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EPA REGISTRATION DIVISION CO MPANY NOTICE OF FILING FOR

PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER

EPA Registration Division contact: [PV Shah, (703) 308-1846]

INSTRUCTIONS: Please utilize this outline in p reparing the pesticide petition. In

cases where the outline element does not apply, please insert “NA-Remove” and

maintain the outline. Please do not chan ge the margins, font, or format in your pesticide petition. Simply replace the instructions that appear in green , i.e., “ [insert company name] ,” with the information specific to your action.

TEMPLATE:

[Rhodia Inc.]

[Insert petition number]

EPA has received a pesticide petition ([insert petition number]) from [Rhodia Inc., c/o SciReg, Inc.], [12733 Di rector’s Loop, Woodbridge, VA 22192] proposing, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 180.

(Options (pick one)

1. by establishing a tolerance for residues of

2. to establish an exemption from the requirement of a tolerance for

[ dimethyl esters of glutaric acid (CAS No. 1119-40-0), succinic acid (CAS No. 106-65-0), and adipic acid (CAS No. 627-93- 0), herein referred to as DME, under 40 CFR 180.910 when used as an inert ing redient in pesticide formulations. ] EPA has determined that the petition contains data or information regarding the elements set forth in section 408 (d)(2) of F DDCA; however, EPA has not fully evaluated the sufficiency

of the submitted data at this time or whethe r the data supports gran ting of the petition. Additional data may be needed before EPA rules on the petition.

[This tolerance exemption petition summa rizes and relies upon available data for DME; data for dibasic esters (DBE) und er CAS No. 95481-62-2; and data on the individual components, dimethyl glutarat e (DMG), dimethyl adipate (DMA), and dimethyl succinate (DMS). In addition, da ta on the metabolites, dicarboxylic acids (glutaric acid, succinic acid, a nd adipic acid), is included in this petition to support the toxicological profile of DME. Data fr om chronic and subchronic oral toxicity

studies conducted with monosodium succinate is also presented. In the absence of experimental data on succinic acid, th ese studies conducted with monosodium2

succinate are considered a relevant surrogat e as its most likely bioavailable form upon ingestion would be the succinic acid.]

A. Residue Chemistry

1. Plant metabolism.

2. Analytical method. [Rhodia is requesting that DME be exempt from the requirement of a tolerance under 40 CFR 180.910. Therefore, Rhodia believes that an analytical method to determine resi dues in treated crops is not relevant.]

3. Magnitude of residues. [Rhodia is requesting that DME be exempt from the requirement of a tolerance under 40 CFR 180.910. Therefore, Rhodia believes that data on the magnitude of residues in treated crops is not relevant.]

B. Toxicological Profile

1. Acute toxicity. [ Acute Oral Toxicity

DME was tested for acute oral toxicity in Sprague-Dawley rats, according to OECD Guideline No. 401 and in compliance wi th OECD Good Laboratory Practice.

The test article, a liquid, was administered in a single oral dose at 2,000 mg/kg, at a dose volume of 1.835 mL/kg, to five males and five females. Examinations for mortality and clinical signs were perfor med daily during the 14-day study period. All surviving animals were euthanized and ne cropsied at the end of the observation period.

No deaths occurred. The general behavior and body weight gain were not affected by treatment. No clinical signs were recorded during the study. At necropsy, a macroscopic examination revealed no a bnormalities. Therefore, the oral LD 50 for DME was greater than 2,000 mg/kg.

Acute Dermal Toxicity

DME was tested for acute dermal toxicity in Sprague Dawley rats, according to OECD Guideline No. 402 and in compliance wi th OECD Good Laboratory Practice.

The test article, a liquid (specific gravity: 1.09), was administered in a single dermal dose of 2,000 mg/kg, at a dose volume of 1.835 mL/kg to a clipped area of the skin (5 x 7 cm) of five males and five female s, for 24 hours under an semi-occlusive dressing. Examinations for mortality, clinic al signs (with special attention given to the treatment sites), and bo dy weight gain were performed during the 14-day study period. All surviving animals were euthan ized and necropsied at the end of the observation period and subjected to macroscopic examination.

No deaths occurred. No clinical signs were recorded during the observation period.3

No signs of dermal irritation were observed at the application site in any of the rats. The general behavior and body weight gain were not affected by treatment. At necropsy, a macroscopic examination reveale d no abnormalities. Therefore, the

acute dermal LD 50 for DME was greater than 2,000 mg/kg.

Primary Eye Irritation

DME was tested for acute ocular irritation with three male New-Zealand White rabbits, according to OECD Guideline No. 405 and in compliance with OECD Good Laboratory Practice.

The test article, a liquid, was instilled in a single dose of 0.1 mL, into the conjunctival sac of the left eye of each animal. The right eye remained untreated and served as a control. The eyes were not rinsed. Ocular examinations were performed at 1, 24, 48, and 72 hours after instillation.

A slight corneal opacity was observed on d ay 2 only (24 hours after instillation) in

one of the animals. No iritis was observed throughout the observation period in any of the animals. Conjunctival redness was obse rved at 1 hour in all animals (score 2 or 3), and in two animals on day 2 (score 1) . Chemosis (score 1 or 3) was observed in two of three animals only at 1 hour after in stillation. In addition, transient discharge was noted at 1 hour. All effects cleared by day 3 (48 hours after instillation). Based on the reported scores, the average eye irri tation index was calculated to be 11 on day 1, and 3 on day 2. Therefore, with a maximum mean total score (MMTS) of 11/110 on day 1 (1-hour after in stillation) and full reversib ility of the effects by day 3, DME was considered minimally irritating in this study.

Primary Dermal Irritation

DME was tested for acute skin irritation on three male New Zealand White rabbits according to OECD Guideline No. 404 and in compliance with OECD Good Laboratory Practice.

The test article, a liquid, was applied as su ch on a dry compress, in a single dermal

dose of 0.5 mL to a 6 cm 2 clipped area of the skin for four hours under a semiocclusive dressing. Cutaneous examinat ions were performed at 1, 24, 48, and 72 hours after removal of dressing.

No cutaneous reactions were observed in any of the animals, throughout the observation period. Therefore, DME is not considered a skin irritant.

Dermal Sensitization

DME was assessed for skin sensitization potential in female CBA/CaOlaHsd mice, using the Local Lymph Node Assay, acco rding to OECD Guidel ine No. 429 and in compliance with OECD Good Laboratory Practice.4

The test item was found soluble in the recommended vehicle acetone/olive oil (4/1, v/v) at the maximum concen tration of 50% (w/v), and was also used undiluted (100%) as supplied.

A preliminary test was conducted in two mice with the selected concentrations of 10, 25, and 50% (w/v) and 100% (undiluted) to a ssess potential local irritation. After 24 hours, no irritating effects were observed at these concentrations following a single application. The highest concentration retained for the main assay was therefore 100% (undiluted).

In the main assay, five treated groups of four animals each receive d the test item at the concentrations of 5, 10, 25, or 50% (w/v ) prepared individually in the vehicle acetone/olive oil (4/ 1, v/v), or 100% (undiluted). One negative control group of four animals received the vehicle alone [acetone/o live oil (4/1, v/v)]. The positive control was assessed in a recent independent stud y, in which groups of four animals

received the reference item α -Hexylcinnamaldehyde (HCA), a moderate sensitizer, at 5, 10, or 25% (w/v) in a cetone/olive oil (4/1, v/v).

During the induction phase, the test item , vehicle, or reference item was applied over the external surface of both ears (25 μl per ear) for three consecutive days (Day 1, 2, and 3). After two days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl-thymidine ( 3 H-TdR) on Day 6 following a single injection in the caudal vein of each animal of 250 μL of 77.01 μCi/ml 3 H-TdR (i.e., 19.3 μCi; specific activity of 2 Ci/mmol). The auricular lymph nodes were pooled for each experimental group. The measurement of dpm values were used to calculate stimulation indices [SI = (dpm treated group/dpm control group)].

No deaths occurred during the study period . No clinical signs were observed during the study. The body weights of the treated and control animals, recorded prior to the first application and prior to necropsy, were within the range commonly recorded for animals of the strain and age.

On the second application day (Day 2), a slig ht to moderate ear erythema (grade 1 or 2 on a scale of 4) was observed at both dosing sites in all mice of the two highest dose groups (50% and undiluted), persisting for a total of two days (Day 2 and 3). In addition, in those two groups, the size of the draining lymph nodes was obviously small compared to those of the control group.

No significant lymphoproliferation was obs erved with the test substance at any of the doses (SI remained ≤ 1.0 in all treated groups). In contrast, a dose response and a significant lymphoproliferation at two doses were observed with the reference

substance α -Hexylcinnamaldehyde. SI values were 2.4, 3.6, and 11.2 for concentrations of 5, 10, and 25% (w/v), respectively.5

Calculation of the EC3 value could not be performed for the test substance because no test concentration produced a stimulation index of 3 or higher.

Under these experimental conditions, DME was not found to induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.]

2. Genotoxicty. [ Bacterial Reverse Mutation Test

The mutagenic potential of DBE was assessed in the Salmonella typhimurium Ames

test.

Salmonella typhimurium strains TA1535, TA1537, TA 1538, TA98, and TA100 were tested in the presence and in the absence of metabolic activation system from liver fraction of rats (S9 mix) at doses up to 5000 μg/plate.

The test substance, DBE, did not induce any noteworthy increases in the number of

revertants which could be considered as relevan t, either with or without S9 mix, in any of the five tester strains.

In Vitro Mammalian Chromosome Aberration Test

The mutagenic potential of DBE was assessed in an in vitro mammalian chromosome aberration test according to OECD Guideline No. 473.

Primary human lymphocytes were used in the presence and in the absence of metabolic activation system from liver fraction of Ar oclor 1254-induced rats (S9 mix). The test article was suspended in DMSO. Cells were exposed to 0.00%, 0.04%, 0.10%, 0.3%, 0.4%, and 0.6 % (v/v), corresponding to 0.0, 0.44, 1.1, 3.3, 4.4, and 6.6 mg/mL, respectively (samples in duplicate: one male and one female donor). Forty four to 47 hours after culture initiation, the medium was replaced with treatment

medium and the cultures were incubated for three hours at 37°C and rinsed with Phosphate Buffered Saline (PBS). Fresh me dium containing 5-bromodeoxyuridine (BrdU) was then added and incubation continued for 24 to 26 hours, with

Colcemid ® (0.1 ug/mL) present during the fi nal two hours to arrest cells in metaphase. Cells were then harvested, s lides prepared, stained (Giemsa), and evaluated. An additional confirmatory scoring was carried out in a different laboratory. Results were statistically analyzed.

Negative and positive controls respo nded adequately. The study was therefore

considered valid.

Under non-activated conditions, DBE was cy totoxic at test concentrations above 0.3% (v/v). DBE was somewhat more cytotoxic under activated conditions with significant cell cycle del ay already at 0.3% (v/v).6

Under non-activated conditions, no st atistically significant increases or

concentration-related trends in chromoso me aberrations were observed in either

trial. Reduced mitotic indices at the hi gher DBE levels indicated that cytotoxic

concentrations had been achieved.

With S9 activation, statistically signif icant increases in the proportion of chromosomally-abnormal cells were seen at 0.3 and 0.4% DBE in Trial 1, and at 0.4% DBE in Trial 2. Additionally, the prop ortion of cells having more than one aberration was significantly in creased at the 0.4% level in Trial 1. In both trials, statistically significant concentration-related trends were seen in the proportion of abnormal cells (p < 0.001, Trials 1 and 2) and the proportion of cells with more than one aberration (p < 0.01, Trial 1; p < 0.05, Trial 2). Mitotic indices were markedly reduced at the higher test concentrations . Therefore, DBE exhibited clastogenic activity in human lymphocytes under S9 activated conditions.

In Vivo Mammalian Erythrocyte Micronucleus Test

The mutagenic potential of DBE wa s assessed in a mouse bone marrow

micronucleus test similar to OECD Guideline No. 474.

Fifteen male and 15 female Cr1:CD ® (ICR)BR mice per dose level were exposed nose-only to 0, 5.5, 11, or 19 mg/mL DBE in aerosol air once for six hours. Concurrent with the initiation of these expo sures, five male and five female mice were dosed by intraperiton eal injection with 20 mg/kg cyclophosphamide, the positive indicator. The mice were observed for clinical signs of toxicity during and immediately following exposure. Surviving mice were weighed and observed daily until sacrifice. Approximately 24, 48, and 72 ho urs after initiation of exposure, five mice per sex per dose level were sacrificed by CO 2 asphyxiation.

Immediately following sacrifice, three bone marrow smear slides per animal were prepared. One thousand polychromatic eryth rocytes (PCE) per animal were scored for the presence of micronuclei. The ratio of PCEs to normochr omatic erythrocytes (NCE) was determined for each animal based on 1,000 total erythrocytes.

While significant depression of the PCE to NCE ratios was seen in the 24-hour 11 mg/L and 19 mg/L DBE-treated females, there were no statistically significant differences in the proportion of micronuc leated PCEs between DBE-treated mice and the concurrent negative control gro ups at any sampling interval, and no statistically significant concentration-relat ed trends were present. In comparison, both the cyclophosphamide-treated males and females showed significant increases in micronucleated PCEs compared to the concurrent air controls. Under the conditions of this assay, DBE did not indu ce micronuclei in bone marrow of mice.7

In Vivo Mammalian Erythrocyte Micronucleus Test

The potential induction of micronuclei by DMG in bone marrow cells of the Fischer 344 rat was assessed according to OECD Guideline No. 474 (Mammalian Erythrocyte Micronucleus Test) a nd EPA Test Guideline OPPTS 870.5395 (Mammalian Erythrocyte Micronucleus Test).

Rats were treated for two six-hour periods of whole body inhalation exposure to the test substance at dose levels of 0.5, 1.0, and 2.0 mg/L expressed in terms of the weight of test substance per unit volume of chamber air. A positive control group was dosed orally, by gastric intubation , with cyclophosphamide at 20 mg/kg bodyweight.

Incidences of weight loss were recorded th roughout the test, but these were small and not considered to be significant. Clin ical signs for animals treated with DMG included partially closed eyes, smacking mout h, lethargy, piloerection, oily fur, and brown staining around snout and jaws. No ad verse clinical signs were noted for the negative control treated animals over the duration of the test.

Bone marrow smears were obtained from five male and five female animals in the negative control and each of the test subs tance groups 24 hours after completion of the second exposure period and from the positive control group 24 hours after dosing. One smear from each animal was e xamined for the presence of micronuclei in 2,000 immature erythrocytes. The prop ortion of immature erythrocytes was assessed by examination of at least 1,000 ery throcytes from each animal. A record of

the incidence of micronucleated matu re erythrocytes was also kept.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in rats treated with DMG. The positive control produced large, highly significant increa ses in the frequency of micronucleated immature erythrocytes.

In conclusion, DMG did not show any evid ence of causing chromosome damage or bone marrow cell toxicity when administered by whole body inhalation exposure in

this in vivo test procedure.

In Vivo Mammalian Chromosome Aberration Test

The mutagenic potential of adipic acid was assessed in an in vivo mammalian chromosome aberration test in Sprague-Dawley rats. In the acute tests, fifteen males per dose level were administered one dose of the test substance by oral gavage at dose levels of 0, 3.75, 37.5, or 375 mg/kg (Acu te Test I) and 0 or 5000 mg/kg (Acute Test II). In the subacute tests, five male s per dose level were administered five doses of the test substance 24 hours apart vi a oral gavage. The dose levels were 0, 3.75, 37.5, or 375 mg/kg (Subacute Test I) and 0 or 2,500 mg/kg (Subacute Test II). Rats8

which had been given triethylene melamine ( TEM) served as positive controls in the acute tests and rats given saline served as the negative controls for the acute and subacute tests. In the acute tests, anim als were sacrificed at 6, 24, or 48 hours after

administration of the test substance. In the subacute tests, animals were sacrificed

six hours after the last dose. Four hours a fter the last test subs tance administration (2 hours prior to sacrifice) each animal was given 4 mg/kg of Colcemid ®
intraperitoneally in order to arrest the bone marrow cells in C-mitosis. At sacrifice, the bone marrow from one femur per animal was collected and processed to slide. The chromosomes of each cell were counted and only 50 diploid metaphase spreads per animal were analyzed for chromoso mal aberrations. Mitotic indices were obtained by counting at least 500 cells.

In the Acute Test I, neither the varie ty nor the number of aberrations differed significantly from the negative controls. The expected severe chromosomal damage was observed in the positive control gro up. The mitotic indices were within normal limits. In the Subacute Test I, the 37.5 mg /kg level contained one cell with a reunion and one cell that was polyploid, and the 375 mg/kg level contained three cells with breaks and one fragment. These were consid ered to be within normal limits of historical controls. The negative contro l group and the 3.75 mg/kg group contained

no aberrations. In the Acute and Subacute Test II, neither the variety nor the number of aberrations differed significantl y from negative controls. Therefore, no clastogenic effects above normal were observed in rats in vivo following a single oral dose of adipic acid or five consecutive doses. ]

3. Reproductive and developmental toxicity. [ One-Generation Reproduction Toxicity - rat

The effects of DBE on fertility were asse ssed by the inhalation route in a onegeneration study (equivalent to an OECD Guideline No. 415 study) in rats, exposed to test concentrations of 0, 0.16, 0.4, or 1.0 mg/L for 14 weeks pre-breeding, and for 8 weeks through breeding, gestation and lact ation. No relevant effects occurred in mating performance, fertility, gestatio n duration, litter size, development or viability, and lactation performance up to th e highest concentration tested. A slight decrease in pup and parental weight was obse rved in the highest dose group. Gross pathology and organ weights in pups showed no noticeable changes. The NOEC for reproductive toxicity was therefore 1.0 mg/L.

Developmental Toxicity - rat

DBE were tested in a developmental toxicity study on Crl:CD ® BR rats after inhalation exposure using a protocol similar to OECD Guideline No. 414.

Pregnant Crl:CD ® BR rats (7-8 per group) were exposed to either 0.16, 0.4, or 1.0 mg/L DBE by inhalation for six hours/day from Days 7 through 16 of gestation in whole-body inhalation chambers. A contro l group of pregnant rats was exposed simultaneously to air only.9

Parental examinations included clinical a nd cage side observations, body weight determination, and determination of feed consumption. Gross pathologic examination was performed on Day 21G and included counting of the corpora lutea and determination of number and positi on of all live, dead, and resorbed conceptuses in the uterus. Liver and gravi d/empty uteri were weighed. All fetuses were weighed and examined for external al terations. About one-half of the live

fetuses were examined for visceral alteration s. All fetuses were sexed and examined

for alterations of the sk eleton after maceration.

A suppression of both food consumption and the rate of body weight gain were seen in the 0.4 and 1.0 mg/L groups during the firs t six days of exposure. Staining on the fur and perineal area was seen in ra ts exposed to 1.0 mg/L and liver weight decreases, although not statistically signif icant, occurred in the two high exposure groups. None of the reproductive paramete rs were altered in any of the groups and

no fetal effects were detected.

It was concluded that DBE exert no advers e effect on prenatal development in the rat following inhalation exposures as high as 1.0 mg/L during the period of organogenesis.

Developmental Toxicity - rabbit

DMG was administered by inhalation (who le-body, daily exposure, 6-hour/day) to groups of 22 mated Hra:(NZW)SPF rabbi ts. Animals were exposed on Gestation days 7 to 28. Target chamber concentrations were 0, 30, 100, 300, or 1,000 mg/m 3

. Maternal body weight, food consumption, and clinical observation data were recorded daily. Animals were euthanized on day 29 of gestation; dams were subjected to a gross external and intern al postmortem examin ation. Gravid uteri were removed, weighed, and dissected and the uterine contents were described. Each live fetus was weighed, sexed, and exam ined for external, visceral, and skeletal

alterations.

At 1,000 mg/m 3 , one animal was found dead on Gestation Day 13 and one animal

was sacrificed in extremis on Gestation Day 22

. Among survivors, there were compound-related reductions in maternal bo dy weight gains and food consumption and there were compound-related clinical ob servations (ocular discharge and wet fur). At 300 mg/m 3 , compound-related effects incl uded reduced maternal body weight gains and increased clinical obser vations (ocular discharge). There was no evidence of compound-related matern al toxicity at 30 or 100 mg/m 3

.

Under the conditions of the study, there was no evidence of developmental toxicity at any level tested. Thus, the NOEC for mate rnal toxicity was considered to be 100 mg/m 3 and the NOEC for developmental t oxicity was considered to be 1,000 mg/m 3

.10

Developmental Toxicity - rat

A developmental toxicity study was conducted with glutaric acid in CD ® rats. Twenty five mated females per dose leve l were given daily oral doses via gavage of the test substance at 0, 125, 400, or 1,300 mg /kg on Gestation Days 6-15. Animals were observed daily for signs of toxi city and body weights were recorded periodically during gestation. On Gestat ion Day 20, the females were sacrificed and examined grossly. Reproductive status wa s determined. Corpora lutea, implantation sites, number of fetuses (live and dead), and resorptions (early and late) were recorded. All fetuses were weighed and examin ed externally. Fetuses of 1/3 of the

litters were examined for visceral abnormalit ies. Fetuses in the remaining 2/3 of the

litters were examined for skeletal abnormalities.

No adverse effects were observed on body weight or clinical signs in rats at 125 mg/kg. At 400 mg/kg, no effect on body we ight was observed, but salivation, rales, and nasal discharge were observed. At 1,300 mg/kg, one animal died on Gestation Day 10 and one animal was sacrificed earl y on Gestation Day 13. Clinical signs observed at 1,300 mg/kg included salivation, ra les, nasal discharge, slight inactivity, labored breathing, decreased body temperatu re, soft stools, and staining around the mouth, nares, and anogenital area. Mean body weight gains were decreased at 1,300 mg/kg during the dosing period. The mean bo dy weight gains post-dosing (Gestation Days 15-20) were normal compared to control, indicating a reversible effect on body weight after test substance withdrawal.

No adverse effects on pregnancy and no teratogenic effects were observed at any level tested. A significant increase in th e number of resorptions at 1,300 mg/kg was observed compared to control. The num ber or resorptions was within normal expected limits; therefore, the increase was not considered biologically meaningful.

The maternal NOAEL and LOAEL were 125 and 400 mg/kg, respectively. The developmental NOAEL for this study was 1,300 mg/kg.

Developmental Toxicity - rabbit

A second developmental toxicity study wa s conducted with glutaric acid in New Zealand White rabbits. Eighteen mated fema les per dose level were given daily oral doses via gavage of the test substance at 0, 50, 160, or 500 mg/kg on Gestation Days 6-18. Clinical signs, body weights, and food consumption were recorded. On Gestation Day 29, the females were sacrificed and examined grossly. Reproductive status was determined. Corpora lutea, impl antation sites, number of fetuses (live and dead), and resorptions (early and la te) were recorded. All fetuses were weighed and given external, visceral, and skelet al examinations for abnormalities.

No test substance-related mortality was ob served. No changes in body weights or clinical signs were observed in females at any level tested. No adverse effects on pregnancy, and no embryotoxic or teratogeni c effects were observed. Therefore, the maternal and developmental NOAEL for this study was > 500 mg/kg.11

Developmental Toxicity - rat

A developmental toxicity study was conducted with adipic acid in Wistar rats. Virgin adult females were mated with young adult males, and observation of a vaginal sperm plug was considered Day 0 of ge station. Twenty females per dose level were given daily oral doses via gavage of the test substance at 0, 2.9, 13, 62, or 288 mg/kg on Gestation Days 6-15. Clinical sign s, body weights, and food consumption were recorded. On Gestation Day 20, all anim als were subjected to cesarean section, and the number of implantation sites, resorption sites, and live and dead fetuses were recorded. The urogenital tract of each fe male was examined in detail for gross anatomical normality. The body weights of the liver pups were recorded, and all fetuses were examined grossly for the presence of external congenital abnormalities.

Fetuses of 1/3 of the litters were examined for visceral abnormalities. Fetuses in the remaining 2/3 of the litters were exam ined for skeletal abnormalities.

The administration of up to 288 mg/kg of adipic acid to pregnant rats for 10 consecutive days had no effect on nidation , or on maternal or fetal survival. The

number of abnormalities seen in either soft or skeletal tissues of the test groups was similar to the number occurring spontaneou sly in controls. Therefore, the maternal and developmental NOAEL for this study was > 288 mg/kg.

Developmental Toxicity - rabbit

A second developmental toxicity study wa s conducted with adipic acid in Dutchbelted rabbits. On Day 0, each doe was given an injection of 0.4 mL of human chorionic gonadotropin. Three hours later, each doe was inseminated artificially with 0.3 mL of diluted semen from a proven donor buck. On Gestation Days 6-18, females were given daily oral doses via gavage of the test substance at 0, 2.5, 12, 54, or 250 mg/kg. Clinical signs, body weight s, and food consumption were recorded. On Gestation Day 29, the females w ere sacrificed and examined grossly. Reproductive status was determined. Corpor a lutea, implantation sites, number of fetuses (live and dead), and resorptions (early and late) were recorded. All fetuses were weighed and given external, viscera l, and skeletal examinations for

abnormalities.

Treatment of pregnant rabbits with up to 250 mg/kg of adipic acid had no significant effect on nidati on, or on maternal or fetal survival. The number of

abnormalities seen in either soft or skeletal tissues of the test groups were similar to the number occurring spontaneously in controls. The maternal and developmental NOAEL for this study was > 250 mg/kg .]

4. Subchronic toxicity. [ 14-day Oral – rat

DBE was tested in a 14-day or al toxicity study on Crl:CD rats. Groups of 10 male rats received feed containing DBE at con centrations of 0 (control), 10,000, 20,000, or 50,000 ppm for 14 consecutive days. All rats were weighed daily during the test period and observations for clinical signs were made at the same time. Food12

consumption for the test period was recorded . On the 14th test day, half of the rats of each test group were sacrificed. The remai ning half of each group were weighed every other day, observed for a 14-day recove ry period, and sacrificed 14 days later. All rats were examined grossl y, selected tissues were weig hed, and selected tissues and organs evaluated histologically.

Oral administration of DBE to rats over 14 days produced no deaths. No gross or microscopic pathologic changes that could be attributed to exposure of DBE were detected. No clinical signs other than init ial and sporadic weight loss were observed. The rate of body weight gain was reduced in rats of the mid- and high-dose group with lower food intake by these rats. Duri ng the recovery period, test rats in these two groups gained slightly more weight th an the controls although the high-dose rats weighed approximately 7% less at the end of the recovery period.

Except for the observed slight weight gain effects, under the conditions of this test, DBE appeared to exhibit a low toxi city in this 14-day feeding study.

14-day Dermal – rat

DBE was tested in a 14-day derm al toxicity study on Crl:CD ® (SD)IGS BR albino rats in accordance with OECD Guideline No. 410 and U.S. EPA (40 CFR Part 792) and OECD [C(97)186/Final] Good Laboratory Practice.

The test substance was applied seven days per week for two weeks to the shaved intact dorsal skin of each rat. The application sites were covered with gauze, occluded with plastic wrap and secured with non-irritating tape for a period of six hours per exposure. Following the exposure period, the application sites were gently washed with a solution of non-irritating soap and disposable paper towels to remove residual test article. Each test article gr oup consisted of 10 males and 10 females. Dosage levels were 100, 300, and 1,000 mg/kg/day. A concurrent control group received deionized water on a comparable regimen. The animals were examined twice daily for mortality and moribundity. Clinical and dermal observations were performed daily, prior to and following expo sure, and once each day of the recovery period. Detailed physical examinations w ere performed weekly. Ophthalmological examinations were performed prior to dosing and during study weeks 1 and 3. Clinical pathology evaluations (hematol ogy, serum chemistry, and urinalysis) were performed prior to the scheduled primary necropsy. Complete necropsies were performed for all animals and selected or gans weighed from animals at the primary and recovery necropsies. Select tissues were examined microscopically.

There were no test article or treatment-rel ated deaths or clinical observations following administration of DBE for 14 co nsecutive days. Minimal to mild dermal

irritation was observed for all treatment le vels near the end of the first week or during the second week of dosing. Scabbing of the treated skin was observed at necropsy for one male in each of the DBE treatment groups and dark red areas on

the treated skin was observed for one fe male in the 1,000 mg/kg/day DBE group;13

however, microscopic findings did not indica te a generalized reaction of the treated

skin to DBE. There were no other test ar ticle-related microscopic findings. Body weights and food consumption were unaffect ed by treatment with DBE. There were no test article-related changes in hematology, serum chemistry, urinalysis, ophthalmoscopy, neurobehavior, or organ we ight parameters. Based on these study results, the NOEL for systemic toxicity for dermal application of DBE for 14 consecutive days was considered to be 1,000 mg/kg/day.

90-day Inhalation – rat

Groups of 40 male and 40 female Crl:CD ® BR rats were exposed six hours/day, five days/week to 20, 76, or 390 mg/m 3 of DBE vapor. Ten males and 10 females per group were exposed to the test substance for seven weeks and 20 males and 20 females per group (19 males and 19 females in the 76 mg/m 3 group) were exposed to the test substance for 13 weeks. After the 13 week exposure phase of the study, 10 male and 10 female rats from each group underwent a 6-week recovery period. A similarly comprised control group was exposed to air only. Exposures were

conducted in 1.4 m 3 stainless steel and glass NYU-styl e inhalation chambers with air flows of approximately 300 liters per minute.

Rats were weighed weekly throughout the test period and observed daily for clinical signs of toxicity. Animals were sacrificed after 7 weeks (10 anim als/sex/group) or 13 weeks of treatment (20 an imals/sex/group). The remai ning 10 animals/sex/group were sacrificed after undergoing a six week recovery period. At sacrifice, samples for serum chemistry, hematology, and urinalysis were collected. A gross pathological exam was conducted and the hear t, kidneys, liver, lungs, spleen, brain, thymus, and testis were weighed. The nasal cavities from all rats were examined microscopically.

Degeneration of the olfactory epithelium was observed in male and female rats that were exposed to 76 or 390 mg/m 3 of DBE for seven weeks. One control female showed minimal degeneration of the olfact ory epithelium after 7 weeks. After 13 weeks of exposure, there was degeneration of the olfactory epithelium in female rats in all exposure groups although minimal at the lowest dose and in male rats exposed to 76 or 390 mg/m 3 . Minimal degeneration of the olfactory epithelium was also observed in one female from the control gr oup after the recovery period. At the end of the 6-week recovery period, nasal effects in DBE-exposed rats included disorganization of the olfactory epitheliu m, decreased numbers of neuronal cells, and respiratory metaplasia. These effe cts are related to tissue repair and regenerative processes and were noted at all DBE exposure concentrations in the male and female recovery rats. Clinical si gns in DBE-exposed rats were similar to those of the controls; however, female rats exposed to 390 mg/m 3 of DBE had slightly depressed rates of weight gain compared to controls during the exposure period. During the recovery period, the weigh t-gain rate of these rats was similar to those of the controls. Liver weights in female rats exposed to 390 mg/m 3 were decreased compared to those of the controls. This effect was not present in rats at14

the end of the recovery period. A slight dec rease in serum sodium concentration was observed in male and female rats exposed to 76 or 390 mg/m 3 DBE for 13 weeks. After the 6-week recovery period, so dium concentrations in the 390 mg/m 3 group males and females remained slightly low, but the sodium concentrations in the other affected groups were no longer different from those of the control group. The NOEL in male rats exposed to DBE was 20 mg/m 3 . A NOEL was not establ ished in females.

90-day Inhalation – rat

A 90-day inhalation toxicity study on DM S, DMG, and DMA was performed in the rat under conditions equivalent to OECD No. Guideline 413 and under U.S. EPA (40 CFR Part 792) and OECD [ENV/MC/CHEM(98)17] Good Laboratory Practice.

Six groups of male and six groups of female Crl:CD ® (SD)IGS BR rats each were exposed via inhalation to 0, 10, 50, or 400 mg/m 3 DMG, 400 mg/m 3 DMS, or 400 mg/m 3 DMA six hours per day, five days a week over a 90-day period. The exposure period was followed by an approximately 1-month recovery period. Rats were weighed once per week and clinical signs were taken prior to exposure on days the rats were weighed. Group clinical signs were observed during each exposure, and the alerting response to an auditory stim ulus was checked during each exposure in

rats visible from the front of the chamber. After each exposure, rats were checked for the alerting response to an auditory st imulus prior to rem oval from chambers and were observed for clinical signs imme diately as they were returned to their cages. Food consumption was determined on a weekly basis. Samples for hepatic, lung, and nasal (levels II and III) cell pro liferation (CP) were collected from five rats/sex/group approximately two weeks after initiation of the study and approximately 90 days after study initiati on. A clinical pathology evaluation was conducted on 10 rats/sex/group designated for subchronic toxicity evaluation approximately 45 and 90 days after initiati on of the study. Approximately 90 days after study initiation, rats designated fo r the clinical pathology evaluation were sacrificed for pathological examination and evaluation of male reproductive endpoints, including sperm motility, sperm number, and sperm morphology. A neurobehavioral test battery, consisting of functional observational battery assessments and motor activity, was conduc ted on 10 rats/sex/group designated for

behavioral evaluation and rec overy prior to test substance administration to obtain baseline measurements, and during test weeks 4, 8, 13, and 18 (recovery). Approximately 90 days after study initia tion, six rats/sex/group designated for neuropathology were sacrificed and evaluated. After approximately one month of recovery, rats designated for neuropathologi cal evaluation at this time point were sacrificed for evaluation of this endpoint. The estrous cycle of female rats was determined for the last 21 days of expo sure in rats designated for subchronic toxicity evaluation. Following 90 days of exposure, blood was collected via the tail vein from 10 rats/sex/group and serum was subjected to hormonal analyses. In male rats, serum luteinizing hormon e, follicle stimulating hormone, and

testosterone concentrations were measured . In female rats, serum estradiol and progesterone concentrations were measured.15

No compound-related effects were observed on mortality, clinical signs, hematology, neurobehavioral endpoints, sperm motility or morpho logy, or estrous cycle parameters.

Male rats exposed to nominal 400 mg/m 3 DMG or 400 mg/m 3 DMA had lower mean body weights and mean body weight gains during the study. In addition, male and female rats exposed to 400 mg/m 3 DMG had lower food consumption, and male rats exposed to 400 mg/m 3 DMA had lower food efficiency during the study. Compoundrelated effects were observed in the noses of male and female rats exposed to 400 mg/m 3 of DMG, DMS, or DMA for 90 days. Th ese effects consisted primarily of degeneration/atrophy of the olfactory muco sa of the dorsal meatus and of the dorsomedial aspect of the dorsal endoturbinate. Less commonly, focal respiratory metaplasia of the olfactory mu cosa of the dorsal meatus was also present. Lesions were minimal to mild in severity and o ccurred in higher incidences in the DMG group. Degeneration/atrophy of the olfa ctory mucosa occurred in recovery animals in the same locations as was apparent at the 90-day sacrifice in animals exposed to DMG, DMS, and DMA. The lesions were usually focal and minimal in severity. The incidence of lesions in female recovery groups was higher in the DMG and DMS groups compared to the DMA group, while in male recovery groups, incidences were somewhat higher for the DMA group.

Male rats exposed to 400 mg/m 3 DMS and DMA showed significant increased CP in the liver at day 14 compared to controls. No significant effects were observed in the liver from males evaluated at 90 days or from females evaluated at either time point. Female rats exposed to 400 mg/m 3 DMA had significantly g reater CP in the lung relative to controls at days 14 and 90. No effects on CP were observed in the lung from male rats. These liver and lung findings were considered not to bear any toxicological relevance.

Male rats exposed to 400 mg/m 3 DMG and DMA showed significantly greater CP in the nose level II compared to controls at day 90. Female rats exposed at 400 mg/m 3
DMG had significantly greater CP in the no se level II at day 14. Male rats exposed to 400 mg/m 3 DMG had significantly greater CP in the nose level III at day 90. CP in the nose level III of female rats exposed to 400 mg/m 3 DMG was significantly greater than controls on day 14. Female rats exposed at 400 mg/m 3 DMS had significantly greater CP in the nose le vel III compared to controls on day 90.

In male rats exposed to DMG, serum testosterone concentrations were statistically significantly decreased at concentrations of 50 and 400 mg/m 3 (59 and 50% of control, respectively). Similarly, serum LH concentrations were decreased in a dosedependent manner and were statistically significantly decreased at 400 mg/m 3 (71% of control). Serum concentrations of FS H were not affected by DMG treatment. In female rats, DMG exposure did not alter serum estradiol or progesterone concentrations. In male rats exposed to DMS or DMA, no significant alterations in serum hormone concentration were observed. In female rats, DMS caused a16

statistically significant decrea se in serum estradiol concen trations (43% of control); serum progesterone concentrations were no t affected. In female rats, DMA exposure

did not alter serum estradiol or progesterone concentrations.

There was a treatment-related increase in epididymal sperm counts (per cauda epididymis and per gram cauda epididym is) following exposure to DMG and the number of sperm per cauda and per gram cauda epididymis was significantly increased at 50 and 400 mg/m 3 (124-131% of control). Epididymal sperm counts were similar to control at 10 mg/m 3 DMG. In male rats exposed to DMS, epididymal sperm counts (per cauda epididymis and per gram cauda epididymis) were significantly increased (153 and 141% of control, respectively). Although not statistically significant, a similar trend of increased epididymal sperm counts was observed in the DMA group (124 and 114% of control for sperm per cauda and per gram cauda epididymis, respectively) that was considered compound-related.

In conclusion, under the conditions of this study, the NOEC for systemic toxicity following repeated exposure to DMG was 10 mg/m 3 , based on the decreases in

serum testosterone and serum LH concen trations and increased epididymal sperm counts at concentrations of 50 mg/m 3 and above. NOECs for DMS and DMA were not established, as there were effects in the only concentration (i.e., nominal 400 mg/m 3 ) of these materials tested. The NOEC for respiratory local toxicity was 50 mg/m 3 DMG, based on olfactory mucosa degen eration and/or atrophy at the highest concentration of 400 mg/m 3

.

90-day Oral – rat

A subchronic oral feeding study was conducted with dicarboxylic acids according to OECD Guideline No. 408. The dicarboxylic acid mixture tested was composed of 4% adipic acid, 16% glutaric acid, 5% su ccinic acid, and up to 4% nitric acid at dose levels of 0, 300, 1,000, and 3,000 mg/kg. Fifteen male and fifteen female Sprague Dawley rats were assigned to each dose level and were administered vehicle (deionized water) or test substance da ily by oral gavage for 90 days. Animals were observed daily, body weight and food consumption were recorded weekly. After 13 weeks of treatment, hematology, chemistry, and urinalysis parameters were analyzed for 10 rats/sex/group. Ophthalmoscopic exams were also conducted on all animals at 13 weeks. At termination, a ll animals were sacrificed and necropsied. Select organ weights and tissues were co llected. Microscopic examination was performed for tissues collected on a ll high dose and control animals.

At the high dose level (3,000 mg/kg), treatme nt related deaths were observed in 2/5

males and 1/15 females. Animals at this dose level also had an increased incidence of labored breathing and rales. Reduced body weight was noted in males and females, and statistically significant reductions in food consumption were noted in males only at 3,000 mg/kg. Body weight gain was slight ly reduced only in females (not statistically significant) and statistically significant reduced food consumption was noted only in males at 1,000 mg/kg. Statis tically significant increased leukocytes17

were noted in males and reduced urine pH in males and females at 3,000 mg/kg and reduced urine pH in males only at 1,000 mg /kg. No effects were noted in animals at 300 mg/kg. Organ weights and tissues examin ed microscopically were normal in all groups. In conclusion, the No Observ ed Adverse Effect Level (NOAEL) for administration of dicarboxylic acids daily for 13 weeks to rats was 300 mg/kg and

the Lowest Observed Adverse Effect Level (LOAEL) was 1,000 mg/kg).

90-day Oral – rat

A subchronic oral feeding study simila r to OECD Guideline No. 408 was conducted with glutaric acid. Fifteen male and fi fteen Sprague-Dawley rats per dose group were exposed to 0, 0.5, 1.0, or 2.0% glutaric acid in feed for 90 days. Mortality, clinical observations, and body weights were recorded. Food consumption was

calculated for five males and five female s per group. Hematology, blood chemistry, and urinalysis parameters were evaluated fo r five males and five females from the high dose group (2%) and control group (0%) at Days 45 and 84. At sacrifice, animals were examined grossly and organ weights were collected. Hematoxylin and

eosin fixed tissues were examined microsc opically from 10 males and 10 females from the high dose group (2 %) and control (0%) group.

No treatment-related mortality was no ted and food consumption was normal. Statistically significant reduced body weight gain was observed in females at the 2%

dose level. Males at 2% also had reduced bo dy weight gain although not statistically significant. No statistica lly significant differences were noted in hematology, blood chemistry, and urinalysis results. There were no histopathological findings or organ weight changes attributed to the test s ubstance. Therefore, the NOAEL for glutaric

acid was ≥ 1% and the LOAEL was 2%.

90-day Oral – rat

A thirteen week repeated dose study was conducted with monosodium succinate in Fischer F344 rats. The test substance was dissolved in distilled water, and ten male and ten female rats per dose level were administered the test substance at 0.3, 0.6, 1.25, 2.5, 5, or 10% ad libitum in their drinking water for 13 weeks. Rats were observed daily for mortality and clinical signs. Body weight was measured every other week. After 13 weeks of treatment, all surviving rats were euthanized and necropsied. Animals were examined gr ossly and tissues were collected for microscopic examination. Sa mples for hematology and clinical chemistry were also

collected at termination.

All of the rats in the 10% group died during the first four weeks of the study, following a severe suppression of body weight gain. No fatalities were observed in the other dose groups until termination. Suppression of body weight gain was

observed at ≥ 2.5%. The volume of drinking wat er consumed was very small in the highest dose groups, although it was la rger in the 5% group than in the other groups. No dose-related effects were observed in the clinical pathology evaluation.18

Rats that died during the study were seve rely emaciated. However, test articlerelated lesions were not noted in any of the tissues examined microscopically, although atrophy was observed. On the basis of body weight depression, the

maximum tolerated dose of monosodium succinate was determined to be approximately 2-2.5% when admini stered in the drinking water.

90-day Oral – dog

A subchronic oral feeding study was conducted with glutaric acid in beagle dogs. Four male and four female dogs per dose level were administered the test substance daily in feed for 90 days at dose levels of 0, 1, 3, and 5% (Days 1-10) then at 0, 0.5, 1, and 2% (Days 11-90). Dogs were observed for mo rtality, clinical signs, body weights and food consumption. Samples for hemato logy, blood chemistry, and urinalysis were collected at Days 45 and 85. At terminal sacrifice, organ weights were recorded

and tissues were collected for microscopic examination.

Body weight loss was observed in males and females at the 5% and 3% dose levels, respectively, after 10 days. Reduced food cons umption was also observed in parallel. Overall, the low- and mid- dose animals ha d weight gains comparable to controls. Five of the eight high dose dogs did no t gain weight. No other treatment-related effects were noted. Therefore, the NOAEL was ≥ 2% and the LOAEL was 3% .]

5. Chronic toxicity. [ Carcinogenicity

No experimental carcinogenicity study is available on DME. The two principal chemical structures present in DME, th at is, DMG and DMS, were assessed using the knowledge-based expert system DE REK for Windows (software version DfW 9.0.0,14_08_2006). The expert system DEREK has been designed for the qualitative prediction of the possible toxicity of ch emicals and the following endpoints were searched for this structure: carcinogenici ty, genotoxicity, HERG channel inhibition, irritation, respiratory sensitization, sk in sensitization, thyroid toxicity, and miscellaneous endpoints. The toxicity asse ssments are based on the presence of specific substructures.

The structures, DMG and DMS, did not tr igger any alerts with any degree of likelihood in the expert system DEREK. Furt her, the dimethyl esters are expected to undergo enzymatic hydrolysis upon inge stion by ubiquitous carboxylesterases (Satoh et al., 2002 and Hosokawa, 2008) to form the corresponding dicarboxylic acids and methanol. No specific findings w ere detected in the repeated dose toxicity studies available by the oral route on the dicarboxylic acids, or in the chronic/carcinogenicity studies conducte d with the monosodium succinate and adipic acid.

Finally, considering the absence of finding s in the genotoxicity assays available in

vitro and in vivo on DME or on the individual dies ters, there is a low concern for a carcinogenicity potential that would be relevant to the foreseen uses.19

Chronic Oral Toxicity/Carcinogenicity – rat

A two-year carcinogenicity study was co nducted with monosodium succinate in Fischer F344 rats. Fifty males and fifty females per dose group were administered

the test substance ad libitum in their drinking water at do se levels of 0, 1, or 2%. The solutions were replaced with freshly prepared solution three times per week and the

amount of solution consumed was measured to calculate the intake of the test substance. After 104 weeks of treatment, the rats underwent a recovery period in which they were administered distilled wa ter for 9 weeks. Animals were observed daily for clinical signs and mortality. Body weight was recorded weekly for the first 13 weeks and then once every four weeks thereafter. Rats that died or were euthanized during the study underwent a necropsy that included macroscopic

examination and collection of tissues for mi croscopic examination. At week 113, all surviving rats were euthanized and necro psied. A macroscopic examination was

conducted and tissues were collected for microscopic examination.

The intake of monosodium succinate was 0, 196, and 437 mg/rat/day at 0, 1, and 2%, respectively, for males and 0, 146, and 309 mg/rat/day at 0, 1, and 2%, respectively, for females.

In males, the mortality rate in the contro l group was slightly higher than that in the other two groups throughout the ex periment. A dose-dependent decrease in growth rate was apparent in both sexes. In males, the overall tumor incidence was almost 100% in all groups, including the control group. In females, it was approximately 77-82% in all three gr oups. In both sexes, there were no statistically significant differences betw een the control and treated groups in

overall tumor incidences and mean survival times.

Tumors were found in many organs or tissues in all groups including the controls. In males of all groups, tumors of the te stes were the most frequent, followed by those of the hematopoietic organs, thyroi d, adrenals, mammary, prostate, pancreas, and pituitary. Tumors of the uterus, pituitary, hematopoietic organs, mammary gland, thyroid, and adrenals were the most common in females. Tumors were also detected in other organs or tissues, but th e incidences were very low. None of the treated groups showed a significant increa se in the incidence of any tumors over that in the corresponding controls, while th e incidence of endometrial stromal polyp in the females given the 2% dose was sign ificantly lower than that in the control group. The incidence of C-cell adenoma/ca rcinoma of the thyroid in the females given 2% was higher than that in the contro ls, although marginally not significant, and a positive trend was noted in the occurrence of this tumor by an age-adjusted statistical test. Histologicall y, all tumors, except prostate tumors, observed in this study were similar to those that are known to occur spontaneously in this strain of

rats. Prostate tumors were observed in all male groups, including the control group, at incidences much higher than those repor ted by others. Histol ogically, the prostate

tumors were all intraductal adenomas/carci nomas. In addition to these tumors, many kinds of non-neoplastic lesions, such as myocardial fibrosis, bile duct20

proliferation, and chronic nephropathy were observed in all groups including controls, and no other specific lesions were de tected in any treated groups of either sex. Therefore, it was concluded that monosodium succinate had no carcinogenic activity in F344 rats when administered continuously in the drinking water for 2

years .

Chronic Oral Toxicity/Carcinogenicity – rat

A two year chronic feeding study was conduc ted with adipic acid in rats. Twenty male and nineteen females per dose group were fed either the basal laboratory diet or basal diet with adipic acid added at concentrations of 0, 0.1, 1.0, 3.0, or 5.0% (males) and 0 or 1.0% (females) ad libitum for two years. Body weight, food consumption, and general appearance were recorded weekly. Whenever possible, animals that died were examined, and gr oss pathology was performed. After two years, surviving rats were euthanized, se lect organs were weighed, and tissues were taken for microscopic examination. Rats were also examined for the incidence of

tumors.

The average test substance consumed per rat per day was 1.69, 174.88, 506.07, and 813.62 mg for the 0.1, 1.0, 3.0, and 5.0% dose groups, respectively. There were no significant differences in body weight gains in females and in males treated with 0.1 or 1.0% adipic acid. During the rapid grow th period, the weight gains of males in the 3.0 and 5.0% dose groups were signif icantly less than the control groups. Throughout the latter half of the st udy, the average body weights were not remarkable, although the 5.0% dose group was consistently the lowest. There were no significant differences in food consum ption for females. There was a slight, but consistent, reduction in food consumptio n in males of the 5.0% dose group. The following clinical signs were observed among males in all groups, including controls: wheezing, blood-tinged crust about the noses and eyes, and body sores. The incidence of these findings did not appe ar to be significantly different among the groups although a lower incidence of signs indicative of respiratory infection and body sores occurred in the 5.0% adipic acid group. Clinical signs noted in females of the control and test article gr oups included blood-tinged crust about the eyes and noses, unthriftiness, and body sores. The incidence of lung pathology and tumor growth appeared to be equally distributed among all groups, including the controls. There were no significant diff erences in organ weights or gross or microscopic pathology. Soft edematous testes were noted at least as frequently in males of the control group as in those in the adipic acid treatment groups. Two of the surviving female control rats and one experimental animal had ovarian tumors; ovarian cysts were noted in both control and experimental rats. Therefore, it can be

concluded that a NOAEL for males and females is 1% .]

6. Animal metabolism. [NA-Remove.]21

7. Metabolite toxicology

. [ Dimethyl esters are hydrolyzed by carboxylesterases to form naturally occurring dicarboxylic acids (glutaric acid, succinic acid, and adipic acid). Mammalian carboxylesterases ar e expressed in a number of tissues in human adults and in rats; therefore, the dime thyl esters are likely to be hydrolyzed to the respective dicarboxylic acids in tissu es such as the gastrointestinal tract and liver. For that reason, the toxicological pr operties of these metabolites were also considered and presented in the toleran ce exemption petition to support the toxicological profile of DME .]

8. Endocrine disruption

. [There is no available evid ence demonstrating that DME is an endocrine disruptor in humans.]

C. Aggregate Exposure

1. Dietary exposure. [ In examining aggregate exposure, section 408 of the

Federal Food, Drug, and Cosmetic Act (FF DCA) directs EPA to consider available information concerning exposures from the pesticide residue in food and all other nonoccupational exposures, including drinki ng water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses).

It is anticipated that DME will be used as a solvent/co-solvent in EC formulations at a concentration no higher than 40%. DME is not expected to be persistent in the

environment based on a PBT assessment of its components, DMG, DMS, and DMA and of its primary metabolites, glutaric acid , succinic acid, and adipic acid. Further, DME has a low toxicity profile as demo nstrated through acute, subchronic, reproductive, developmental, and mutagenicity testing. Therefore, dietary exposure

to DME should not be of concern.

DME will not be applied directly to water. Agricultural practices minimize spray drift and typical end-use product labels proh ibit direct application to water. DME is readily biodegradable and is not expected to be persistent in the environment. Therefore, use of DME as a solvent/co-solve nt in EC formulations does not pose a risk to drinking water.

The intended use of DME is as a solven t/co-solvent in agricultural pesticide formulations applied to food/feed crops. Therefore, exposure to nonoccupational use of DME in gardens, lawns, or buildings ( residential and other indoor uses) is not expected to occur.

Overall, DME is considered to be a low toxicity substance with a reasonable certainty of no harm from dietary exposu re and all other nonoccupational sources of exposure. ]

i. Food. [NA-Remove.]

ii. Drinking water. [NA-Remove.]22

2. Non-dietary exposure

. [NA-Remove.]

D. Cumulative Effects

[ Section 408(b)(2)(D)(v) of the FF DCA requires that, when considering whether to establish, modify, or revoke a tolerance, the Agency consider available information concerning the cumu lative effects of the partic ular pesticide’s residues

and other substances that have a common mechanism of toxicity.

Rhodia has not found DME to share a common mechanism of toxicity with any other substances, and DME does not appear to produce a toxic metabolite produced by other substances.

Resultant risks, separately and/or combined with other substances that may have a common mechanism of toxicity, should be low for DME. Rhodia does not expect any

adverse cumulative effects associated with the use of DME. ]

E. Safety Determination

1. U.S. population. [ DME exhibits a low toxicity pr ofile, as demonstrated by the acute, subchronic, chronic/carcinoge nicity, reproductive, and developmental

studies summarized above. In a chromosome aberration test, DBE was positive at high concentrations in the presence of S9 metabolic activation; however, all other in

vitro and in vivo genotoxicity studies conducted wi th DBE and DMG were negative. It is possible that in the presence of S9 metabolic activation, the esters were hydrolyzed and the dicarboxylic acids releas ed, making the pH more acidic. This is known to cause false positive effects in cytogenicity assays.

When DME is used on raw agricultura l commodities in accordance with good agricultural practice, it is expected to me et EPA’s reasonable certainty of no harm requirement. Based on the low toxicity prof ile of DME, there is no reason to believe that adverse effects on the U.S. populatio n will result from the use of this inert ingredient in pesticide formulations.

2. Infants and children

. [ DME exhibits a low toxicity profile, as demonstrated by the acute, subchronic, chronic/carcinoge nicity, reproductive, and developmental

studies summarized above. While some ho rmonal variations were observed in the 90-day inhalation toxicity study with DM G, there were no compound-related effects on sperm motility or morphology, or estrou s cycle parameters in that study, and no specific alerts were identified in the reproductive (one-generation) and developmental toxicity studies with D BE by the inhalation route or with dicarboxylic acids by the oral route. There was no specific neurotoxicity study available, but no neurobehavioral effects were observed in the 90-day repeat dose toxicity study conducted with DMG, DMS, and DMA.23

When DME is used on raw agricultura l commodities in accordance with good agricultural practice, it is expected to me et EPA’s reasonable certainty of no harm requirement. T here is no reason to believe th at infants and children will be disproportionately at risk to the use of DME in pesticide formulations .]

F. International Tolerances

[ There are no known international tolera nces or tolerance exemptions for

DME .]