = 38 55) in the following microscopic liver lesions were observed: hyperplastic nodule( s) (includes both foci of cellular alteration and adenoma) in males at 2500 (39% vs 20%) and 10,000 ppm (36%) and in females at 10,000 ppm (15% vs 3%); and necrosis (severity and type unspecified) in the 10,000 ppm males (36% vs 7%). Centrilobular hepatocyte hypertrophy was observed (% treated vs % controls) at the terminal sacrifice (n = 38 55) in males at 2500 (18% vs 0%) and 10,000 ppm (98%) and in females at 10,000 ppm (46% vs 0%) and in the dead and moribund males (n = 25 40) at 2500 (44% vs 0%) and 10,000 ppm (60%). Increased (p 0.05 or 0.01) liver/ gall bladder weights were observed at 10,000 ppm in males in both absolute and relative to body weights and in females in relative to body weight.

Other signs of toxicity were evident. Distal tail tip sloughing and/ or discoloration was observed at 10,000 ppm in males at Weeks 13 104 and in females at Weeks 5 and 13 104. Macroscopically, tip of tail missing/ sloughed was observed at the terminal sacrifice in the 10,000 ppm males (31% vs 5%) and females (61% vs 11%) and in the dead and moribund 10,000 ppm females (46% vs 2%). The toxicological significance of these findings was unclear. Minor decreases (p 0.05 or 0.01) in body weights were observed in the 10,000 ppm treatment groups at 19 Weeks 13 104 in both sexes. Overall body weight gains (calculated by the reviewers) were decreased in the 10,000 ppm males ( 25%) and females ( 31%). The LOAEL is 2500 ppm for males (equivalent to 366 mg/ kg/ day) based on gross liver nodules/ masses, hyperplastic nodules in the liver and centrilobular hepatocyte hypertrophy and 10,000 ppm for females (equivalent to 1915 mg/ kg/ day [limit dose]) based on decreased body weights, increased relative liver/ gall bladder weights, hyperplastic nodules and centrilobular hepatocyte hypertrophy. The NOAEL is 200 ppm (equivalent to 28 mg/ kg/ day) for males and 2500 ppm (equivalent to 450 mg/ kg/ day) for females.

Liver samples were first evaluated using the term hyperplastic nodule which did not clearly distinguish neoplasia from non neoplasia. Reevaluation was conducted to make this distinction, and no significant differences were observed between the treatment groups and the concurrent controls. However positive trends (p< 0.05) were observed (% treated vs % controls) in focus/ foci of cellular alteration in males, hepatocellular neoplasm( s) (including adenoma, sarcoma, carcinoma, leukemia, and lymphoma) in females, and singular hepatocellular adenoma in females. Focus/ foci of cellular alteration were observed in males at 2500 (11.3% vs 5.0%) and 10,000 ppm (24.1%) and females at 10,000 ppm (12.5% vs 3.8%) beginning at Week 57. Singular hepatocellular adenoma was observed in the 10,000 ppm females (7.5% vs 2.5%) beginning at Week 77. Hepatocellular neoplasm( s) were observed in the 10,000 ppm females (8.8% vs 2.5%). A carcinoma in the 10,000 ppm treatment groups was first observed at Week 65. The incidence of carcinomas were within historical control ranges for each sex, while the incidence of adenomas were increased by 3.21% in the 10,000 ppm females. A dose dependent increase in adenomas was not observed in males. The Health Effects Division Carcinogenicity Peer Review Committee (CPRC) concluded that hexazinone should be classified as a Group D (not classifiable as to human carcinogenicity)( 7/ 27/ 94).

Under the conditions of this study, evidence of carcinogenic potential was equivocal: a positive trend test for neoplasia was observed in female mice, but no significant difference was determined by pair wise comparison.

The submitted study is classified as acceptable for guideline 870 4200 carcinogenicity study in mice.

Discussion of Tumor Data: A dose dependent increase in adenomas was not observed in males. Under the conditions of this study, evidence of carcinogenic potential was equivocal: a positive trend test for neoplasia was observed in female mice, but no significant difference was determined by pair wise comparison.

Adequacy of the Dose Levels Tested: Adequate dose levels were used in male and female mice to test for the carcinogenic potential of hexazinone. Liver toxicity was seen at the top dose level in males and females.

3. Classification of Carcinogenic Potential HED's Carcinogenicity Peer Review Committee classified hexazinone as a Group D Chemical 20 (not classifiable as to human carcinogenicity) (7/ 27/ 94). This classification was based on the following weight of evidence considerations. In rats, females showed no evidence for carcinogenicity; males showed a significant trend only for thyroid adenomas. In mice, the evidence of carcinogenicity was equivocal: a positive trend test for liver tumors was observed in female mice, but no significant difference was seen by pair wise comparison (CPRC Report dated July 27, 1994).

IV. MUTAGENICITY Adequacy of data base for Mutagenicity: The data base for mutagenicity is considered adequate based on pre 1991 mutagenicity guidelines. There is low concern for mutagenic potential of hexazinone.

Hexazinone was found to be positive for mutagenicity in one chromosomal aberration assay (in vitro cytogenics) (MRID# 00130709), but negative in the remaining studies. It is concluded that the test material was clastogenic in both of the non activated trials and was also clastogenic in the one adequate S9 activated trials. Under both test conditions, concentrations providing evidence of clastogenicity induced an acceptable level of cytotoxicity (> 50% relative cell survival). Thus, the findings can not be considered to be a secondary effect of cytotoxicity. Nevertheless, the outcome of the induced structural damage (i. e., primarily chromatid and chromosome breaks) is unclear since these types of structural aberrations would not likely be passed on to daughter cells. Because unambiguous positive results were achieved, it was concluded that the study provided adequate evidence that INA 3674 112 (hexazinone, technical) is clastogenic in an acceptable study.

Gene Mutation 1. Guideline 870.5100, Reverse mutation in Salmonella EXECUTIVE SUMMARY: In a reverse gene mutation assay in bacteria (MRID 40826201), strains TA98, TA100, TA1535, TA1537 and TA1538 of S. typhimurium were exposed to Striazine 2,4( 1H, 3H) dione, 3 cyclohexyl 6 dimethylamino 1 methyl (95% a. i.) in ethanol at concentrations of 200, 400, 600, 800 and 1000 : g/ plate without mammalian metabolic activation (S9 mix) and at concentrations of 400, 800, 1200, 1600 and 2000 : g/ mL with S9 mix. The S9 fraction was obtained from Aroclor 1254 induced male Crl: CD( SD) BR rat liver.

The maximum concentrations of S triazine 2,4( 1H, 3H) dione, 3 cyclohexyl 6 dimethylamino1 methyl tested produced little or no cytotoxicity, were not limited by solubility and were not a limit dose for the assay. No statistically significant increases in the number of revertants per plate or positive linear dose response were seen. The solvent and positive controls induced acceptable responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as Unacceptable. It does not satisfy the requirement for FIFRA Test 21 Guideline [OPPTS 870.5100 (§ 84 2)] for in vitro mutagenicity [bacterial reverse gene mutation] data and should have used higher doses.

2. Guideline 870.5300, Gene mutation at HGPRT locus EXECUTIVE SUMMARY: In a mammalian cell gene mutation assay at the HGPRT locus (MRID No. 00076956), Chinese hamster CHO K1 BH4 cells cultured in vitro were exposed to INA 3674 112, (Lot No. 7612 5E6E, 95% a. i.) in ethanol in two trials. Concentrations used in Trial 1 were 2.0, 11.1, 13.1, 13.9 and 14.3 mM under nonactivated conditions and concentrations of 2.0, 7.9, 8.9, 9.3 and 9.9 mM under activated conditions (S9 mix). Concentrations used in Trial 2 were 2.0, 5.9, 11.1, 13.1 and 13.9 mM without S9 mix and concentrations of 2.0, 7.9, 8.9, 9.3 and 9.9 mM with S9 mix. The S9 fraction was obtained from Aroclor 1254 induced male Charles River CD® rat livers. INA 3674 112 was tested up to cytotoxic concentrations. In Trial 1, the cultures treated at 14.3 mM were not plated for mutation determination due to cytotoxicity and in both Trials 1 and 2, those cultures treated at 13.9 mM were excluded from analysis because no mutants were seen. No statistically significant increases in mutant frequency over solvent control values were seen with or without S9 mix in either Trial 1 or 2. The expected marked increase in the mutation were seen with the positive controls. There was, however, no indication that INA 3674 112 induced a mutagenic effect either in the presence or the absence of S9 activation.

This study is classified as acceptable. It satisfies the requirement for FIFRA Test Guideline OPPTS [870.5300 (§ 84 2)] for in vitro mutagenicity (mammalian forward gene mutation) data.

Chromosomal Aberrations 3. Guideline: 870.5375: In vitro mammalian cytogenics (chromosomal aberrations) in Chinese hamster CHO cells.

EXECUTIVE SUMMARY: In a mammalian cell cytogenetics assay (MRID No. 00130709), Chinese hamster ovary CHO K1 BH4 cell cultures were exposed to INA 3674 112 (Hexazinone, 95% a. i.) in ethanol in two separate trials. Exposure was for two hours with activation and for 10 hours without activation. Cells were harvested 10 hours after the start of treatment. In Trial 1, cells were treated at concentrations of 1.58, 3.94, 15.85 and 19.82 mM without metabolic activation (S9 mix) and at concentrations of 0.32, 3.17, 7.93 and 15.85 mM with S9 mix. In Trial 2, cells were treated at concentrations of 1.58, 3.94, 7.93 and 15.85 without S9 mix and at concentrations of 0.32, 3.17, 7.93 and 15.85 mM with S9 mix. The S9 fraction was obtained from Aroclor 1254 induced CD rat livers.

INA 3674 112 was tested up to cytotoxic concentrations. Based on the results of a preliminary cytotoxicity test, upper concentrations of 23.78 mM without S9 mix and 47.56 mM with S9 mix were selected for the first cytogenetic assay but these concentrations proved excessively cytotoxic and were not scored for chromosomal aberrations. Without S9 activation, statistically significant increases (p< 0.01) in structural aberrations per cell (excluding gaps), lesions per cell and percent abnormal cells were seen at 15.85 mM (Trials 1 and 2) and 19.82 mM (tested in Trial 1 only). Relative percent survival (RPS) at this level was . 50%. The percent abnormal cells averaged over all cultures from both trials was 28.0% and 21.5% at 19.82 and 15.85 mM, respectively, compared to the solvent control values of 2.0% (0.5% ethanol in Trial 2) and 7.0% (0.75% ethanol in Trial 1). The percent abnormal cells in positive control cultures was 18% in both Trial 22 1 (4.83 mM EMS) and Trial 2 (6.44 mM EMS). In the presence of S9 mix, no statistically significant increases in chromosomal aberration induction were seen in Trial 1; however, very low positive control values indicated a problem with the S9 mix. Statistically significant increases were seen at 15.85 mM in Trial 2; RPS at 15.85 mM was 75%. There was a statistically significant dose related trend for all three parameters. The statistically significant (p <0.01) increases at 15.85 mM remained when the data from Trial 1 and 2 were combined (average of 20% abnormal cells compared to 10% for the solvent control). The predominant aberrations with or without S9 mix were chromatid and isochromatid breaks. Solvent and positive controls (except the positive control in Trial 1 with S9 mix) induced the appropriate responses. INA3674 112 was positive for the induction of structural chromosomal aberrations in both the presence and absence of S9 mix.

This study is classified as Acceptable/ guideline and satisfies the requirement for FIFRA Test 4. Guideline 870.5385: In vivo cytogenics assay in rat bone marrow cells EXECUTIVE SUMMARY: In a mammalian cell cytogenetics assay (MRID 00131355), in bone marrow cells of Sprague Dawley CD rats, three rats/ dose/ sex/ harvest time were exposed to H# 14,555 in corn oil ( assumed 100% a. i.) at doses of 100, 300 and 1000 mg/ kg by oral gavage. Bone marrow cells were harvested at 6, 12, 24 and 48 hours post treatment.

The highest dose tested (1000 mg/ kg) was lethal. A major limitation of this study was the number of animals treated and the number of cells analyzed per animal. At most, three rats/ sex/ dose/ harvest time were treated with, at most, 50 cells per rat analyzed. Few or no analyzable cell were available from many rats. Positive control values were significantly (p= 0.03) increased. There was no evidence that H# 14, 14,555 induced an increase in the incidence of chromosomal aberrations in the bone marrow cells of treated animals.

This study is classified as Unacceptable. The number of cells analyzed and the number of rats treated was insufficient. The study does not satisfy the requirement for FIFRA Test Guideline [OPPTS 870.5385 (§ 84 2)] for in vivo cytogenetic mutagenicity data.

5. Guideline 870.5395: Micronucleus assay in mouse bone marrow EXECUTIVE SUMMARY: In a Crl: CD 1 (ICR) BR mouse bone marrow micronucleus assay (MRID 45124401), 5 mice/ sex/ dose/ harvest time were treated orally with Hexazinone 25L (Lot No. 9912033, 25% Hexazinone a. i. (24.5% by analysis) and 75% inert ingredients) at doses of 1000, 2000 and 3000 mg/ kg. Bone marrow cells were harvested at 24 and 48 hours posttreatment and examined for micronucleated polychromatic erythrocytes (MPCEs). The vehicle was Milli Q ® water.

Signs of toxicity noted at 3000 mg/ kg included: death, convulsions, half shut eyes, head tilt, irregular respiration, lethargy, low carriage, pallor, prostration, uncontrollable spinning, shovelnosing straining up on toes and tremors. Micronuclei were scored in bone marrow from mice treated at 3000 mg/ kg and from the solvent and positive controls. Mice from the two lower dose 23 groups were not evaluated for micronuclei induction. No statistically significant increases in the frequency of MPCEs or in the PCE/ NCE ratio over the solvent control values were seen in either sex at either the 24 or 48 hour harvest time. The solvent and positive control values were appropriate and within the testing laboratory's historical control ranges. There was no evidence that Hexazinone 25L induced a clastogenic or aneugenic effect in bone marrow at any harvest time.

This study is classified as Acceptable/ Guideline. It satisfies the requirement for FIFRA Test Guideline [OPPTS 870.5395 (§ 84 2)] for in vivo cytogenetic mutagenicity data.

Other genetic Studies 6. Guideline 8760.5550: Unscheduled DNA synthesis assay in rat hepatocytes EXECUTIVE SUMMARY: In an unscheduled DNA synthesis assay (MRID 00130708), primary rat hepatocyte cultures were exposed to INA 3674 112 (Lot No. 7612 5E6E, 95% a. i.) in ethanol for 18 hours at concentrations of 1 x 10 5 , 1 x 10 4 , 1 x 10 3 , 1 x 10 2 , 0.1, 1.0, 10.0 and 30.0 mM in Trial 1 and at concentrations of 1 x 10 5 , 1 x 10 4 , 1 x 10 3 , 1 x 10 2 , 0.1, 1.0, 5.0, 10.0 and 30.0 mM in Trial 2.

INA 3674 112 was tested up to the highest achievable concentration in the solvent. Two slides per dose, 25 cells per slide were evaluated for UDS induction in Trial 1. One slide per dose, 25 cells per slide were evaluated in Trial 2. The author did not report that the slides were coded prior to analysis. The average net nuclear grain counts of test material treated cells in Trial 1 were all less than zero with the exception of one slide at 1 x 10 5 mM (0.1 ± 9.6) and one slide at 1.0 mM (1.6 ± 5.2). The average net nuclear grain count was below zero for all test material concentrations in Trial 2 with the exception of 0.1 mM where the average net nuclear grain count was 0.0 ± 2.9. The criterion for a positive response was an average net nuclear grain count of at least five in two experiments at any tested concentration. The results were thus negative. The number of cells in repair was not reported. The solvent and positive (DMBA) controls induced the appropriate responses. 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 requirement for FIFRA Test Guideline; OPPTS 870.5550 [§ 84 2] for other genotoxic mutagenicity data. Compliance statements were not provided. V. HAZARD CHARACTERIZATION Hexazinone is a herbicide used to control a broad spectrum of weeds including woody plants in alfalfa, rangeland, pastures, woodlands, pineapple, sugarcane and blue berries. Non crop areas include ornamental plants and forests. Hexazinone is used as a pre emergent, post emergence herbicide as well as by direct spray and soil applications. There are no non occupational (residential) uses.

Hexazinone has low acute toxicity by the oral (Category III), dermal (Category IV ) and inhalation routes (Category III). Primary eye irritation is severe, causing corneal opacity and moderate irritation in unwashed eyes (Category I). It causes mild skin irritation and is classified Category IV for skin irritation. It is not a skin sensitizer in the Guinea pig. 24 Body weight decrement and liver toxicity were the most frequent effects shown in studies with hexazinone. Liver toxicity was seen in the chronic dog and mouse studies. Body weight decrement was seen in the chronic rat studies and the studies on reproduction.

In a reproduction study, pup weight decrement occurred at the same dose as parental body weight decrement. No reproductive effects were seen in the study other than pup weight decrement. The rat prenatal study showed fetal weight decrement and possibly renal malformations but no increased susceptibility. The new rabbit study showed fetal weight decrement at the same dose causing maternal weight decrement. Thus, no offspring susceptibility is seen in rat or rabbit prenatal studies or the rat reproduction study.

The 21 day dermal study in the rabbit showed no systemic toxicity and mild dermal irritation at the limit dose.

The chronic study in dogs showed severe body weight decrement, changes in liver related clinical chemistry values, and microscopic lesions.

Body weight decrement was seen in a chronic carcinogenicity studies in rats and mice. Liver toxicity was seen in mice. The mouse carcinogenicity study showed an increased trend for liver carcinomas, but no pair wise significant increases. The rat study showed no carcinogenic potential. Based on these studies in rats and mice, hexazinone was classified in a group D, not classifiable as a carcinogen.

Mutagenicity is of low concern. A reverse mutation assay was unacceptable, but the potential concern is mitigated by an negative in vivo mutation assay and the remaining negative studies on potential genetic damage.

Rat metabolism studies showed that hexazinone was rapidly absorbed and excreted and essentially no difference in the metabolism of males and females at high or low dose levels. Almost no parent hexazinone was recovered in urine or feces. Two major metabolites were recovered from feces and urine, in addition to lesser amounts of a third metabolite and small amounts conjugated products from urine. 25 VI. DATA REQUIREMENTS The HIARC requested a 28 day inhalation study with hexazinone as confirmation. There was concern for potential inhalation exposure based on the use pattern. A 1976 21 day inhalations study in rats (MRID# 00063972) was considered unacceptable as documented.

VII. ACUTE TOXICITY Acute Toxicity of Hexazinone Guideline No. Study Type MRIDs # Results Toxicity Category 81 1 Acute Oral/ Rat 41235004 (1989) LD50 = 1200 mg/ kg III 81 2 Acute Dermal/ Rabbit 00104974 LD50 >5278 mg/ kg IV 81 3 Acute Inhalation 41756701 (1990) LC50 > 3.94 mg/ L( 4 hour) 1 III 81 4 Primary Eye Irritation 00106003 (1982) Irreversible corneal opacity, Severe I 81 5 Primary Skin Irritation 00106004 (1982) Mild IV 81 6 Dermal Sensitization 41235005 (1989) NA Not a skin sensitizer 81 8 Acute Neurotoxicity Not conducted 1 These effects are consistent with an unreviewed and unacceptable three week inhalation study with dust [( MRID# 00063972 (1976); summary only submitted] reporting no significant toxic effects at 2.5 mg/ L, the only dose tested. 26 VII. Summary of Toxicological Dose and Endpoints for HEXAZINONE Exposure Scenario Dose (mg/ kg/ day) UF /MOE Hazard Based Special FQPA Safety Factor Endpoint for Risk Assessment Dietary Risk Assessments Acute Dietary females 13 50 years of age NOAEL = 400 UF = 100 Acute RfD = 4.0 mg/ kg/ day 1x Developmental Toxicity  Rat LOAEL is 900 mg/ kg/ day based on decreased male and female fetal weight, kidneys with no papilla (malformation) and misaligned sternebrae (variation)

Acute Dietary general population including infants and children An appropriate endpoint attributable to a single dose was not identified in the oral studies for general population, including the rat and rabbit developmental studies.

Chronic Dietary all populations NOAEL= 5.0 UF = 100 Chronic RfD = 0.05 mg/ kg/ day 1x Chronic one year feeding  Dog LOAEL = 41.24 male; 37.57 female mg/ kg/ day based on severe body weight decrement and clinical chemistry changes including elevated aspartate aminotransferase and alkaline phosphatase.

Incidental Oral Short Term (1  30 Days) Residential Only There are no residential uses at the present time and therefore, endpoints were not selected.

Incidental Oral Intermediate Term (1  6 Months) Residential Only There are no residential uses at the present time and therefore, endpoints were not selected.

Non Dietary Risk Assessments Dermal Short Term (1  30 days) No hazard was identified, therefore quantification of risk is not required. No systemic toxicity was seen at the limit dose following repeat dermal application, and there were no concerns for pre and/ or post natal toxicity.

Residential Not Applicable Occupational Not Applicable Dermal Intermediate Term 1 (1  6 Months) Oral NOAEL= 5.0 Chronic one year feeding  Dog LOAEL = 41.24 male; 37.57 female mg/ kg/ day based on severe body weight decrement and clinical chemistry changes including elevated aspartate aminotransferase and alkaline phosphatase. Residential MOE= Not Applicable Not Applicable Occupational MOE= 100 Not Applicable Exposure Scenario Dose (mg/ kg/ day) UF /MOE Hazard Based Special FQPA Safety Factor Endpoint for Risk Assessment 27 Dermal Long Term 1 (1  6 Months) Oral NOAEL= 5.0 Chronic one year feeding  Dog LOAEL = 41.24 male; 37.57 female mg/ kg/ day based on severe body weight decrement and clinical chemistry changes including elevated aspartate aminotransferase and alkaline phosphatase. Residential MOE= Not Applicable Occupational MOE= 100 Not Applicable Inhalation Short Term 2 (1  30 days) Oral NOAEL= 100 Developmental Toxicity  Rabbit LOAEL = 50 mg/ kg/ day based on decreases in maternal food consumption and dose related body weight decrement and fetal weight decrement. Residential MOE= Not Applicable Not Applicable Occupational MOE= 100 Not Applicable Inhalation Intermediate Term (1  6 Months) Oral NOAEL= 5.0 Chronic one year feeding  Dog LOAEL = 41.24 mg/ kg/ day, male; 37.57 mg/ kg/ day, female, based on severe body weight decrement and clinical chemistry changes including elevated aspartate aminotransferase and alkaline phosphatase. Residential MOE =Not Applicable Not Applicable Occupational MOE= 100 Not Applicable Inhalation Long Term (> 6 Months) Oral NOAEL= 5.0 Chronic one year feeding  Dog LOAEL = 41.24 mg/ kg/ day, male ;37.57 mg/ kg/ day, female, based on severe body weight decrement and clinical chemistry changes including elevated aspartate aminotransferase and alkaline phosphatase. Residential MOE= Not Applicable Not Applicable Occupational MOE= 100 Not Applicable Cancer Classification: D  Not Classifiable as to human carcinogenicity 1 Since an oral NOAEL was selected 12.5% dermal absorption factor should be used for route to route exposures. 2 Absorption via the inhalation route is assumed to be equivalent to oral absorption.