Triazole acetic acid. The acute toxicity of triazole acetic acid is also low, based on an acute oral toxicity study in the rat ( Category IV).

Table 11.1. Acute Toxicity Profile for Triazole Alanine Test Material [% ai] Guideline Number Study Type MRID Number Results Toxicity Category Triazole alanine [ not specified] 870.1100 Acute oral  rat 00138119 ( TXR 004766 Acc. 252132) 00133356 LD50 (   +     ) > 2000 mg/ kg Unacceptable/ supplementary III Triazole alanine [" analytically pure"] 870.1100 Acute oral  rat 00138120 ( TXR 004766 Acc. 252132) 00133357 LD50 (   +     ) > 5000 mg/ kg Acceptable/ guideline IV 48 of 94 Table 11.1. Acute Toxicity Profile for Triazole Alanine Test Material [% ai] Guideline Number Study Type MRID Number Results Toxicity Category Triazole alanine [" analytically pure"] 870.1100 Acute oral  mouse 00138120 ( TXR 004766 Acc. 252132) 00133357 LD50 (   +     ) > 5000 mg/ kg Acceptable/ guideline IV Triazole alanine [ 99%] 870.1100 Acute oral  dog 00138130 ( TXR 004766 Acc. 252132) Both dogs vomited a portion of the test material ( 5000 mg/ kg) within 4 hours of dosing. No other signs were observed in the male. Slight salivation, slightly unsteady gait, and decreased food consumption and body weight were seen in the female.

Unacceptable Not determined Triazole alanine [" analytical pure"] Non GDLN Acute intraperitoneal  rat 00138120 ( TXR 004766 Acc. 252132) 00133357 LD50 (   +     ) > 5000 mg/ kg Acceptable/ non guideline N/ A Triazole alanine 870.1200 Acute dermal  rat ND ND ND Triazole alanine 870.1300 Acute inhalation  rat ND ND ND Triazole alanine 870.2400 Acute eye irritation  rabbit ND ND ND Triazole alanine 870.2500 Acute dermal irritation  rabbit ND ND ND Triazole alanine 870.2600 Skin sensitization  guinea pig ND ND ND ND = Study not done Table 11.2. Acute Toxicity Profile for Triazole Acetic Acid Test Material [% ai] Guideline Number Study Type MRID Number Results Toxicity Category Triazole acetic acid [> 99%] 870.1100 Acute oral  rat 45596802 LD50 (   +   ) > 5000 mg/ kg IV Triazole acetic acid 870.1200 Acute dermal  rat ND ND ND Triazole acetic acid 870.1300 Acute inhalation  rat ND ND ND Triazole acetic acid 870.2400 Acute eye irritation  rabbit ND ND ND Triazole acetic acid 870.2500 Acute dermal irritation  rabbit ND ND ND Triazole acetic acid 870.2600 Skin sensitization  guinea pig ND ND ND ND = Study not done <IP_ADDRESS> Subchronic, Chronic, and Other Toxicity Triazole alanine. The toxicity of triazole alanine in the rat was examined in 14, 28, and 90 day oral toxicity studies, a developmental toxicity study, and a two generation reproduction study. Of these five studies, adverse effects in adults were only observed in the 90 day feeding study. 49 of 94 These effects included decreased leukocytes in males and decreased triglycerides at the LOAEL ( 370/ 400 mg/ kg/ day, males/ females), with decreased body weight and body weight gain ( males), decreased leukocytes ( both sexes), and decreased triglycerides ( both sexes) observed at 1510/ 1680 mg/ kg/ day ( males/ females).

No treatment related effects were seen in the 14 day drinking water study, in which male rats were exposed to triazole alanine at dose levels up to ~ 1500 mg/ kg/ day. Because hematological and clinical chemistry parameters ( including those decreased in the 90 day study) were not measured in this study, it is unknown if they would be affected by two weeks exposure to triazole alanine. The decreases in body weight and body weight gain seen at the high dose in the 90 day rat study were not observed until week 3, so it is not unexpected that body weight measurements remained comparable to controls in this two week study.

No treatment related effects were seen in the 28 day oral toxicity study, in which rats were exposed to triazole alanine via oral gavage at dose levels up to 400 mg/ kg/ day. This study was classified unacceptable due to the lack of analytical data on the test compound and incomplete hematological, clinical chemistry, and histopathological analyses. Leukocyte counts, which were measured in this study, were unaffected following 28 days exposure to triazole alanine by gavage ( vehicle = Cremophor EL), although they were slightly decreased after one month of dietary exposure in the 90 day feeding study. Triglycerides, which were decreased in the 90 day study, were not evaluated in this study. The decreased body weights and body weight gains observed on week 3 of the 90 day study were not seen in this study, which is not unexpected given that the highest dose tested in the 28 day study was just 400 mg/ kg/ day vs. 1510 mg/ kg/ day in the 90 day study.

Systemic maternal/ paternal toxicity was not seen in the developmental toxicity or two generation reproduction studies in rats at dose levels up to ~ 1000 mg/ kg/ day. Reproductive toxicity was also not noted. Because hematology and clinical chemistry are not examined in developmental or reproduction studies, it is unknown if they were affected as in the 90 day feeding study. Changes in body weights observed in the 90 day study were not seen in either of these studies; however, the highest dose levels tested in the developmental and reproduction studies were lower than those which produced body weight effects in the 90 day study.

Differences in toxicity due to increased duration of exposure to triazole alanine could not be confirmed since treatment related effects were only seen in the 90 day study and since hematological and clinical chemistry parameters were not measured in the shorter duration studies.

Differences between routes of exposure to triazole alanine could not be examined because dermal and inhalation studies were not available.

Differences in the toxicity of triazole alanine across mammalian species were examined by comparing the 90 day feeding studies in the rat and the dog. ( Studies using the mouse or rabbit were not available. The lack of a rabbit study is a particularly important data gap because the rabbit is the most sensitive species to free triazole. Mortality and clinical signs of neurotoxicity were seen in adult rabbits following a single gavage dose of 45 mg/ kg 1,2,4 triazole in a rabbit 50 of 94 developmental study [ MRID 46492903]). As stated previously, treatment related effects in the 90 day rat study include decreased leukocytes ( males) and triglycerides ( females) at the LOAEL ( 370/ 400 mg/ kg/ day in males/ females), as well as decreased leukocytes and triglycerides in both sexes and decreased body weights and body weight gains in males at the highest dose tested ( 1510/ 1680 mg/ kg/ day in males). Triazole alanine is less toxic in dogs. The only treatmentrelated effect seen in the 90 day dog study was a 10% decrease in female food consumption near the limit dose ( 902 mg/ kg/ day), with no effects seen in males.

Increased quantitative and qualitative susceptibility of the offspring was seen in the developmental toxicity rat study, and increased quantitative susceptibility was seen in the twogeneration reproduction study. In the developmental toxicity study, increased incidences of skeletal findings were seen in the offspring at the mid and high doses, while no treatment related effects were seen in the dams up to the limit dose. These skeletal findings include unossified odontoid processes at 300 and 1000 mg/ kg/ day, with partially ossified transverse processes of the 7th cervical vertebra ( bilateral), unossified 5th sternebra, and partially ossified 13th thoracic centrum observed only at 1000 mg/ kg/ day. In the reproduction study, mean litter weights were decreased approximately 10 20% for both generations at ~ 1000 mg/ kg/ day, compared to controls, but parental body weights were unaffected at all treatment levels.

No mutagenic potential was noted in the following acceptable mutagenicity studies: 1. bacterial reverse mutation assays, 2. in vitro mammalian cell gene mutation tests conducted in BALB/ 3T3 and CHO cells, 3. mammalian erythrocyte micronucleus tests conducted using mice and Chinese hamsters, and 4. bacterial DNA damage and repair tests. An increased number of transformed colonies was seen in an in vitro mammalian cell gene mutation test conducted in baby hamster kidney cells ( MRID 00132914); however, this study was classified unacceptable/ inconclusive due to severe toxicity, excessive concentrations, and lack of information regarding test material purity. Mutagenic potential was also seen in a mammalian erythrocyte micronucleus test conducted using mice ( MRID 0013912); however, this study was classified unacceptable/ inconclusive because effects were seen only at 24 hours and not at 48 or 72 hours. Finally, triazole alanine inhibited protein biosynthesis in microorganisms ( TXR 005841); however, this study was classifed unacceptable due to inconsistent results, a lack of individual data, not enough dose levels tested, and no explanation of incubation times used.

Triazole acetic acid. In a 14 day toxicity feeding study in rats, no treatment related effects were observed up to the highest dose tested ( 788/ 703 mg/ kg/ day in males/ females). No other studies are available to determine the target organs and critical effects of triazole acetic acid; to compare toxicity across species, routes of exposure, or duration of exposure; or to examine potential developmental, reproductive, or neurological toxicity. This short term study is comparable to the 14 day oral triazole alanine study, in which no effects were seen in male rats up to 1491 mg/ kg/ day, and to the 28 day oral triazole alanine study, in which no effects were seen in male or female rats up to 400 mg/ kg/ day. However, since no effects were seen in the available shortterm studies, a difference in toxicity between triazole alanine and triazole acetic acid could not be evaluated. 51 of 94 Mutagenicity was not seen in an acceptable/ guideline bacterial reverse mutation assay using triazole acetic acid or in the available mutagenicity studies for triazole alanine. Therefore, neither compound appears mutagenic.

11.1.3 Dose Response For triazole alanine, greater toxicity was seen at higher doses. At the mid dose ( LOAEL) in the 90 day feeding study, decreased leukocytes were seen in males only and decreased triglycerides were seen only in females ( 370/ 400 mg/ kg/ day, males/ females). However, at the high dose, decreased leukocytes and triglycerides were each seen in both sexes, along with decreased body weight and body weight gain in males ( 1510/ 1680 mg/ kg/ day, males/ females). In the rat developmental toxicity study, more types of skeletal findings were observed in offspring at the high dose ( 1000 mg/ kg/ day), compared to the mid dose ( 300 mg/ kg/ day).

For triazole acetic acid, dose response could not be evaluated because no treatment related effects were observed in the available toxicity studies.

Table 11.3. Subchronic, Chronic, and Other Toxicity Profile for Triazole Alanine Guideline Number Study Type/ Classification MRID Number Doses Results Nonguideline 14 Day drinking water toxicity in rodents  rat Acceptable/ nonguideline 00138121 ( TXR 004766 Accession # 252132) 00133358 0, 3000, 10000 ppm M: 0, 448, 1491 mg/ kg/ day F: not tested NOAEL = 1491 mg/ kg/ day ( M) LOAEL = > 1491 mg/ kg/ day ( M)

870.3050 28 Day oral toxicity in rodents  rat Unacceptable 00138122 ( TXR 004766 Accession # 252132) 00133359 0, 25, 100, 400 mg/ kg/ day NOAEL = 400 mg/ kg/ day ( M/ F) LOAEL = > 400 mg/ kg/ day ( M/ F)

Many of the required hematology, clinical chemistry, and microscopic pathology parameters were not examined. Dosages administered to the test animals were not analytically quantitated. 52 of 94 Table 11.3. Subchronic, Chronic, and Other Toxicity Profile for Triazole Alanine Guideline Number Study Type/ Classification MRID Number Doses Results 870.3100 90 Day oral toxicity in rodents  rat Acceptable/ nonguideline 00164107 ( TXR 005532 005094) 0, 1250, 5000, 20000 ppm M: 0, 90, 370, 1510 mg/ kg/ day F: 0, 160, 400, 1680 mg/ kg/ day NOAEL = 90/ 160 mg/ kg/ day ( M/ F) LOAEL = 370/ 400 mg/ kg/ day ( M/ F) based on decreased leukocyte counts in males and decreased triglycerides in females.

At 1510/ 1680 mg/ kg/ day ( M/ F), decreased body weight ( M), body weight gain ( M), leukocytes ( M& F), and triglycerides ( M& F) were seen.

There was no assessment of motor activity, grip strength, or sensory reactivity of the test animals. There was no indication of how often fresh batches of food containing the test material were given to the animals. 870.3100 90 Day oral toxicity in rodents  mouse ND ND ND 870.3150 90 Day oral toxicity in nonrodents  dog Acceptable/ guideline 00164106 00154946 ( TXR 004469 005841 Accession # 256058) 0, 3200, 8000, 20000 ppm M: 0, 144, 322, 850 mg/ kg/ day F: 0, 150, 345, 902 mg/ kg/ day NOAEL = 850/ 345 mg/ kg/ day ( M/ F) LOAEL = > 850/ 902 mg/ kg/ day ( M/ F) based on 10% decreased food consumption in females only.

No treatment related effects were seen in males. 870.3200 21/ 28 Day dermal toxicity ND ND ND 870.3250 90 Day dermal toxicity ND ND ND 870.3465 90 Day inhalation toxicity ND ND ND 870.3700 Prenatal developmental toxicity in rodents  rat Acceptable/ guideline 00138128 ( TXR 004766 & 005155 Accession # 252132)

00132915 00147889 ( TXR 0005155 Accession # 252132) 0, 100, 300, 1000 mg/ kg/ day Maternal NOAEL = 1000 mg/ kg/ day LOAEL = > 1000 mg/ kg/ day Developmental NOAEL = 100 mg/ kg/ day LOAEL = 300 mg/ kg/ day based on increased incidence of skeletal findings ( unossified odontoid process).

At 1000 mg/ kg/ day, increased incidences of partially ossified transverse processes of the 7th cervical vertebra ( bilateral), unossified 5th sternebra, and partially ossified 13th thoracic centrum were also seen. 53 of 94 Table 11.3. Subchronic, Chronic, and Other Toxicity Profile for Triazole Alanine Guideline Number Study Type/ Classification MRID Number Doses Results 870.3700 Prenatal developmental toxicity in nonrodents ND ND ND 870.3800 Reproduction and fertility effects  rat Acceptable/ guideline 00164112 ( TXR 005841 & 008292 Accession # 252132)

41326803 ( TXR 008292)

41326804 ( TXR 008292) 0, 500, 2000, 10000 ppm M ( F0/ F1): 0, 50/ 47, 213/ 192, 1098/ 929 mg/ kg/ day F ( F0/ F1): 0, 51/ 49, 223/ 199, 1109/ 988 mg/ kg/ day Parental/ Systemic NOAEL = 929/ 988 mg/ kg/ day ( M/ F) LOAEL = > 929/> 988 mg/ kg/ day ( M/ F)

Reproductive NOAEL = 929/ 988 mg/ kg/ day ( M/ F) LOAEL = > 929/> 988 mg/ kg/ day ( M/ F)

Offspring NOAEL = 192/ 199 mg/ kg/ day ( M/ F) LOAEL = 929/ 988 mg/ kg/ day ( M/ F) based on reduced mean litter weights in both generations. 870.4100 Chronic toxicity ND ND ND 870.4200 Carcinogenicity ND ND ND 870.4300 Combined chronic toxicity/ carcinogenicity ND ND ND 870.5100 Bacterial reverse mutation assay Acceptable/ guideline 00138123 00132911 ( TXR 004469 004562 004766 Accession # 252132) 20, 100, 500, 2500, 12500 ug/ plate for Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 ( ± S9) Not mutagenic in bacteria ( Salmonella typhimurium, ± S9) under conditions of this assay.

870.5100 Bacterial reverse mutation assay Acceptable 00164111 ( TXR 005841) 20 5000 µ g/ plate for Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 ( ± S9). Not mutagenic. 54 of 94 Table 11.3. Subchronic, Chronic, and Other Toxicity Profile for Triazole Alanine Guideline Number Study Type/ Classification MRID Number Doses Results 870.5300 In vitro mammalian cell gene mutation test ( Baby hamster kidney cells)

Unacceptable 00132914 00139576 ( TXR 004562 004766 Accession # 252132)  S9: 0.5, 1, 2, 4, 8 mg/ ml + S9: 1, 2, 4, 8, 16 mg/ ml Inconclusive.  S9: LD39 = 8mg/ ml ( HDT). Transformation frequency was 39 vs 10 for control. An increase in number of transformed colonies.

+ S9: LD50 = 5.2 mg/ ml. Transformation frequency was 28 vs 0 for the control at 16 mg/ ml. Increase in number of transformed colonies, but in the presence of severe toxicity and excessive concentrations. In addition, the lack of information on the test material purity raises concerns regarding the validity of the response. 870.5300 In vitro mammalian cell gene mutation test ( BALB/ 3T3 cells)

Acceptable 00147892 ( TXR 005155 Accession # 257997) 62.5, 125, 250, 500, 1000 ug/ ml ( ± S9) Not mutagenic. Triazole alanine did not induce cell transformations at levels up to 1000 µ g/ ml. The HDT of 1000 µ g/ ml is the highest level recommended for this test system. The increase in cell transformation frequency in the presence of S9 was not significant. 870.5300 In vitro mammalian cell gene mutation test ( CHO cells)

Acceptable 00164108 41326801 ( TXR 005841 008292) 500, 1000, 2000, 4000, 6000, 8000, 10000 µ g/ ml Not mutagenic with or without S9 activation. Viability of cells at 10000 µ g/ ml was 66 67% relative to control. At 10000 µ g/ ml, a 3 fold increase was within the accepted spontaneous range for this cell line. Stability of compound was confirmed. 870.5375 In vitro mammalian chromosome aberration test ND ND ND 870.5395 Mammalian erythrocyte micronucleus test ( mice)

Unacceptable 00132912 00138125 00138131 ( TXR 004562 004766 Accession # 252131) 8000 mg/ kg, single oral dose to 15 mice/ sex Inconclusive because effects were only seen at 24 hours. No clinical or cytotoxice effects. TA caused slight induction of micronuclei in bone marrow PCEs at 24 hrs, but not at 48 or 72 hrs. No data for the negative control at these two intervals. 55 of 94 Table 11.3. Subchronic, Chronic, and Other Toxicity Profile for Triazole Alanine Guideline Number Study Type/ Classification MRID Number Doses Results 870.5395 Mammalian erythrocyte micronucleus test ( mice)

Acceptable/ guideline 00133361 00138124 ( TXR 004562 004766 Accession # 252132) 2500 or 5000 mg/ kg, by intraperitoneal injection Not mutagenic. Under the conditions of this test, triazole alanine did not cause induction of micronuclei in mouse bone marrow PCE stem cells when administered by i. p. injection at 2500 or 5000 mg/ kg. The effects of the positive control were clearly demonstrated. 870.5395 Mammalian erythrocyte micronucleus test ( Chinese hamster)

Acceptable 00164110 41326802 ( TXR 005841 008292) 5000 mg/ kg ( limit dose)

Preliminary test: 200, 1000, 5000 mg/ kg Not mutagenic. Based on scoring 1000 polychromatic erythrocytes per animal for the incidence of micronuclei, TA was comparable to the control. The best 5 slides per sex were scored. 870.5500 Bacterial DNA damage test Acceptable/ guideline 00132913 00138126 ( TXR 004469 004562 004766 Accession # 252131) 62.5, 125, 250, 500, 1000 µ g/ plate ( ± S9)

E. coli p3478 ( polA) & E. coli W3110 ( polA+) Not mutagenic. No measurable inhibition areolae were found for E. coli polA or polA+ strains ( ± S9). Under the conditions of this assay, triazole alanine ( ± S9) did not elicit measurable DNA damage.

870.5550 Bacterial DNA repair test ( rat hepatocytes)

Acceptable/ guideline 00164109 ( TXR 005841) 0.08, 0.4, 2, 10 mg/ ml Induced with Arochlor 1254 Not mutagenic. No evidence that unscheduled DNA synthesis was induced.

Nonguideline Potential inhibition of protein biosynthesis in microorganisms Unacceptable No MRID TXR 005841 Accession # 265203 265209 1, 10, 100, 500 ppm for E. coli, S. cerevisiae, and A. flavus TA inhibited protein synthesis in all three microorganisms in the presence of both radiolabeled phenylalanine and alanine after 1 hr of incubation. Growth curve of E. coli indicated that maximum period of protein synthesis was ~ 1 hr of incubation. Unacceptable due to lack of individual data, not enough doses of TA tested, no explanation of incubation times used, inconsistent results. 56 of 94 Table 11.3. Subchronic, Chronic, and Other Toxicity Profile for Triazole Alanine Guideline Number Study Type/ Classification MRID Number Doses Results 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ nonguideline 45607001 ~ 10.1 mg/ day* radiolabeled, 5 days by oral gavage, to 1 rat/ sex * Approx. 48 49 mg/ kg/ day, based on given body weights Triazole alanine is readily absorbed and excreted within 48 hours following 5 daily doses of ~ 10 mg/ day of either compound. At 168 hours, radioactivity recovery was ~ 92 98% of the administered dose. Absorption of the administered doses ( urine and cage wash) at 168 hours was ~ 71 81%. Urinary excretion accounted for ~ 55 63% of the administered radioactivity; fecal excretion accounted for ~ 11 27% of the administered radioactivity. Minor gender related quantitative differences were observed, but because only 1 rat/ sex/ compound was tested, these are likely due to individual variability. Tissue burdens were negligible at 72 hrs post dosing. Triazole alanine, triazole acetic acid, free triazole, plus three additional unknown metabolites were detected in the urine and feces. The investigators propose that one of these unknown degradates is a pyruvic acid derivative. 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ nonguideline 00138118 ( TXR 004766 Accession # 252132) 00133354 0.5 or 56 mg/ kg 14Ctriazole alanine, single dose by oral gavage, to 4 rats/ sex/ dose For both dose levels, > 90% of the administered dose was excreted in urine at 168 hours, ~ 4 5% in feces, and < 1% in volatiles, with most excretion in the first 24 hours. At the low dose, no radioactivity was detected in tissues. At the high dose, 0.002 0.02 ppm of TA equivalents were detected in tissues, mostly in the liver and kidneys. TA made up 72% and 80.3%, and N acetyl TA made up 19.4% and 12.7% of the radioactivity recovered from the urine of low dose males and females, respectively. TA made up 83.4% and 86.4%, and N acetyl TA made up 10.6% and 7.7% of the radioactivity recovered from the urine of high dose males and females, respectively. 57 of 94 Table 11.3. Subchronic, Chronic, and Other Toxicity Profile for Triazole Alanine Guideline Number Study Type/ Classification MRID Number Doses Results 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ nonguideline Continuation of MRID 00138118 00139575 ( TXR 004766 Accession # 252132) 00133355 56 mg/ kg 14C triazole alanine ( TA), single dose by oral gavage, to 4 rats/ sex/ dose Urine and feces samples included the first 24 hours after exposure. Triazole alanine is completely absorbed and rapidly excreted. The major route of excretion is urine. Most of the radiolabel recovered in the urine sample from the first 24 hours was unchanged TA ( 72 86%), with 8 19% of the urinary radioactivity as N acetyl TA. Two or three minor unknown metabolites (< 3% each) were also found in the various urine samples. About 50% of the radiolabel recovered in the feces was TA, 16% was recovered as N acetyl TA, and 30% was not identified but wasn't found in the urine. 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ nonguideline 00164114 ( TXR 005841) 0.56, 54.4 or 993.7 mg/ kg 14C triazole alanine, single dose by oral gavage, to 2 rats/ sex/ dose Radioactivity recoveries at 168 hours were 101.2%, 90.7%, and 92.1% for the low, mid, and high doses, respectively. There were no major sex differences in excretion patterns. The major route of excretion was urine: 81 82% of the administered dose for males and 84 85% for females. Absorption and excretion was rapid: within 24 hours after dosing, 76 82% of the administered dose in males and 71 91% in females was eliminated in the feces and urine. Tissue residues were low (< 1% for both sexes). 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ nonguideline Continuation of MRID 00164114. 00164115 ( TXR 005841) 0.56, 54.4 or 993.7 mg/ kg 14C triazole alanine, single dose by oral gavage, to 2 rats/ sex/ dose Urine samples included the first 24 hours after exposure. Triazole alanine made up 82 93% of the radioactivity recovered in the first 24 hours urine sample, and Nacetyl triazole alanine made up 13 30%. The significance of an apparent decrease in the percentage of N acetylation at the high dose is questionable. 58 of 94 Table 11.3. Subchronic, Chronic, and Other Toxicity Profile for Triazole Alanine Guideline Number Study Type/ Classification MRID Number Doses Results 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ nonguideline 00138132 ( TXR 004766 Accession # 252132) Metabolism: 5 mg/ kg 14Ctriazole alanine, by oral gavage or i. v. injection, to 5 males/ exposure route Autoradiography: 10 mg/ kg 14C triazole alanine, by i. v. injection, to 6 males Absorption and excretion was rapid, with most excretion in the urine. At 48 hours after oral exposure, 94.5% and 3.5% of the administered radiolabel was recovered in the urine and feces, respectively. At 48 hours after i. v. exposure, 92.4% and 2.1% of the administered radiolabel was recovered in the urine and feces, respectively. Tissue residues were low, with the highest residues found in the kidney and liver. Following oral administration, radioactivity increased in the plasma and a maximum was attained within 40 minutes. Radioactivity declined in a biphasic manner with half lives of 3 and 8 hours. Following iv administration, radioactivity decreased in triphasic manner with half lives of 15 minutes, 4.4 hours, and 12.4 hours. Whole body autoradiography indicated radioactivity distribution in all tissues and organs except the compacts of the bone. ND = Study not done Table 11.4. Subchronic, Chronic, and Other Toxicity Profile for Triazole Acetic Acid Guideline Number Study Type/ Classification MRID Number Doses Results Nonguideline 14 Day oral toxicity in rodents  rat Acceptable/ non guideline 45596801 0, 100, 1000, 8000 ppm M: 0, 10.6, 102.8, 788.3 mg/ kg/ day F: 0, 10.1, 97.2, 703.5 mg/ kg/ day NOAEL = 788.3/ 703.5 mg/ kg/ day ( M/ F) LOAEL = > 788.3/> 703.5 mg/ kg/ day ( M/ F)

870.3050 28 Day oral toxicity in rodents ND ND ND 870.3100 90 Day oral toxicity in rodents ND ND ND 870.3150 90 Day oral toxicity in nonrodents ND ND ND 870.3200 21/ 28 Day dermal toxicity ND ND ND 870.3250 90 Day dermal toxicity ND ND ND 870.3465 90 Day inhalation toxicity ND ND ND 870.3700 Prenatal developmental toxicity in rodents ND ND ND 59 of 94 Table 11.4. Subchronic, Chronic, and Other Toxicity Profile for Triazole Acetic Acid Guideline Number Study Type/ Classification MRID Number Doses Results 870.3700 Prenatal developmental toxicity in nonrodents ND ND ND 870.3800 Reproduction and fertility effects ND ND ND 870.4100 Chronic toxicity ND ND ND 870.4200 Carcinogenicity ND ND ND 870.4300 Combined chronic toxicity/ carcinogenicity ND ND ND 870.5100 Bacterial reverse mutation assay Acceptable/ guideline 45596803 20, 80, 320, 1280 and 5120 ug/ plate for Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 ( his ) No mutagenic activity in bacteria ( Salmonella typhimurium) in the presence or absence of S9 activation.

870.5300 In vitro mammalian cell gene mutation test ND ND ND 870.5375 In vitro mammalian cell chromosome aberration test ND ND ND 870.5395 Mammalian erythrocyte micronucleus test ND ND ND 870.5550 Unscheduled DNA synthesis in mammalian cells in culture ND ND ND 60 of 94 Table 11.4. Subchronic, Chronic, and Other Toxicity Profile for Triazole Acetic Acid Guideline Number Study Type/ Classification MRID Number Doses Results 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ non guideline 45596804 45596805 0.58, 58.63, or 1034.69 mg/ kg radiolabeled, single dose by oral gavage. Triazole acetic acid was readily absorbed and excreted within 48 hours following a single oral dose of 0.58, 58.63, or 1034.69 mg/ kg. Average absorption at 168 hours was nearly complete (~ 93 103%, ~ 102 104%, and ~ 92 101% of the administered dose for the males and females in the low, mid, and highdose groups, respectively) and did not appear to be saturated at the highest dose. Urinary excretion accounted for ~ 90 104% of the administered radioactivity, whereas fecal excretion accounted for only ~ 1 7% of the administered radioactivity in all treatment groups. Excretory patterns did not exhibit gender related variability. Because radioactivity in tissues was very low, neither triazole acetic acid nor its metabolites appear to undergo significant sequestration. The urinary metabolite was identified as triazole acetic acid, indicating that the parent compound was excreted intact without being metabolized in the rat. 61 of 94 Table 11.4. Subchronic, Chronic, and Other Toxicity Profile for Triazole Acetic Acid Guideline Number Study Type/ Classification MRID Number Doses Results 870.7485 Metabolism and pharmacokinetics  rat Acceptable/ non guideline 45607001 ~ 10.5 mg/ day* radiolabeled, 5 days by oral gavage, to 1 rat/ sex * Approx. 50 51 mg/ kg/ day, based on given body weights Triazole acetic acid is readily absorbed and excreted within 48 hours following 5 daily doses of ~ 10.5 mg/ day. At 168 hours, radioactivity recovery was ~ 101 102% of the administered dose. Absorption of the administered doses ( urine and cage wash) at 168 hours was ~ 69 91%. Urinary excretion accounted for ~ 66 75% of the administered radioactivity. Fecal excretion accounted for ~ 11 32% of the administered radioactivity. Minor gender related quantitative differences were observed, but because only 1 rat/ sex/ compound was tested, these are likely due to individual variability. Tissue burdens were negligible at 72 hrs post dosing. The sole component identified in extracts of feces and urine from radiolabeled triazole acetic acid fed rats was triazole acetic acid, which indicates excretion without metabolic conversion of triazole acetic acid. ND = Study not done 11.2 FQPA Hazard Considerations 11.2.1 Adequacy of the Toxicity Data Base The toxicology database for triazole alanine is considered incomplete in terms of endpoint studies and dose response information to characterize potential pre and/ or post natal risk for infants and children. Acceptable developmental toxicity and two generation reproduction studies in the rat were evaluated, but a developmental toxicity study in the rabbit was not available.

No studies on triazole acetic acid, including rat or rabbit developmental toxicity studies or a twogeneration reproduction study in the rat, were available to characterize any potential pre and/ or post natal risk for infants and children.

11.2.2 Evidence of Neurotoxicity There is no evidence that exposure to triazole alanine results in neurotoxicity. No clinical signs of neurotoxicity, changes in brain weights, changes in brain gross or microscopic pathology, or any other neurotoxic effects were observed in the short term rat studies, the subchronic rat and 62 of 94 dog feeding studies, the rat developmental toxicity study, or the two generation reproduction study. Slight salivation and a slightly unsteady gait were seen in the female dog tested in an acute oral dog study; however, the neurotoxicological significance of this effect is questionable, since the effects were seen at an extremely high dose ( 5000 mg/ kg), and similar effects were not seen in the male dog. Spastic gait, piloerection, lethargy, and diarrhea were initially seen in rats tested in an acute intraperitoneal study; however, the significance of these effects is also questionable because the rats were exposed to an extremely high dose ( 5000 mg/ kg), and similar effects were not seen at 5000 mg/ kg in three acute oral studies of triazole alanine in rats.

While the available repeated dose studies show no evidence for triazole alanine to adversely affect the nervous system, there is some concern that this is due to an incomplete database, rather than a true inability of the compound to produce neurotoxicity. For instance, no acute or subchronic neurotoxicity tests or an evaluation of perfused brain tissue are available for triazole alanine. Tremors, decreased brain weight, and cerebellar degeneration were seen in the 90 day mouse study on 1,2,4 triazole; however, no mouse studies on triazole alanine are available for comparison. Clinical signs of neurotoxicity were seen in adults in the developmental rabbit study on 1,2,4 triazole, including decreased motor activity, head tilt, lacrimation, drooping eyelids, diarrhea, salivation; however no rabbit studies are available on triazole alanine. Cerebellar lesions and decreased brain weights were seen at the highest dose tested in the twogeneration reproduction study on free triazole; however, brains were not microscopically examined in the triazole alanine reproduction study.

11.2.3 Developmental Toxicity Studies <IP_ADDRESS> Developmental Toxicity Study in Rats For triazole alanine, increased quantitative and qualitative susceptibility was seen in the developmental toxicity study in rats. ( See section A 1.3.1 of the appendix for the executive summary of MRIDs 00138128 and 00147889). In this study, increased incidences of skeletal findings were seen in the offspring at the mid and high doses, while no treatment related effects were seen in the dams up to the limit dose. The skeletal findings included unossified odontoid processes at 300 and 1000 mg/ kg/ day, with partially ossified transverse processes of the 7th cervical vertebra ( bilateral), unossified 5th sternebra, and partially ossified 13th thoracic centrum observed only at 1000 mg/ kg/ day.

A developmental toxicity study in rats was not submitted for triazole acetic acid.

<IP_ADDRESS> Developmental Toxicity Study in Rabbits A developmental toxicity study in rabbits was not submitted for either triazole alanine or triazole acetic acid.

11.2.4 Reproductive Toxicity Study For triazole alanine, increased quantitative susceptibility was seen in the two generation reproduction study in rats. ( See section A 1.4.1 of the appendix for the executive summary of 63 of 94 MRIDs 164112, 41326803, and 41236804). In this study, mean litter weights were decreased approximately 10 20% for both generations at ~ 1000 mg/ kg/ day, compared to controls, but parental body weights were unaffected at all treatment levels tested. The lowest dose associated with decreased body weights and body weight gains in adult rats was 1510 mg/ kg/ day, from the 90 day toxicity study.

A two generation reproduction study in rats was not submitted for triazole acetic acid.

11.2.5 Additional Information from Literature Sources No relevant specific information on the toxicity of triazole alanine or triazole acetic acid was noted in the open scientific literature.

11.2.6 Pre and/ or Postnatal Toxicity <IP_ADDRESS> Determination of Susceptibility Increased quantitative and qualitative susceptibility of the offspring was seen in the developmental toxicity rat study, and increased quantitative susceptibility was seen in the twogeneration reproduction study. In the developmental toxicity study, increased incidences of skeletal findings were seen in the offspring at the mid and high doses, while no treatment related effects were seen in the dams up to the limit dose. These skeletal findings include unossified odontoid processes at 300 and 1000 mg/ kg/ day, with partially ossified transverse processes of the 7th cervical vertebra ( bilateral), unossified 5th sternebra, and partially ossified 13th thoracic centrum observed only at 1000 mg/ kg/ day. In the reproduction study, mean litter weights were decreased approximately 10 20% for both generations at ~ 1000 mg/ kg/ day, compared to controls, but parental body weights were unaffected at all treatment levels.

<IP_ADDRESS> Degree of Concern Analysis and Residual Uncertainties for Pre and/ or Postnatal Susceptibility The toxicology database for triazole alanine is incomplete with respect to pre and post natal toxicity because a rabbit developmental toxicity study was not available, and the existing twogeneration reproduction study, completed in 1986, did not meet the current guideline protocol. Evidence of increased qualitative and quantitative susceptibility in the offspring was seen in the rat developmental toxicity study in the form of skeletal findings observed in the offspring at dose levels that produced no treatment related effects in the dams. Evidence of increased quantitative susceptibility was also seen in the two generation reproduction study as a reduction in mean litter weights for both generations at dose levels that produced no treatment related effects in the parents. However, the concern is low for the increased susceptibilities seen in these studies, as well as for the residual uncertainties regarding the data gaps, because the doses currently used for dietary, residential, and occupational exposure risk assessments are similar or lower than the doses that caused the effects.

For triazole acetic acid, the toxicology database is incomplete with respect to pre and post natal toxicity because no rat or rabbit developmental toxicity studies or a two generation reproduction 64 of 94 study in the rat are available. Increased qualitative and quantitative susceptibility was seen for both triazole alanine and 1,2,4 triazole. Therefore, there is residual uncertainty for increased pre and/ or postnatal susceptibility following exposure to triazole acetic acid, but this uncertainty is addressed by retaining the 10X database uncertainty factor.

11.3 Recommendation for a Developmental Neurotoxicity Study At this time, a developmental neurotoxicity study on the triazole conjugates is not required.

11.3.1 Evidence that supports requiring a Developmental Neurotoxicity Study At present, there is no evidence to support requiring a Developmental Neurotoxicity Study.

11.3.2 Evidence that supports not requiring a Developmental Neurotoxicity Study There was no evidence of neurotoxicity in any of the studies available in the toxicology database for triazole alanine or triazole acetic acid, including the short term rat studies, the subchronic rat and dog feeding studies, the rat developmental toxicity study, and the two generation reproduction study.

<IP_ADDRESS> Rationale for the UFDB The database uncertainty factor is 10X, based on the lack of a developmental toxicity study in rabbits for both triazole alanine and triazole acetic acid, lack of a chronic study in rats ( including additional neuropathological and neurobehavioral measurements) with triazole alanine, and lack of a combined 90 day/ subchronic neurotoxicity study in rats for triazole acetic acid.

11.4 Hazard Identification and Toxicity Endpoint Selection Because no effects were seen in the available acute oral toxicity studies ( 14 day oral studies in rats and a bacterial reverse mutation assay), a difference in toxicity between triazole alanine and triazole acetic acid could not be evaluated. In the absence of data to suggest otherwise, the endpoint selection discussed in the following sections applies to all triazole conjugates ( i. e., triazole alanine, triazole acetic acid, triazole lactic acid, and triazole pyruvate).

11.4.1 Acute Reference Dose ( aRfD)  Females ( 13 to 49 years of age)

Study Selected: Prenatal developmental toxicity in rodents  rat MRID Number: 00138128 and 00147889 Dose and Endpoint for Establishing aRfD: 100 mg/ kg/ day ( NOAEL), based on increased incidence of skeletal findings ( unossified odontoid process) at 300 mg/ kg/ day ( LOAEL). Uncertainty Factor( s): 1000 ( 10X for interspecies extrapolation, 10X for intraspecies variations, and 10X for database uncertainty). Comments about Study/ Endpoint/ Uncertainty Factor: Adverse effects seen in offspring from an oral developmental study are appropriate for setting an acute dietary endpoint for women of 65 of 94 childbearing age. There is some uncertainty as to whether effects on ossification are due to a single dose. Delays in ossification can indicate an effect on growth, which would be seen with repeated exposure, but no change in fetal body weights was observed in this study. However, in the absence of a developmental rabbit study, which is the most sensitive species for free triazole, this dose and study were selected for this risk assessment. In accordance with current FQPA policy, a 1000X uncertainty factor is applied, which includes a 10X database UF to account for the lack of developmental toxicity studies on triazole alanine and triazole acetic acid in rabbits.

Acute RfD = 100 mg/ kg/ day ( NOAEL) ÷ 1000 ( UF) = 0.1 mg/ kg/ day 11.4.2 Acute Reference Dose ( aRfD)  General Population In 2003, the Triazole Peer Review Committee ( PRC) determined that the acute endpoint for the triazole conjugates should be based on the developmental toxicity study in rats discussed previously, with an uncertainty factor of 300X to include a 3X database UF ( TXR No. 0052011). However, reevaluation of the triazole alanine database showed that no appropriate endpoints ( i. e., effects seen in adult animals following a single, oral dose) were available to set an acute reference dose for the general population.

11.4.3 Chronic Reference Dose ( cRfD)

Study Selected: 90 Day oral toxicity in rodents  rat MRID Number: 00164107 Dose and Endpoint for Establishing cRfD: 90 mg/ kg/ day ( NOAEL), based on decreased leukocyte counts in males at 370 mg/ kg/ day ( LOAEL). Uncertainty Factor( s): 1000 ( 10X for interspecies extrapolation, 10X for intraspecies variations, and 10X for database uncertainty). Comments about Study/ Endpoint/ Uncertainty Factor: For females, the NOAEL is 160 mg/ kg/ day, and the LOAEL is 400 mg/ kg/ day, based on decreased triglycerides. Out of the available toxicology studies on the triazole conjugates, the two generation reproduction study and the 90 day studies were the longest in duration. Chronic studies in rats, mice, or dogs were not available. Additionally, rabbits, which are more sensitive than rats to free triazole, were not used in any of the available toxicology studies on the triazole conjugates. Although no adverse effects were seen in adults in the reproduction study, fewer endpoints are measured in this study compared to the subchronic feeding studies. Treatment related effects were seen in both the 90 day dog and rat studies, but the 90 day rat study was chosen because rats were more sensitive to triazole alanine than dogs. The 10X database uncertainty factor used for the chronic endpoint is to account for the lack of a developmental toxicity study in rabbits for both triazole alanine and triazole acetic acid, the lack of a chronic study in rats ( including additional neuropathological and neurobehavioral measurements) with triazole alanine, and a lack of a combined 90 day/ subchronic neurotoxicity bridging study on triazole acetic acid.

In 2003, the Triazole Peer Review Committee ( PRC) determined that the chronic endpoint for the triazole conjugates should be based on the developmental toxicity study in rats discussed above, with an uncertainty factor of 300X to include a 3X database UF ( TXR No. 0052011). This decision was made prior to the current FQPA policy; to the receipt of the 90 day/ subchronic 66 of 94 neurotoxicity rat study, developmental rabbit toxicity study, and reproduction study on 1,2,4 triazole; and to the reevaluation of the triazole alanine studies. The reanalysis of the triazole alanine studies resulted in a change in chronic dose and endpoint selection. The subchronic rat study is a more appropriate basis for a chronic endpoint than a developmental study. The change in FQPA policy and receipt of additional free triazole studies resulted in an increased database UF.

Chronic RfD = 90 mg/ kg/ day ( NOAEL) ÷ 1000 ( UF) = 0.09 mg/ kg/ day 11.4.4 Incidental Oral Exposure The endpoint for short and intermediate term incidental oral exposure is based on changes in hematology observed in the 90 day oral feeding study in rats ( MRID 00164107), which is discussed earlier in section 11.4.3. The oral route of exposure used in this study and the type of effects seen are considered appropriate for short or intermediate term incidental oral endpoints. Additionally, the NOAEL from the 90 day study is protective of the offspring toxicity observed in the developmental rat study. The 90 day duration of the study is most appropriate for an intermediate term endpoint. However, due to the lack of studies and of parameters measured in the available studies and because the effects seen in the 90 day rat study could occur with shortto long term exposure, this study was used as the basis for residential and occupational endpoints for all durations.

11.4.5 Dermal Absorption No dermal absorption studies on triazole alanine or triazole acetic acid are available in the database. Therefore, there are no data to support a dermal absorption factor other than 100%.

11.4.6 Dermal Exposure ( Short, Intermediate, and Long term)

In the absence of dermal studies, the endpoint for short, intermediate, and long term dermal exposure is the same as for short and intermediate term incidental oral exposure. See section 11.4.4 for more details.

11.4.7 Inhalation Exposure ( Short, Intermediate, and Long term)

In the absence of inhalation studies, the endpoint for short, intermediate, and long term inhalation exposure is the same as for short and intermediate term incidental oral exposure. See section 11.4.4 for more details.

11.4.8 Margins of Exposure and Levels of Concern The levels of concern ( LOCs) for residential and occupational exposure and risk assessment are given below. Each LOC is based on the conventional uncertainty factor of 100X ( 10X for intraspecies variation and 10X for interspecies extrapolation), as well as a 10X database uncertainty factor for the lack of a developmental toxicity study in rabbits for both triazole alanine and triazole acetic acid, the lack of a chronic study in rats ( including additional 67 of 94 neuropathological and neurobehavioral measurements) with triazole alanine, and a lack of a combined 90 day/ subchronic neurotoxicity study in rats for triazole acetic acid.

Table 11.5. Summary of Levels of Concern for Residential and Occupational Risk Assessments for Triazole Alanine and Triazole Acetic Acid. Duration of Exposure Route of Exposure Short Term ( 1 30 Days) Intermediate Term ( 1 6 Months) Long Term (> 6 Months) Occupational Exposure Dermal 1000 1000 1000 Inhalation 1000 1000 1000 Residential Exposure Incidental Oral 1000 1000 1000 Dermal 1000 1000 1000 Inhalation 1000 1000 1000 11.4.9 Recommendation for Aggregate Exposure Risk Assessments Because the same endpoints are used for all exposure routes, oral, dermal, and inhalation exposures can be aggregated.

11.4.10 Classification of Carcinogenic Potential There are no available cancer bioassay studies on 1,2,4 triazole. 1,2,4 triazole and its conjugate ( triazole alanine), however, are not mutagenic. For a further discussion of carcinogenicity and triazole metabolites, see Section 4.4.11.

11.5 Special FQPA Safety Factor The special FQPA safety factor has been removed ( i. e., reduced to 1X) for the current risk assessment on the triazole conjugates because the currently selected endpoints, together with a 10X database uncertainty factor, are protective of increased susceptibility of infants and children seen in the available studies. Although increased qualitative and quantitative susceptibility of the offspring was seen in the developmental toxicity and two generation reproduction studies in rats ( see section 11.2.6), the currently selected dietary, residential, and occupational endpoints are all based on NOAELs that are protective of these adverse effects. Additionally, no evidence of neurotoxicity was seen in the available toxicology database, so a developmental neurotoxicity study is not recommended at this time. Finally, while the current recommendation is to reduce the special FQPA safety factor to 1X, residual uncertainty is accounted for by a 10X database uncertainty factor. This 10X database uncertainty factor is retained for the lack of developmental toxicity ( rabbit) studies with triazole alanine and triazole acetic acid, lack of a chronic rat study ( with additional neuropathological and neurobehavioral endpoints) with triazole alanine, and lack of a combined 90 day/ subchronic neurotoxicity rat study for triazole acetic acid.

Future receipt of additional, required toxicology studies on triazole alanine ( or the other triazole conjugates) may warrant removal of the database uncertainty factor ( i. e., reduction to 1X). At 68 of 94 that time, the need for a 10X special FQPA safety factor based on the newly available toxicity data will be re evaluated.

Table 11.6. Summary of Toxicological Doses and Endpoints for Triazole Conjugates to be Used in Human Health Risk Assessments. Exposure Scenario Dose Used in Risk Assessment and UF Special FQPA SF * and Level of Concern for Risk Assessment Study and Toxicological Effects Acute Dietary ( females 13 49) NOAEL = 100 mg/ kg/ day UF = 1000 Acute RfD = 0.1 mg/ kg/ day FQPA SF = 1 aPAD = acute RfD FQPA SF = 0.1 mg/ kg/ day Prenatal developmental toxicity in rodents  rat LOAEL = 300 mg/ kg/ day based on increased incidence of skeletal findings ( unossified odontoid process). Acute Dietary ( general population, including infants and children) None None No appropriate dose and endpoint could be identified for these population groups.

Chronic Dietary ( all populations) NOAEL = 90 mg/ kg/ day UF = 1000 Chronic RfD = 0.09 mg/ kg/ day FQPA SF = 1 cPAD = chronic RfD FQPA SF = 0.09 mg/ kg/ day 90 Day oral toxicity in rodents  rat LOAEL = 370/ 400 mg/ kg/ day ( M/ F) based on decreased leukocyte counts in males and decreased triglycerides in females.

Incidental Oral ( all durations) NOAEL = 90 mg/ kg/ day Residential LOC for MOE = 1000a Occupational = NA 90 Day oral toxicity in rodents  rat LOAEL = 370/ 400 mg/ kg/ day ( M/ F) based on decreased leukocyte counts in males and decreased triglycerides in females. Dermal ( all durations) NOAEL = 90 mg/ kg/ day ( dermal absorption rate = 100%) Residential LOC for MOE = 1000a Occupational LOC for MOE = 1000a 90 Day oral toxicity in rodents  rat LOAEL = 370/ 400 mg/ kg/ day ( M/ F) based on decreased leukocyte counts in males and decreased triglycerides in females. Inhalation ( all durations) NOAEL = 90 mg/ kg/ day ( inhalation absorption rate = 100%) Residential LOC for MOE = 1000a Occupational LOC for MOE = 1000a 90 Day oral toxicity in rodents  rat LOAEL = 370/ 400 mg/ kg/ day ( M/ F) based on decreased leukocyte counts in males and decreased triglycerides in females. Cancer ( oral, dermal, inhalation) Classification: Not determined. Evaluate by RfD approach.

UF = uncertainty factor, FQPA SF = Special FQPA safety factor, NOAEL = no observed adverse effect level, LOAEL = lowest observed adverse effect level, PAD = population adjusted dose ( a = acute, c = chronic) RfD = reference dose, MOE = margin of exposure, LOC = level of concern, NA = Not Applicable * Refer to Section 11.5 a Additional 10x database uncertainty factor for lack of developmental toxicity ( rabbit) studies with triazole alanine and triazole acetic acid, a chronic rat study ( with additional neuropathological and neurobehavioral endpoints) with triazole alanine, and a combined 90 day/ subchronic neurotoxicity rat study for triazole acetic acid. 69 of 94 11.6 Endocrine Disruption EPA is required under the FFDCA, as amended by FQPA, to develop a screening program to determine whether certain substances ( including all pesticide active and other ingredients) " may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other such endocrine effects as the Administrator may designate." Following recommendations of its Endocrine Disruptor and Testing Advisory Committee ( EDSTAC), EPA determined that there was a scientific basis for including, as part of the program, the androgen and thyroid hormone systems, in addition to the estrogen hormone system. EPA also adopted EDSTAC's recommendation that the Program include evaluations of potential effects in wildlife. For pesticide chemicals, EPA will use FIFRA and, to the extent that effects in wildlife may help determine whether a substance may have an effect in humans, FFDCA authority to require the wildlife evaluations. As the science develops and resources allow, screening of additional hormone systems may be added to the Endocrine Disruptor Screening Program ( EDSP).

In the available toxicity studies on triazole alanine and triazole acetic acid, no estrogen, androgen, and/ or thyroid mediated toxicity was observed.

When additional appropriate screening and/ or testing protocols being considered under the Agency's EDSP have been developed, triazole conjugates may be subjected to further screening and/ or testing to better characterize effects related to endocrine disruption.

12.0 Public Health Data None.

13.0 Exposure Characterization/ Assessment 13.1 Dietary Exposure/ Risk Pathway ( M. Doherty, D322239, 12/ 20/ 05)

13.1.1 Residue Profile <IP_ADDRESS> Residues in Food Through a joint effort by the U. S. Triazole Task Force ( USTTF) and USDA's Pesticide Data Program ( PDP), monitoring data depicting residues of TA and TAA are available for apples, peaches, wheat flour, bananas, eggs, peanut butter, soybeans, finished water, strawberry, milk, grapes, and tomato. For all foods addressed by these assessments, including those with monitoring data, an anticipated residue was derived by converting the tolerance value for each parent triazole derivative fungicides to its TA equivalent using molecular weight conversion factors. For the acute assessment, the highest anticipated residue was used for a given food; for the chronic assessment, the average anticipated residue was used. For foods with monitoring data, the greater of the maximum monitoring data ( maximum TA plus maximum TAA) or the anticipated residue was used in the assessment. For all commodities except those of peanut and cereal grain, the anticipated residues were used. For peanut commodities the maximum monitored residue value from peanut butter was used. For cereal grain commodities, the 70 of 94 maximum monitored residue value from wheat flour was used. The assessments include default processing factors from DEEM Version 7.81. The Agency was recently made aware of an issue with the analytical method for TA in soybeans in which the method underestimates residues by 4 to 12 fold. That issue is still being resolved. In order to ensure that this assessment does not underestimate exposure to TA/ TAA via soybean, all inputs for soybean were multiplied by 10 in both the acute and chronic analyses. This is a very conservative, higher end deterministic assessment.

Table 13.1. Summary of Input Residue Values for the Acute and Chronic Dietary Analyses of Triazole Alanine and Triazole Acetic Acid. DEEM Input Value, ppm Anticipated Residue, ppm Food Acute Chronic Acute Chronic Max. Monitored Residue, ppm1 Remarks Pome Fruit ( Apple) 0.53 0.23 0.53 0.21 0.23   Artichoke 0.54 0.43 0.54 0.43     Asparagus 0.80 0.03 0.80 0.03     Banana 2.16 0.57 2.16 0.32 0.57   Dry Bean/ Pea 0.23 0.12 0.23 0.12     Succulent Bean/ Pea 0.54 0.22 0.54 0.22     Blueberry 0.46 0.30 0.46 0.30     Caneberry 1.08 0.87 1.08 0.87     Canola 0.05 0.02 0.05 0.02     Carrot 0.09 0.09 0.09 0.09     Leafy Petioles 2.28 2.28 2.28 2.28     Barley 0.55 0.55 0.046 0.31   From wheat flour Oats 0.55 0.55 0.05 0.03   From wheat flour Rice 3.19 1.11 3.19 1.11     Rye 0.55 0.55 0.05 0.04   From wheat flour Wheat 0.55 0.55 0.05 0.03   From wheat flour Wheat Flour 0.55 0.55 0.05 0.03 0.55   Wild Rice 3.19 1.11 0.23 0.23   From rice Citrus Group 0.46 0.23 0.46 0.23     Coffee 0.005 0.005 0.005 0.005     Field Corn 0.55 0.55 0.05 0.03   From wheat flour Sweet Corn 0.55 0.55 0.05 0.03   From wheat flour Cotton 1.01 0.26 1.01 0.26     Cranberry 0.46 0.24 0.46 0.24     Cucurbits 0.11 0.08 0.11 0.08     Currant 1.62 1.04 1.62 1.04     Elderberry 0.46 0.46 0.46 0.46     Grape 2.53 0.82 2.53 0.82 0.29   Raisin 2.53 0.82 2.53 0.82     Hops 15.20 7.60 15.20 7.60     Lychee 0.76 0.76 0.76 0.76     Mango 0.10 0.10 0.10 0.10     Mayhaw 0.38 0.19 0.38 0.19     Bulb Vegetables 0.14 0.09 0.14 0.09     Okra 0.51 0.51 0.51 0.51     Peanut 2.32 2.32 0.09 0.04   From peanut butter Peanut Butter 2.32 2.32 0.09 0.04 2.32   Peppers 0.54 0.54 0.54 0.54     Peppermint 1.62 1.62 1.62 1.62     71 of 94 Table 13.1. Summary of Input Residue Values for the Acute and Chronic Dietary Analyses of Triazole Alanine and Triazole Acetic Acid. DEEM Input Value, ppm Anticipated Residue, ppm Food Acute Chronic Acute Chronic Max. Monitored Residue, ppm1 Remarks Pineapple 1.59 0.82 1.59 0.82     Sorghum 0.55 0.55 0.09 0.05   From wheat flour Soybean 9.1 3.3 0.91 0.33 0.12 10X the anticipated residue to account for method issue. Spearmint 1.62 1.62 1.62 1.62     Stone Fruit no cherry 1.08 0.95 1.08 0.74 0.95   Cherry 2.70 1.53 2.70 1.53     Strawberry 0.68 0.48 0.68 0.48 0.13   Sugar Beet 0.14 0.07 0.14 0.07     Sugar Beet Molasses 0.14 0.07 0.14 0.07     Sunflower 0.03 0.03 0.03 0.03     Tomato 0.16 0.16 0.16 0.16 0.09   Turnip 0.20 0.20 0.20 0.20     Turnip Greens 2.28 2.28 2.28 0.04   From Leafy petiole Tree Nuts 0.19 0.11 0.19 0.11     Almond 0.19 0.11 0.19 0.11     Pecan 0.05 0.05 0.05 0.05     Pistachio 0.05 0.04 0.05 0.04     Poultry Meat 0.03 0.02 0.03 0.02     Poultry Fat 0.03 0.02 0.03 0.02     Poultry Meat Byprod. 0.03 0.02 0.03 0.02     Poultry Liver 0.03 0.02 0.03 0.02     Egg 0.03 0.02 0.03 0.02 0.016   Cattle Meat 0.53 0.13 0.53 0.13     Cattle Fat 0.53 0.15 0.53 0.15     Cattle Meat Byprod. 0.53 0.14 0.53 0.14     Cattle Liver 1.68 0.63 1.68 0.63     Cattle Kidney 0.91 0.29 0.91 0.29     Milk 0.11 0.04 0.11 0.04 0.01   1 Residue data for TA/ TAA from USDA Pesticide Data Program or U. S. Triazole Task Force monitoring data. Values are the combined maxima for TA and TAA for monitored foods and do not reflect the maximum combined residue of TA and TAA for a particular sample.

<IP_ADDRESS> Residues in Drinking Water As noted in Section 6, there is not sufficient information available to model potential residues of the triazole conjugates in drinking water. As a surrogate for modeled concentrations of TA and TAA, HED has used the modeled estimates for 1,2,4 T ( Table 6.2) in the TA/ TAA dietary exposure assessment, multiplying the values by 2.26 to correct for differences in molecular weight. Given the infrequent and very low residue levels reported in the monitoring data ( Table 13.1), the use of 1,2,4 T residue estimates in drinking water is highly conservative. 72 of 94 13.1.2 Acute and Chronic Dietary Exposure and Risk Table 13.2. Dietary ( Food + Water) Exposure and Risk Estimates for Triazole Alanine and Triazole Acetic Acid. Exposure Estimate, mg/ kg/ day Risk Estimate, % aPADa Population Subgroup aPAD, mg/ kg/ day 95th % ile 99th % ile 99.9th % ile 95th % ile 99th % ile 99.9th % ile Acute Females 13 49 yrs 0.1 0.0274 27 0.0412 41 0.0827 83 Chronic Population Subgroup cPAD, mg/ kg/ day Exposure Estimate, mg/ kg/ day Risk Estimate, % cPADa U. S. Population ( total) 0.09 0.0080 9 All infants (< 1 year) 0.09 0.0160 18 Children 1 2 yrs 0.09 0.0239 27 Children 3 5 yrs 0.09 0.0196 22 Children 6 12 yrs 0.09 0.0121 13 Youth 13 19 yrs 0.09 0.0073 8 Adults 20 49 yrs 0.09 0.0062 7 Adults 50+ yrs 0.09 0.0054 6 Females 13 49 yrs 0.09 0.0060 7 The values for the population with the highest risk for each type of risk assessment are bolded. a Reported to 2 significant figures.

Due to the conservatism of the assessment inputs, HED believes that it is most appropriate to base regulatory recommendations on results at the 95th percentile of exposure for the acute assessment. The results indicate that for both acute and chronic exposure durations and for all population subgroups, risk estimates are well below HED's level of concern.

13.2 Residential ( Non Occupational) Exposure/ Risk Pathway Triazole alanine is formed in plants by the conjugation of 1,2,4 T to serine. The TA may then be further oxidized to form TAA. Because of the nature of this process, HED has assumed that it occurs within the plant itself on not on leaf surfaces. Therefore, the residues are not available for dermal, hand to mouth, or object to mouth exposures and HED has not conducted a residential exposure assessment for the triazole conjugates. Residues of TA and TAA may occur in soil. 1,2,4 Triazole is more toxic than TA/ TAA and exposures to TA/ TAA via soil ingestion are unlikely to exceed those of 1,2,4 T. The assessment for soil ingestion of 1,2,4 T shows that risk estimates are below HED's level of concern; therefore, risk estimates for soil ingestion of TA/ TAA will also be below HED's level of concern.

14.0 Aggregate Risk Assessments and Risk Characterization Dietary exposure is the only pathway of concern for TA/ TAA; therefore, aggregate risk estimates are equivalent to those discussed for dietary risk ( Section 13.1.2). All aggregate risk estimates are below HED's level of concern.

15.0 Cumulative Risk Characterization/ Assessment See Section 8.0. 73 of 94 16.0 Occupational Exposure/ Risk Pathway Triazole alanine and triazole acetic acid are not present in pesticide formulations; therefore, there is no risk from exposure to these chemicals that results from mixing, loading, or application activities. Although TA and TAA are significant plant metabolites, the residues occur inside the plant structures and are not available for dermal contact ( see Section 13.2). Therefore, there is no significant exposure as a result of post application activities and quantitative occupational exposure and risk assessments are not needed for TA and TAA.

17.0 Data Needs and Label Requirements 17.1 Toxicology A 10X database uncertainty factor is retained for the lack of the following studies:

Triazole alanine   Developmental toxicity study in rabbits   Chronic toxicity study in rats, conducted according to current guidelines that include neurobehavioral assessments, with additional neuropathology evaluations conducted according to the neurotoxicity guidelines Triazole acetic acid   Developmental toxicity study in rabbits   Combined 90 day feeding/ neurotoxicity study in rats 17.2 Residue Chemistry   Resolution of concerns regarding the prevalence of conjugated residues of TA and the ability of the analytical method to quantify them.

17.3 Occupational and Residential Exposure   None.

References Allen, J., Wolf, D., George, M. Hester, S., Sun G., Thai, S F., Delker, D., Nelson, G., Winkfield, E. Roop, B., Leavitt, S., Ward, W., and Nesnow, S. ( 2005) Comparable Liver P450 Enzyme Activity and Histopathology in Mice Treated With the Conazole Fungicides: Myclobutanil, Propiconazole and Triadimefon. The Toxicologist. Abstract. No, 2146.

Goetz, A. K., Blystone, C., Ren, H., Schmid, J. E., Nichols, H. P., Bao, W., Best, D. S., Narotsky, M. G., Wolf, D. C., Rockett, J. C., and Dix, D. J. ( 2005) Reproductive Effects of Exposure to Conazole Fungicides in the Male Rat. The Toxicologist. Abstract No 2148. 74 of 94 Hester, S., Thai, S. F., Wolf, D., Ward, W., and Nesnow, S. ( 2005) Comparison of Gene Expression Profiles from Rats Fed Acute Levels of Three Toxicologically Different Conazoles. The Toxicologist. Abstract No. 2149.

Ren, H., Schmid, J. E., Retief, J., Turpaz, Y., Zhang, X., Jones, P. D., Newsted, J. L., Giesy, J. P., Wolf, D. C., Wood, C. R., Bao, W., and Dix, D. J. ( 2005) Profiling Gene Expression in Human H295R Adrenocortical Carcinoma Cells and Rat Testes to Identify Pathways of Toxicity for Conazole Fungicides. The Toxicologist. Abstract No. 2150.

Sun G, Thai SF, Tully DB, Lambert GR, Goetz AK, Wolf DC, Dix DJ, Nesnow S: Propiconazole induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver. Tox Lett 155: 277 287, 2004; and abstracts from the 2005 SOT meeting,

Ward, W., Delker, D., Hester, S., Thai, S F., Allen, J., Jones, C., Wolf, D., and Nesnow, S. ( 2005) Comparison of Hepatic Gene Expression Profiles from Mice Exposed to Three Toxicologically Different Conazoles. The Toxicologist. Abstract No. 2147.

Wolf, D. C., Allen, J., Sun, G., Thibodeaux, J., George, M., Hester, S. D., Thai, S. F., Delker, D., Nelson, G., Winkfield, E., Roop, B., Leavitt, S., Ward, W., and Nesnow, S. ( 2005) Triadimefon Induces Rat Thyroid Tumors Through a Non TSH Mediated Mode of Action. The Toxicologist. Abstract No. 2144.

MRID 45284011. Rakhmatov, R. M., V. B. Danilov, U. A. Madzhidov, and S. A. Gamiyants. ( 1991). Basis data for setting the limiting allowed concentration ( LAC) of 1,2,4 triazole in workplace air. Gigiena i Sanitariya 2: 30 1. [ Abstract only].

MRID 46553701. Heard, N. E. ( 2005). 1,2,4 Triazole Aggregate Exposure Assessment, Syngenta Study T022748 04, May 20, 2005 Attachments:

1. TXR 0052011. Report of the Ad Hoc Triazole HED Peer Review Committee 2. TXR 0052012. Second Report of the Ad Hoc Triazole HED Peer Review Committee 3. Triazole Summaries  Additional Data Summaries/ Literature Articles 75 of 94 Attachment 1: Report of the Ad Hoc Triazole HED Peer Review Committee TXR NO. 0052011 DATE: August 5, 2003 MEMORANDUM SUBJECT: TRIAZOLES  Report of the Ad Hoc HED Peer Review Committee.

FROM: Kathleen Raffaele, Toxicologist Registration Action Branch 3 Health Effects Division ( 7509C)

THROUGH: Stephen Dapson Senior Scientist, Registration Action Branch 3 Health Effects Division ( 7509C)

TO: Bob Tomerlin Registration Division ( 7505C)

cc: Margaret Stasikowski, HED Elizabeth Doyle, OW PC Codes ( include but are not limited to): 004401; 120503; 128993; 128847; 123909; 129011; 128835; 128925; 128857; 125601; 122101; 120603; 128997; 109901; 127201; 128976 On 11/ 4/ 02, an ad hoc HED committee conducted an internal Peer Review of the OPP Triazole Team's 7/ 8/ 02 analysis of the Triazolylalanine Group's ( TAG) 1/ 9/ 02 document entitled " Profile of the Triazole derivative Fungicide Compounds and their Common Metabolites"( MRID# 45575501). The committee, led by Elizabeth Doyle, addressed toxicology and dietary exposure issues associated with this class of fungicides and its metabolites/ degradates of concern. The conclusions of the committee with respect to these issues are presented in this report. 76 of 94 Ad Hoc HED Peer Review Committee Members in Attendance Elizabeth Doyle ( Chair), Karl Baetcke, William Burnam, Vicki Dellarco, Steve Knizner, Nancy McCarroll, Alberto Protzel, Jess Rowland, and Brenda Tarplee ( Executive Secretary).

OPP Triazole Team Members in Attendance Steve Dapson, William Hazel, Rick Loranger, Kathleen Raffaele, Clark Swentzel, and Jean Holmes ( EFED).

Also in attendance were: Rick Keigwin ( RD), Timothy McMahon ( AD)

Chemistry Data Evaluation / Report Presentation: _______________________ Rick Loranger, Chemist _______________________ William Hazel, Chemist Toxicology Data Evaluation / Report Presentation: _______________________ Kathleen Raffaele, Toxicologist 77 of 94 INTRODUCTION On 11/ 4/ 02, an ad hoc HED committee conducted an internal Peer Review of the OPP Triazole Team's 7/ 8/ 02 analysis of the Triazolylalanine Group's ( TAG) 1/ 9/ 02 document entitled " Profile of the Triazole derivative Fungicide Compounds and their Common Metabolites" ( MRID# 45575501). The committee, led by Elizabeth Doyle, addressed toxicology and dietary exposure issues associated with this class of fungicides and its metabolites/ degradates of concern.

I. DIETARY EXPOSURE CONCLUSIONS The conclusions/ recommendations of the Peer Review Committee ( PRC) related to dietary exposure are as follows:

1. The PRC agreed with the OPP Triazole Team that there will be dietary exposure to 1,2,4 triazole and its conjugates, that such exposure could be significant, and that reliable estimates of dietary exposure cannot be made based upon available data.

2. The PRC had no issues with the dietary exposure components of the 7/ 8/ 02 OPP Triazole Team's evaluation of the TAG's 1/ 9/ 02 risk assessment including responses to TAG conclusions 3 and 4. The OPP Triazole Team's responses were as follows ( the last four bullets are responses to TAG conclusions 3 and 4):

3. The TAG continues to exclude 1,2,4 triazole conjugates from dietary exposure which the Agency currently considers to be residues of concern for the triazole fungicides.

4. Exclusion of the triazole conjugates results in a great underestimate of dietary exposure.

5. Even if found to be less toxic than free 1,2,4 triazole, the much higher exposure to the conjugates is expected to result in significant risk.

6. Calculation of free triazole residues based on the amount of parent fungicide at harvest also is expected to greatly underestimate dietary exposure ( the soil serves as a reservoir).

7. Exposure to free and conjugated residues in rotational crops, often with some of the highest residues, is expected to result in significant dietary exposure underestimation.

8. Use of 100% crop treated and 95th percentile of exposure are appropriate assumptions for a Tier 1 assessment but they may not be that conservative due to crop rotation and the soil serving as a reservoir of triazole degradates/ metabolites. 78 of 94 9. With all of these exposure uncertainties, hazard uncertainties, and sources of potentially great exposure underestimation, risk due to exposure to free plus conjugated triazole residues is likely to be much higher than the TAG's estimates for free triazole alone.

10. The TAG states that free triazole formation in livestock commodities and water is compound specific and should be considered only in the context of each individual triazole fungicide's risk. The first statement is likely correct, but the law requires all sources of exposure be considered in aggregate risk assessment.

11. The TAG states that residues of free triazole in plant commodities are very low and occur in < 15% of samples. We agree there are low, infrequent residues in raw crops, but effects of processing on triazole formation are unknown.

12. The TAG further states that plant residues have a low contribution to total dietary load. We believe that insufficient data exist to determine relative exposure contributions of plant, livestock and water residues.

13. The TAG also believes that there is no need to include free triazole in crop residue definitions. We agree that free triazole should not be part of the residue definition for tolerance enforcement purposes due to its numerous sources. However, from a risk assessment perspective, both free triazole and its conjugates need to be included based on toxicological properties.

3. The PRC agreed that monitoring of 1,2,4 triazole and its conjugates in the U. S. food supply would provide representative and rapid data to permit assessment of human dietary risk associated with this class of fungicides needed for risk management decisions on numerous registration actions and several reregistration eligibility decisions. Based on their significance as major food consumption items ( particularly for children), based on properties of free triazole and its conjugates, and based on the need to represent livestock commodities and extensive crop rotation practices, it was highly recommended that survey/ monitoring data be generated on milk, eggs, and soybean protein isolate.

4. The PRC agreed that the TAG be requested to perform cooking and processing studies ( including baking studies to assess flour residues) to determine the potential for, and extent of, conversion of parent fungicides and triazole conjugates to free 1,2,4 triazole at elevated pressure, temperature, and at the pHs of various processed products as well as the fractionation of the various residues in different processed products. In addition, the PRC recommended that the TAG be requested to perform studies in which livestock are fed triazole conjugates to determine distribution of the various residues in tissues and milk and to determine 79 of 94 the likelihood and extent of conversion of triazole conjugates to free 1,2,4 triazole in livestock ( metabolism may be more rapid in the rumen).

II. TOXICOLOGY CONCLUSIONS The conclusions/ recommendations of the Peer Review Committee ( PRC) related to toxicology are as follows:

1. The PRC concluded that, based on LD50 values, the acute toxicity of free triazole and triazole alanine are comparable to the parent compounds.

2. The PRC concluded that the subchronic toxicity data indicate that triazole alanine is less toxic than free triazole; however, there is no data available to evaluate neurotoxic potential.

3. The PRC concluded that there is no need for additional mutagenicity data; available data indicate that free triazole and triazole alanine are not mutagenic.

4. The PRC agreed with the OPP Triazole Team that there is concern for toxicity of free triazole, triazole alanine, and other triazole conjugates 5. The PRC concurred with the OPP Triazole Team that free triazole has not been adequately tested in toxicity studies with the parent triazole fungicides.

6. The PRC determined that no analogies could be made between toxicity of parents and expected toxicity of free triazole and triazole conjugates, based on the following considerations:

° Target tissues and toxicological effects vary across parent compounds, with some overlap but no consistent pattern.

° Carcinogenicity also varies across parent compounds, as does tumor site for carcinogenic parents.

° The relationship between toxicity of free triazole, triazole conjugates, and parent compounds ( based on available limited data), is not consistent; e. g. some parent compounds exhibit toxicity at lower doses than free triazole, others at higher doses.

7. The PRC determined that separate risk assessments should be conducted for free triazole and for triazole conjugates, and selected endpoints and Uncertainty Factors: 80 of 94 ° For free triazole, the developmental toxicity study, with a NOAEL of 30 mg/ kg should be used for both acute and chronic risk assessments; UF= 1000 ( 10x intraspecies, 10x interspecies, 10x database).

° For triazole alanine and other triazole conjugates, the developmental toxicity study, with a NOAEL of 100 mg/ kg, should be used for both acute and chronic risk assessments; UF= 300 ( 10x intraspecies, 10x interspecies, 3x database).

° The developmental endpoints from the rat developmental toxicity studies for free triazole and triazole alanine were selected for use based on the available limited database, and are considered to be protective for all risk assessment scenarios, in the absence of reliable alternative endpoints.

° The use of a smaller database factor ( 3x) for triazole alanine and other triazole conjugates is based on the following considerations: ° Available studies for triazole alanine showed minimal toxicity in adults at doses approaching the limit dose; ° There is no indication of neurotoxicity in the available triazole alanine studies; ° If additional studies were requested for triazole alanine, they would likely also be conducted at doses approaching the limit dose, leading to endpoints higher than that selected above from the developmental toxicity study; ° The absence of a developmental toxicity study in rabbits, given the findings in the developmental toxicity study in rats, supports the use of an additional 3x database factor.

8. The PRC determined toxicology data needs for free triazole and triazole alanine ( following evaluation of requested studies, it is possible that additional data may be needed):

° For free triazole, the following studies should be required: Acute neurotoxicity study in rat Combined 90 day/ subchronic neurotoxicity study in rat Developmental toxicity study in rabbit Reproductive toxicity study in rat ( 2 generation) Two year chronic toxicity/ oncogenicity study in female mice and male rats ° For triazole alanine, requirements for additional studies should be reserved pending re review of previously submitted studies or results of monitoring studies. 81 of 94 III. REFERENCES Triazolylalanine Group ( TAG). January 9, 2002. Profile of the Triazole derivative Fungicide Compounds and their Common Metabolites. MRID# 45575501.

Review of " Profile of the Triazole derivative Fungicide Compounds and their Common Metabolites". Memorandum from C. Swentzel to R. Keigwin dated July 8, 2002. 82 of 94 Attachment 2: Second Report of the Ad Hoc Triazole HED Peer Review Committee TXR NO. 0052012 DATE: August 5, 2003 MEMORANDUM SUBJECT: TRIAZOLES  2nd Report of the Ad Hoc HED Peer Review Committee.

FROM: Kathleen Raffaele, Toxicologist Registration Action Branch 3 Health Effects Division ( 7509C)

THROUGH: Stephen Dapson Senior Scientist, Registration Action Branch 3 Health Effects Division ( 7509C)

TO: Bob Tomerlin Registration Division ( 7505C)

PC Codes ( include but are not limited to): 004401; 120503; 128993; 128847; 123909; 129011; 128835; 128925; 128857; 125601; 122101; 120603; 128997; 109901; 127201; 128976 On 11/ 4/ 02, an ad hoc HED committee conducted an internal Peer Review of the OPP Triazole Team's 7/ 8/ 02 analysis of the Triazolylalanine Group's ( TAG) 1/ 9/ 02 document entitled " Profile of the Triazole derivative Fungicide Compounds and their Common Metabolites"( MRID# 45575501). The committee, led by Elizabeth Doyle, addressed toxicology and dietary exposure issues associated with this class of fungicides and its metabolites/ degradates of concern, including toxicology data needs ( see Memo dated July 10, 2003, TXR# 0052011, K. Raffaele to Bob Tomerlin). On June 16, 2003, the Committee reconvened, to address a data waiver request submitted by the US Triazole Task Force ( USTTF, DP Barcode 289197), requesting that data requirements for the acute neurotoxicity study, the rabbit developmental study, and the chronic/ oncogenicity studies on male rats and female mice be waived. 83 of 94 Ad Hoc HED Peer Review Committee Members in Attendance Elizabeth Doyle, Karl Baetcke, William Burnam, Vicki Dellarco, George Herndon ( substituting for Steve Knizner), Alberto Protzel, Jess Rowland, and Brenda Tarplee ( Executive Secretary).

OPP Triazole Team Members in Attendance Kathleen Raffaele, Paula Deschamp, and Mike Metzger.

Also in attendance were: Timothy McMahon ( AD)

Toxicology Data Evaluation / Report Presentation: _______________________ Kathleen Raffaele, Toxicologist 84 of 94 INTRODUCTION On 11/ 4/ 02, an ad hoc HED committee conducted an internal Peer Review of the OPP Triazole Team's 7/ 8/ 02 analysis of the Triazolylalanine Group's ( TAG) 1/ 9/ 02 document entitled " Profile of the Triazole derivative Fungicide Compounds and their Common Metabolites" ( MRID# 45575501). The committee, led by Elizabeth Doyle, addressed toxicology and dietary exposure issues associated with this class of fungicides and its metabolites/ degradates of concern, including toxicology data needs ( for conclusions from this meeting, see TXR# 0052011). On June 16, 2003, the Committee reconvened, to address a data waiver request submitted by the US Triazole Task Force ( USTTF, DP Barcode 289197), proposing that data requirements for the acute neurotoxicity study, the rabbit developmental toxicity study, and the chronic/ oncogenicity studies on male rats and female mice be waived.

BACKGROUND In their meeting on 11/ 4/ 02, the ad hoc Triazole Peer Review Committee ( PRC) determined toxicology data needs for free triazole. These data needs were transmitted to the US Triazole Task Force ( USTTF) in November, 2002. The OPP Triazole Team met with the USTTF to discuss toxicology data needs on February 20, 2003, and a detailed proposal for generation of toxicology data, including a rationale for waiver of several studies, was submitted by the USTTF on March 28, 2003. This proposal was reviewed by the OPP Triazole Team, and the PRC was reconvened on June 16, 2003 to evaluate the proposed waiver.

CONCLUSIONS The conclusions/ recommendations of the Peer Review Committee ( PRC) regarding the toxicology study waiver requests are as follows:

1. Acute Neurotoxicity Study The PRC determined that the data requirement for the acute neurotoxicity study should be placed in reserve, pending the outcome of the combined subchronic/ neurotoxicity study in rats. Upon receipt of the combined subchronic/ neurotoxicity study, the need for the acute neurotoxicity study will be reassessed. In the absence of data from an acute neurotoxicity study, the PRC concluded that the use of the developmental endpoint from the developmental toxicity study in rats, when used for acute risk assessments in all populations, would be protective for effects that might have been seen in the acute neurotoxicity study.

2. Developmental Toxicity Study in Rabbits The PRC reaffirmed their previous decision that the developmental toxicity study in rabbits should be required. Given the developmental effects seen in the rat study, the potential for developmental toxicity cannot be fully evaluated in the absence of a study in rabbits ( to assess the possibility that the rabbit may be more sensitive than the rat). Although the USTTF argued, 85 of 94 based on results from parent compounds, that the rat is more sensitive to developmental toxicity from triazole derivative compounds, the PRC reaffirmed its previous decision that the toxicity of free triazole cannot be predicted based on results from parent compounds. The PRC also noted that the results from studies in parent compounds were mixed with respect to most sensitive species for developmental effects ( for some compounds, lower NOAELs were seen in rabbit studies).

3. Chronic toxicity/ oncogenicity studies in male rats and female mice The previous recommendation for chronic toxicity/ oncogenicity studies in male rats and female mice was based on the widespread presence and persistance of free triazole in the environment and concerns regarding possible carcinogenicity of free triazole. To perform an adequate assessment of carcinogenicity, the PRC determined that both rat and mouse studies were needed.

The USTTF argued that chronic toxicity studies for free triazole are not needed; they suggested that chronic endpoints could be extrapolated based on subchronic data, since subchronic and chronic endpoints are within an order of magnitude for parent compounds. As mentioned above, the PRC previously concluded that toxicity of free triazole could not be predicted based on results of studies conducted in parent compounds. In addition, the PRC noted that the relationship between subchronic and chronic NOAELs/ LOAELs has much to do with dose selection, so comparisons of NOAELs/ LOAELS between subchronic and chronic studies provides limited information. The PRC also noted that subchronic studies do not evaluate the potential for carcinogenicity, which is seen with many triazole derivative compounds.

The USTTF stated that the parent triazole compounds are not mutagenic or genotoxic, and that available mutagenicity data for free triazole are also negative, mitigating the need for carcinogenicity data. The PRC agreed that available mutagenicity data are negative, but also noted that many triazole derivative compounds are carcinogenic, in the absence of mutagenicity or genotoxicity, limiting the reliance that can be placed on these negative data.

The USTTF stated that there was no indication of increased toxicity over time in the available 30 and 90 day studies with free triazole, and no accumulation of free triazole was seen in available metabolism studies. The PRC concluded that differences in the route of administration ( gavage vs. dietary) and dose selection, and limitations on the endpoints evaluated, preclude direct comparison of results across the 30 and 90 day studies ( both of which were unacceptable, with multiple deficiencies). The PRC also noted that the length of the cited studies was not substantially different, when compared to the duration of chronic studies. Regarding the metabolism data, the PRC noted that cumulative toxicity can occur due to repeated insult, without need for bioaccumulation, therefore the presence or absence of bioaccumulation is not definitive. In addition, the estimated half life of 10 12 h from these available metabolism studies does not preclude the possibility of bioaccumulation. The PRC also noted that the available metabolism studies for free triazole are limited ( single exposure only), and have been classified as Unacceptable, based on multiple deficiencies ( including inadequate methodological information, inadequate characterization of test substance, etc.); the reliability of these data is therefore questionable. 86 of 94 The USTTF indicated that additional endpoints would be included in subchronic rat and mouse studies to address possible cancer issues. Based on review of submitted protocols by the Triazole team, the added endpoints were limited ( retention of liver tissue for possible enzyme analyses [ criteria for performance of analyses was not provided] and analysis of thyroid hormone levels in the subchronic rat study). No mechanistic data were proposed or provided, nor was there any discussion of how proposed enzyme analyses ( if performed) or thyroid hormone analyses would be predictive of carcinogenicity.

In conclusion, the PRC determined that the subchronic studies do not assess carcinogenicity, increased severity of effects, or changes in dose/ response curve, which may occur with long term continuous exposure. In the absence of chronic toxicity/ oncogenicity studies, long term risk from exposure to free triazole, including possible carcinogenic effects, cannot be evaluated. The requirement for chronic toxicity/ oncogenicity studies in male rats and female mice was reaffirmed. 87 of 94 Attachment 3: Triazole Summaries  Additional Data Summaries/ Literature Articles 1. O. Hockwin and A. Wegener. ( 1989). Final Expert Opinion on the In Vivo examination of the lens using slit lamp microscope and Schiempflug photography and post mortem biochemistry of the lenses from Bayer Study T 3 027 392 in Beagle Dogs. Department of Exp. Ophthalmology of the Rheinische Friedrich Wilhelm University Bonn, Sigmund Freud Street 25, 5300 Bonn 1, Germany. Laboratory Project ID AC Report No. 109988. MRID 45284013. Unpublished.

This document contains information regarding a study evaluating ocular effects in Beagle dogs during exposure to several Bayer compounds, including tebuconazole ( referred to in the document as HWG 1608) and triazole ( 1,2,4 Triazole). The document contains no information regarding the study protocol, exposure levels, or clinical signs in the exposed animals. Administered doses are not described in the document, although a listing on one page seems to indicate doses of 100 mg/ kg for both triazole and tebuconazole. The report does contain detailed information regarding the ocular findings, including in vivo examinations and in vitro studies of changes in lens biochemistry. Statistically significant changes were found on some parameters for both tebuconazole and triazole. Given the lack of procedural information, the results are difficult to interpret.

Unless additional information can be provided for this study ( for example, clinical signs or necropsy data, and a more adequate explanation of the procedures), the data available do not provide information usable in assessing risk from triazole exposure.

2. J. Thyssen and G. Kimmerle. ( 1976). 1,2,4 Triazole Occupational Toxicology Study. Bayer AG, Department of Toxicology, Friedrich Ebert Strasse 217 333, D 42096 Wuppertal, Germany. Bayer AG Report No. 5926. MRID 45284004. Unpublished.

This document contains short summaries of several acute toxicity studies on 1,2,4 triazole ( studies are described separately below). Very little information was provided on the specific procedures used, nor was there analytical data on the test substance.

Acute oral toxicity: 1,2,4 triazole was administered by gavage to Wister rats ( size of most dose groups was 15/ sex, but there was some variation among doses). Doses ranged from 250 2500 mg/ kg for males, 100 2500 mg/ kg for females; there were 9 dose levels/ sex, with dose intervals usually varying by 250 mg/ kg. Results were presented as the number of animals/ group who died or showed clinical signs, as well as a range of days on which death occurred for each group. An LD50 was also calculated separately for each sex.

For males, clinical signs were not seen at 250 mg/ kg, but were seen in all animals for all other dose groups ( starting at 500 mg/ kg). For females, clinical signs were not seen at 100 mg/ kg, but were seen in all animals for all dose groups starting at 250 mg/ kg. Clinical signs were not listed for individual animals, but the summary listed the following clinical signs: reduction in general well being, sedation, and breathing disorders; at higher doses, lying in the abdominal or side position ( doses at which these occurred was not stated, time of occurrence was given as " within 88 of 94 an hour of administration and ... for a maximum of up to 13 days after administration" [ study report, p. 6]). The lowest dose at which death occurred was 1250 mg/ kg for both sexes, with the death occurring on the day of dosing. The oral LD50 was similar for both sexes ( 1650 mg/ kg for males, 1648 mg/ kg for females).

Dermal toxicity: 1,2,4 triazole ( moistened with Cremophor EL) was applied to the shaven dorsal skin of male and female Wistar rats. After 24 h, substance was washed off ( with soap and water). Animals were observed for 14 days. The summary stated that studies were carried out using the occlusive dressing method ( not further described, although a citation was provided). Group size varied from 5 to 20, with most groups including 10 animals/ sex. The dose range was 1000 5000 mg/ kg for males ( 4 dose groups) and for females ( 6 dose groups). Clinical signs were seen in all animals from all treatment groups. Again, individual data were not provided but the summary stated that the symptoms were similar to those seen in the oral studies. Death was seen at the 2500 mg/ kg dose in both sexes; time of death varied from 1 9 days after treatment, with earlier deaths occurring after exposure to higher dose levels. The calculated dermal LD50 was somewhat lower for females ( 3129 mg/ kg) than for males ( 4200 mg/ kg).

Inhalation: No effects were observed in the inhalation studies ( using 5 male rats and 5 male mice), but the summary also states that " no substance vaporized or atomized in the 4 and 6 hr. experiments", so it is not clear that any exposure actually occurred.

Dermal irritation: 1,2,4 triazole was applied to cellulose patches ( 1.5 cm2, 500 mg/ patch). The patches were applied for 24 h to the hairless skin of the ears of 2 rabbits. Treated skin was observed for 7 days following removal of patches. The summary stated that the ' skin revealed no changes'.

A similar study was also performed using similar treatment on the human forearm ( specific procedure and dose not provided), with exposure from 2 8 h and 7 day observation periods ( a total of six subjects, 1 female and 5 male). The summary stated that treated skin was ' physiologically normal' after removal of the dressing and for the remainder of the observation period.

Ocular irritation: 1,2,4 triazole was applied to the conjuctival sac of the left eye ( 50 mg/ animal) of 2 rabbits. Intense reddening and very intense swelling of the eye and conjuctivae were observed immediately after application and at 24 h. After 5 days, one animal had returned to normal but the other still had some redness and swelling of the conjuctivae. Effects on the cornea and iris were also observed during the first 2 days after application. The test substance was classified as a severe eye irritant.

Summary: The report concluded that 1,2,4 triazole was moderately acutely toxic by the oral and dermal route, and that the central nervous system was affected at high oral and dermal doses.

3. P. K. Chan, P. M. Fisher, and R. D. Morrison. ( 1981). 1,2,4 Triazole Acute range finding studies. Rohm and Haas Toxicology Department, 727 Norristown Rd., Spring House, PA 19477. Rohm and Haas Report No. 81R 0057. MRID No. 45284006. Unpublished. 89 of 94 and K. R. Procopio and J. D. Hamilton. ( 1992). 1,2,4 Triazole Acute range finding studies. Rohm and Haas Company, Toxicology Department, 727 Norristown Rd., Spring House, PA 19477. Rohm and Haas Report No. 81R 057A. MRID No. 45324301 ( Supplemental submission to EPA MRID No. 45284006). Unpublished.

Both of the above cited documents provide information regarding the same series of acute toxicity studies on 1,2,4 triazole, with the second submission including additional information not provided in the first submission.

Acute oral toxicity: 1,2,4 triazole ( Sample No. TD 81 112, Lot No. 113296, 92.8% pure), in 0.5% methylcellulose, was administered by gavage to male rats ( Crl: CDBR, 3/ group) at 0.5 or 5 mg/ kg. At 5.0 mg/ kg, all rats died within 10 min. of dosing; at 0.5 mg/ kg, all rats survived and no clinical signs were noted.

Acute dermal toxicity: 1,2,4 triazole ( lot number as above), was moistened with saline and applied to shaven intact skin of rabbits ( 2 males/ dose level) at 0.2, 2.0, and 5.0 g/ kg. Application sites were occluded. After 24 hours, covers were removed and site was wiped with paper towels. Animals were observed for 14 days; skin irritation was also recorded.

All animals treated with 2.0 or 5.0 g/ kg died; the higher dose animals died on days 1 and 2, the lower dose animals on day 3 and 4. Observed clinical signs at those doses included passiveness, scant droppings, soft feces, tremor, ataxia, salivation, abdominal breathing, gasping, and nasal discharges. No clinical signs were seen in animals treated at 0.2 g/ kg. Skin irritation was noted as well defined erythema and very slight edema. It was not clear at which dose( s) these irritant effects were observed.

Skin irritation: 1,2,4 triazole, 0.5 g/ patch, was moistened with saline and applied to the skin of 2 male rabbits. Two patches were applied to each rabbit, 1 on intact skin and 1 on abraded skin. Application sites were occluded, under a gauze lined patch and an impervious cuff, for 24 h, after which the patches were removed and the site wiped with paper towels.

No edema was observed for either site on either rabbit. Erythema, scored 1 or 2, was observed on one or both sites for both rabbits. Based on these results, 1,2,4 triazole was rated as ' slightly irritating' to skin.

Eye irritation: 1,2,4 triazole, 0.1 g/ eye, was applied to the conjunctival sac of the left eye of two male rabbits. Eye irritation was evaluated according to the method of Draize, for up to 14 days. Effects were seen on the cornea, iris, and conjunctivae starting at 4 h. By day 7, corneal and conjunctival effects had cleared. Iridal effects had cleared by day 14. Based on these results, 1,2,4 triazole was rated as ' substantially irritating' to the eye.

Summary: Findings in this series of studies were similar to those in the previous report. The only notable difference was that there were no clinical signs reported in the current acute oral 90 of 94 study at 0.5 g/ kg, while clinical signs were seen in both sexes in the previous acute oral study at 500 mg/ kg ( the same dose). We also note that only males were tested in the current study, and that females were found to be more sensitive in the previous report.

4. Wickramaratne, G. A. de S. ( 1987). The Chernoff Kavlock Assay: Its validation and application in rats. Teratogenesis, Carcinogenesis, and Mutagenesis 7: 73 83. MRID No. 45284010.

This document consists of a copy of a literature article reporting on a study intended to validate the use of an abbreviated Chernoff Kavlock assay in rats to predict chemicals likely to cause teratogenic effects ( testing was conducted at Central Toxicology Laboratory, Imperial Chemical Industries PLC, Cheshire, UK). Test substance was administered to pregnant rats ( Wistarderived from gestation days 7 17. Maternal observations consisted of maternal bodyweights on days 1, 7 17, and 22 of gestation; offspring observations included only litter weights of live pups on PNDs 1 and 5, and number of live and dead pups on PNDs 1 and 5.

Triazole, at dose of 25 and 100 mg/ kg, was included as one of the many substances tested in this assay. Authors reported no differences between triazole treated animals and control animals.

The parameters evaluated and reported in this study were very limited; data collected in the rat developmental studies conducted by Bayer are much more complete and would supercede those reported in this study.

5. Wickings, E. J., M. C. Middleton,. and S. G. Hillier. ( 1987). Non Steroidal Inhibition of Granulosa Cell Aromatase Activity in vitro. J. Steroid Biochem. 26 ( 6): 641 646. MRID No. 45284014.

In this published article, a substituted triazole ( R151885 [ 1,1 di( 4 fluorophenyl) 2 1,2,4 triazol 1 yl) ethanol) was evaluated in several in vitro assays for effects on steroid metabolism/ synthesis ( including granulosa cell estradiol production, progesterone production, and testosterone aromatization). A series of structurally related compounds were then evaluated, to determine which structual components of the compound were responsible for aromatase inhibition. Results indicated that imidazolyl or pyridyl moieties, when replacing the triazolyl substituent, produced more potent and specific inhibition.

These results appear to indicate that the triazole moiety was not the active component of this compound, with respect to inhibition of the aromatase enzyme in the assay used in this study.

6. Flucke, W. ( 1978). 1,2,4 Triazole. Determination of Acute Toxicity ( LD50). Bayer AG, Department of Toxicology, Friedrich Ebert Strasse 217 333, D 42096 Wuppertal, Germany. AC Report No. 109972. MRID No. 45284008. Unpublished.

This document consists of a one page report, with no procedural information or explanation of the data. It appears to report on an acute oral LD50 study; doses were administered to male rats only, 10/ dose. Doses ranged from 850 2500 mg/ kg ( 8 dose levels tested). It appears that clinical signs ( not described) were seen in all animals at all doses. The lowest dose at which death 91 of 94 occurred was 1200 mg/ kg, all animals died at 2000 and 2500. The LD50 was calculated to be 1375 mg/ kg ( range 1273 1485 mg/ kg).

The findings from this study appear to be similar to those described above.

7. Rakhmatov, R. M., V. B. Danilov, U. A. Madzhidov, and S. A. Gamiyants. ( 1991). Basis data for setting the limiting allowed concentration ( LAC) of 1,2,4 triazole in workplace air. Gigiena i Sanitariya 2: 30 1. [ Abstract only]. MRID No. 45284011.

This submission consists of a translated Russian abstract that describes very briefly a series of studies conducted for use in setting safe exposure levels ( in air) for 1,2,4 triazole. Little or no procedural information was provided for the various studies. The results reported are described below.  Oral LD50s for mice ( 3650 mg/ kg), rats ( 3080 mg/ kg) and rabbits ( 666 mg/ kg). Signs reported were lethargy and ataxia, with death on the ' first day of observation.'  Inhalation LC50 ( 4 hour) for mice ( 2200 mg/ m3) and rats ( 2050 mg/ m3).  Skin irritation was not seen upon dermal exposure to rats.  Eye irritation ( slight) was seen in rabbits.  Subacute exposure ( dose, route, and duration not specified) to rats demonstrated some potential for cumulative toxicity. Effects seen after repeated oral exposure were described as ' macrovesicular fatty dystrophy of the liver, plethora and acirculatory expansion of vessels in the kidneys with filling of canal loops of glomerulus capillaries by blood, and shallow hemorrhaging.'  Threshold concentration following single inhalation exposure ( duration not specified) was stated as 226.6 ± 10.5 mg/ m3, with findings of ' increased methemoglobin concentration and decreased cholinesterase activity in the blood.'  Effects of aerosol exposure were evaluated in rats and rabbits at doses of 19.8 ± 0.33 mg/ m3 and 4.9 ± 0.08 mg/ m3. Following 3 months exposure ( apparently at the higher dose), decreased levels of urea in urine were seen; following 4 months, decreased cholinesterase and increased methemoglobin were seen in blood. No effects were noted at the lower dose.  Mutagenic ( as represented by chromosome aberrations in rat marrow cells) or ' embryotropic' activity ( not further described) were not found ( studies not described but conducted using inhalation exposure at the above doses).

The abstract concluded that toxic effects of the compound included ' primarily effects on the liver, kidneys, and CNS.' A LAC of 5 mg/ m3 in workplace air was proposed.

8. Saratikov, A. S., E. M. Trofimovich, T. P. Novozheeva, T. A. Zimina, M. R. Ozhegina, and E. L. Morokova. ( 1986). Toxicological and Health Evaluation of 1,2,4 triazole for health standards in aquifers. Gigiena i Sanitariya 51 ( 11): 65. [ Abstract only]. MRID 45284012.

This submission also consists of a translated abstract, describing briefly a series of studies conducted for use in evaluating safe levels of 1,2,4 triazole in aquifers. It was stated that the triazole used for studies was 98% pure. In addition to toxicity, several properties of triazole in aqueous solutions were evaluated. 92 of 94 Acute toxicity in mice and rats was evaluated following intraperitoneal administration of an aqueous solution. Average toxic dose was stated to be 1350 mg/ kg for mice and 1750 mg/ kg for rats, with clinical signs listed as decreased motor activity, excitability, muscle cramp, and ( in some cases) tremors and difficulty breathing.

Irritancy to skin was evaluated using a 25% solution, eye irritation using a 12.5% solution; no irritant effects were noted.

Cumulative properties were also evaluated, and it was stated that the compound has moderatively cumulative properties, but insufficient information was provided to interpret the stated results.

A six month study was performed in male white rats ( strain not specified), using i. p. doses of 0.02, 0.20, 2.0 mg/ kg. Evaluated parameters included body weights, blood cell counts, and a variety of clinical chemistry parameters; apparently no histopathology or necropsy data were collected. Stated results included an increase in urine protein content, changes in leukocytes, and changes in alkaline phosphatase activity at the high dose ( no other description provided). No changes were noted at the two lower doses. An LAC for aquifers was recommended at 4 mg/ dm3.

9. Holzum, B. ( 2000). Developmental toxicity of triazole fungicides, Characterization and risk assessment. Bayer AG, Department of Toxicology, Friedrich Ebert Strasse 217 333, D 42096 Wuppertal, Germany. AC Report No. 109973. MRID No. 45284009. Unpublished.

This submission consists of a copy of a presentation by a Bayer scientist, regarding common developmental toxicity of triazole fungicides, and discussion of classification of developmental effects under the European classification and labelling requirements ( used within the European Community). Several of the slide copies include tables listing the specific types of developmental toxicity seen following exposure to triazole fungicides ( including the doses at which effects were seen maternally and in offspring), with the rat and rabbit studies compared separately.

In rats, the summary notes that common effects generally seen include embryolethality, retarded development, and supernumerary ribs, with urinary tract variations and malformations seen in a subgroup of compounds. In rabbits, the only general effect noted was embryolethality, with a variety of malformations again seen in a subgroup of compounds.

Included in the summary is a reference to developmental toxicity studies using triazole; these studies have been reviewed separately and the results will not be discussed here.

With respect to common toxicity across triazoles, the report authors concluded " we may conclude that, to a certain extent, a typical pattern exists for triazole developmental toxicity in animals." ( p. 9), although they did point out some differences in the patterns of abnormalities across studies. The report author, however, attributes developmental effects to maternal toxicity for this group of compounds ( perhaps acting via maternal hypoxia or adrenal effects); this attribution of causation appears to be relevant in the European system of classification. 93 of 94 10. Menegola, E., M. L. Broccia, F. DiRenzo, and E. Giavini. ( date not provided). Poster Presentation: In Vitro Comparative Study of the Teratogenic Activity of Some Triazoles. University of Milan, Dept. of Biology, Milan Italy. [ Copy of a poster, presentation meeting not specified.] MRID No. 45344602. Unpublished.

This submission consists of a one page copy of a poster. Two substituted triazoles ( Flusilazole and fluconazole) and triazole were evaluated for teratogenic potential in vitro in 48 h whole embryo cultures of 9.5 day old rat embryos. Methodological details regarding the culture conditions ( including the number of embryos evaluated in each condition) and analytical procedures were not provided. The results stated that flusilazole ( 6.25 250 µ M) and fluconazole ( 125 500 µ M) " showed abnormalities at the branchial apparatus level and increased cell death at the branchial mesenchymes" ( hypoplasia of I and II branchial arches and fusion of I II branchial arches) without indication of developmental delays. Exposure to triazole ( 2500 5000 µ M) caused slight developmental delays and severe anemia of the visceral yolk sac. Authors concluded that both mono triazole derivatives showed teratogenic potential, in particular for cranio facial abnormalities, but that triazole was not teratogenic.

We note that insufficient information was provided for us to evaluate the conclusions stated in this document, and that the conditions of exposure for this in vitro study may not represent those that would occur in vivo. We also note that the results from the available developmental toxicity study in rats would supercede findings from this study with respect to evaluating teratogenic effects of triazole.

Old metabolism studies In a series of metabolism/ disposition studies ( MRID 45284018, 45284019, and 45297202), groups of male and female Sprague Dawley rats were given a single intraduodenal ( 1 mg/ kg), intravenous ( 0.1 100 mg/ kg), or oral dose ( 0.4 866 mg/ kg) of 14C labeled triazole ( MRID 45284018   Lot No. not reported, sp. act. 470 µ Ci/ mg, purity   97%; MRID 45284019   Lot No. CL V 70, sp. act. > 67 µ Ci/ mg, purity > 98%; MRID 45297202, Lot No. CFQ 2458, sp. act. 103 µ Ci/ mg, purity   99%). The test material is a common moiety found in several compounds under development by Ciba Geigy at the time these studies were conducted. Urine and feces were collected up to 7 days postdose and expired air was monitored for residual activity. Bile samples were collected up to 24 hours after treatment with the radiolabel. Tissue/ carcass burdens were investigated at selected time intervals up to 7 days postdose.

No treatment related signs of toxicity among any of the test animals were reported. Overall recovery of administered radioactivity ranged from 97 105%. Based upon urinary and biliary excretion and tissue/ carcass burden data, 80%  95% of the administered dose was absorbed. Elimination of administered radioactivity was primarily via the urine, accounting for 80 95% of the dose, and was ~ 95% complete within 48 hours after treatment. Elimination via expired air was negligible (< 0.1%). Fecal excretion typically accounted for < 15% of the administered radioactivity and was essentially complete within 48 hours. Biliary excretion accounted for approximately 10% of the dose during the 24 hr study period, and the results were suggestive of enterohepatic circulation and diffusion/ secretion by the stomach mucosa. 94 of 94 The only compound identified in the urine was the unchanged parent which constituted ~ 95% of the radioactivity. There were possibly three other potential polar metabolites in the urine, each constituted < 3% of the administered dose. Their structures were not elucidated, but all three seem to be more polar than triazole as judged by their delayed migration relative to triazole under normal phase TLC separation conditions. Based on the study results, triazole was nearly completely absorbed and rapidly excreted mostly unchanged in the urine.

These metabolism/ disposition studies in rats are Unacceptable/ Guideline and do not satisfy the requirements for a Metabolism and Pharmacokinetics Study [ OPPTS 870.7485 ( § 85 1)]. None of the studies were described in detail and gave the impression they were more laboratory notes than metabolic study reports. Neither the care and treatment of the animals nor the test material were adequately described. Details concerning dose preparation, such as stability and homogeneity were also lacking. Finally, the potential metabolites in the feces were not identified, particularly in those instances when the feces contributed > 5% of the total isotope recovery ( MRID 45284019). The study can be upgraded to acceptable if it can be shown that the feces also contained unchanged parent compound and if more information were provided for the test material ( description, purity, stability, homogeneity, etc.).