


EPA REGISTRATION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER  

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


[Morse Enterprises Limited, Inc. d/b/a KeyPlex]

[IN 11093]

	EPA has received a pesticide petition (IN 11093) from [Morse Enterprises Limited, Inc. d/b/a KeyPlex], [PO Box 2515, Winter Park, FL 32790] requesting, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 180.920 in order to permit trans-anethole as a fragrance in pesticide formulations at rates of 3% of the formulation when applied pre-harvest to crops.

(Options (pick one)
   
   	1. by establishing a tolerance for residues of NA-Remove

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

	[NA-Remove] in or on the raw agricultural commodity [NA-Remove] at [NA-Remove] parts per million (ppm).  EPA has determined that the petition contains data or information regarding the elements set forth in section 408 (d)(2) of  FDDCA; however, EPA has not fully evaluated the sufficiency of the submitted data at this time or whether the data supports granting of the petition. Additional data may be needed before EPA rules on the petition.

A. Residue Chemistry

	1. Plant metabolism.

	2. Analytical method. [NA-Remove.]

	3. Magnitude of residues. [NA-Remove.]


B. Toxicological Profile

	1. Acute toxicity.  [The oral acute toxicity of trans-anethole in rats and mice (LD50) has been reported as 1510 and 3050 mg/kg, respectively. The dermal acute toxicity of trans-anethole in rabbits (LD50) has been reported as >5000 mg per kg. In an acute eye irritation study in rabbits, no irritation occurred at up to 72 hours of observation. In a skin irritation test on New Zealand White rabbits, it was determined to be not irritating. In a sensitization (non-LLNA) study with guinea pigs, trans-anethole was noted to be a sensitizer.]

	2. Genotoxicty. [A wide variety of genotoxicity studies have been conducted on trans-anethole including bacterial reverse mutation tests, in vitro mammalian gene mutation tests, in vivo micronucleus tests where most have negative results. A few in vitro studies have been noted to have positive results including 3 reverse mutation studies with S. typhimurium and 2 mouse lymphoma gene mutation studies. The overall assessment considering all studies concludes that trans-anethole is not genotoxic.]

	3. Reproductive and developmental toxicity. [In a combined pre-natal/reproductive toxicity study (equivalent to OECD Guideline 414), groups of 10 pregnant Sprague-Dawley rats received oral (gavage) administration of trans-anethole in corn oil at 0, 35, 175 or 350 mg/kg bw/day for 7 days, then in a 7 day mating period (with untreated males), during gestation, parturition and lactation (up to 4 days). Unmated rats continued to be dosed for 25 days after the 7-day mating period. On day 4 of lactation, pups were examined, sacrificed and discarded. Parameters evaluated included: body weight, body weight gain, food consumption, male and female fertility, gestation index, implantation sites, length of gestation, number of stillborn pups, litter size, pup viability, pup weight, and clinical observations of pups. 

Maternal toxicity at the high dose (350 mg/kg bw/day) manifested as significant decreases in body weight and food consumption throughout study. Dams exhibited various types of clinical signs (urine-stained fur, emaciation and pale in appearance). In dams that died necropsy showed, congested lungs, raised yellow area in the liver and hematomas on the vessels supplying the implantation sites). Average gestation duration was increased (number of dams delivering on days 23 and 24 was increased over controls); number of dams with stillborn pups and with all pups dying before postpartum day 4 was significantly increased (P <0.01). At 175 mg/kg bw/day, maternal toxicity was characterized as significant decreases in body weight (GD 6 and 14) and food consumption (premating days 1-8).

Developmental toxicity at the high dose manifested as: significant (p<0.01) decreases in the number of live born pups (75) compared to controls (147); number of stillborn pups (18)  compared to controls (0); number of pups dying on day 1 and days 2-4 (8 and 7 respectively) compared to controls (0 and 0, respectively); viability index (80%) compared to controls (99.3%); number of surviving pups/litter on postpartum day 4 (7.5) compared to controls (14.6); live litter size on postpartum day 4 (12.0) compared to controls (14.6); and pup weight/litter on postpartum day 1 (5.1 g) compared to controls (6.2 g). No other effects were reported at the other doses. External examination of the fetuses revealed no anomalies.

For maternal toxicity, the NOAEL was 35 mg/kg bw/day and the LOAEL was 175 mg/kg bw/day based on decreases in body weight and food consumption.

For developmental toxicity, the NOAEL was 175 mg/kg bw/day and the LOAEL was 350 mg/kg bw/day based on decreases in litter size, pup viability and pup weight.]

	4. Subchronic toxicity. [In a subchronic feeding study, groups of male and female Sprague-Dawley rats (20/sex/dose) were fed diets containing trans-anethole at 0 (control), 150, 300, 600, and 900 mg/kg bw/day for 90-days. Parameters evaluated included: survival, clinical signs of toxicity, body weight, body weight gains, food consumption, food efficiency, ophthalmology, clinical pathology (hematology and clinical chemistry), organ weights, gross and histopathology. No treatment-related effects were seen on survival, clinicals signs, or ophthalmology. Minor changes seen in body weight and food consumptions at the high dose were attributable to palatability. Hematology revealed decreases in the mean cell volume and mean cell hemoglobin in females, and a decrease in platelet counts in both sexes at the high dose. Clinical chemistry showed significant alterations in liver enzymes including increases in gamma glutamyl transferase, alanine aminotransferase and aspartate aminotransferase and decreases in total protein, albumin and glucose levels at 600 mg/kg/day. Relative liver weights were increased at doses above 300 mg/kg/day. Gross necropsy revealed enlarged liver in males and uterine findings in females. Histopathology showed increased incidence of single cell necrosis in males at 600 mg/kg/day. Under the conditions of this study, the NOAEL was 300 mg/kg bw/day and the LOAEL was 600 mg/kg bw/day based on elevation in serum gamma glutamyltranferase in females and single cell necrosis in male rats.]

	5. Chronic toxicity. [In a chronic feeding study, rats were fed daily doses of anethole at 125 or 500 mg/kg for 52 or 16 weeks, respectively. At the higher dose level, slight changes in hepatic cells were observed in males, whereas at the lower dose, no adverse effects were observed.  

In a combined chronic and carcinogenicity study, male and female Sprague-Dawley rats were fed trans-anethole at concentrations of 0.25% (100 and 120 mg/body weight day, female and male, respectively), 0.5% (200 and 250 mg/kg body weight/day, female and male, respectively), and 1% (400 and 550 mg/kg body weight/day, female and male, respectively) for ~2 years (117 to 121 weeks).  Rats were weighed weekly for the first 26 weeks and monthly thereafter. Food and water consumption was recorded daily for first 32 weeks and then for 1 day/month for the remainder of the study. Hematology parameters evaluated were: erythrocyte count, hemoglobin concentration, mean corpuscular volume, hematocrit, mean corpuscular hemoglobin concentration, leukocyte count and differential count. Brain, caecum, heart, kidneys, liver, ovaries, spleen and testes were weighed. All the major organs underwent histopathological evaluations. No treatment related effects were seen on survival, body weight, body weight gain, food consumption, hematology, organ weights or gross necropsy. Histopathology revealed non-neoplastic liver lesions including sinusoidal dilation in males in the 1% group, in both sexes of the 0.5% group, nodular hyperplasia in males in the 0.5% group and in both sexes in the 1% group, and hepatocytic hypertrophy in the females of 0.5 and 1% groups. A small but statistically significant (p<0.005) increase in liver tumors (6/52 benign and 6/52 malignant) were seen in females at 1% when compared to controls (1/52 for both tumors). The malignant tumors were characterized as hepatocellular carcinomas and had a late onset (first tumor seen at 98 weeks), had no effect on survival of the rats, and the incidence (6/52; 11.5%) were within the historical control range (0-14%). Under the conditions of this study, the NOAEL was 100 mg/kg bw/day in males and 120 mg/kg bw/day in females. The LOAEL was 200 mg/kg bw/day in males and 250 mg/kg bw/day in females based on non-neoplastic lesions (nodular hyperplasia and hepatocytic hypertrophy) of the liver.

A Pathology Work Group (PWG) evaluated the liver lesions of the rats from the above noted study. The PWG confirmed the results reported by the study authors, and further concluded that the slightly increased incidence of hepatocellular neoplasms in females at 1% is of no significance to human safety, and that trans-anethole does not constitute a carcinogenic risk to humans.

In another study the carcinogenic potential of some naturally occurring and synthetic alkenyl benzene derivatives in two strains of mice. In the first experiment 51 male and 56 female CD-1 mice received post oral administration of anethole at 5 or 2.5 umol/g body weight twice weekly for 10 weeks. Mice were necropsied at 11-14 months. In the second experiment, 53 male CD-1 mice received intraperitoneal injection of trans-anethole on days 1, 8, 15 and 22 for a total dose of 9.45 umol/mouse and were sacrificed at 12 months. In the third experiment, 50 male B6C3F1 mice received intraperitoneal injection of trans-anethole for a total dose of 4.75 umol/mouse. Mice were sacrificed at 12 months. trans-Anethole showed no hepatocarcinogenic activity in the three experiments.]

	6. Animal metabolism. [Metabolism of trans-anethole in vivo has been well characterized in rodents and in humans, with cytochrome P450-catalyzed oxidation, epoxidation and o-demethylation as the major pathways and glycine conjugates of p-methoxyhippuric and p-methoxycinnamic acid carboxylic acids as the major metabolites in the urine.]

	7. Metabolite toxicology. [trans-Anethole is metabolized into metabolites that are not of toxicological significance.]

	8. Endocrine disruption. [In an in vitro test on yeast conducted to assess androgenic activity of essential oils including trans-anethole, no effects were reported. In an in vitro test with human endometrial cells, trans-anethole was negative in stimulating effects in the cells. This test was repeated and the results were also negative in the second round.  In an in vitro test with yeast, trans-anethole was determined to have estrogenic activity at high concentrations.]

C. Aggregate Exposure

	1. Dietary exposure. [trans-Anethole is currently found in a significant number of food and beverage products as a flavoring agent and in animal feeds. In addition, trans-anethole is naturally found in anise, fennel, thyme, cinnamon, clove bud, nutmeg, pepper, coriander seed, and dill seed. Therefore, the potential for aggregate exposure from dietary routes does exist for trans-anethole. While it is difficult to develop a precise estimate of total human exposure to trans-anethole, its low toxicity and metabolism into innocuous compounds that are readily excreted, indicate that expected exposures are likely to be of low toxicological concern.  The addition of trans-anethole in biopesticide products at 3% w/w of the formulation applied to food crops is not expected to significantly increase the dietary exposure potential of humans.]

	i. Food. [trans-Anethole when used as a fragrance in pesticide formulations at 3% w/w of the formulation applied pre-harvest according to 40 CFR 180.920 will come into contact with food crops. trans-Anethole is currently found in a significant number of food and beverage products in addition to being found naturally in several spices. While it is difficult to develop a precise estimate of total human exposure to trans-anethole, its low toxicity and metabolism into innocuous compounds that are readily excreted, indicate that expected exposures are likely to be of low toxicological concern.]

	ii. Drinking water. [trans-Anethole when used as a fragrance in pesticide formulations at 3% w/w of the formulation applied pre-harvest according to 40 CFR 180.920 is not expected to come into contact directly with drinking water. trans-Anethole is slightly soluble in water (111 mg/L at 25°C) and readily biodegradable. Any potential exposure via drinking water is not expected to be of toxicological concern.]

	2. Non-dietary exposure. [trans-Anethole is currently found in non-dietary products including pesticides (nonfood inert, fragrance uses) and occurs naturally in many plants. Therefore, the potential for aggregate exposure from non-dietary routes does exist for trans-anethole. While it is difficult to develop a precise estimate of total human exposure to trans-anethole, its low toxicity and metabolism into innocuous compounds that are readily excreted, indicate that expected exposures are likely to be of low toxicological concern.  The addition of trans-anethole in biopesticide products at 3% w/w of the formulation applied to food crops is not expected to significantly increase the exposure potential of humans.]

D. Cumulative Effects

	[trans-Anethole has no known common mechanism of toxicity with any other toxic substances.]

E. Safety Determination

	1. U.S. population. [The safety of trans-anethole to humans has been previously evaluated based on its use as a flavoring in foods and beverages and its natural occurrence in many foods. Many toxicity studies are available in the public literature for trans-anethole. Though there is a long history of anise and fennel preparations as herbal medicines, specific therapeutic and beneficial effects of trans-anethole have not been especially well documented although this is an area of active research. trans-Anethole is one of the active principles in culinary spices thought to reduce the risk of specific cancers, i.e., it can act as a chemo preventive agent. It also shows anti-thrombatic activity and protects against ethanol-induced gastric lesions in rats. trans-Anethole is approved by the FDA for use as a food additive under 21 CFR 180.60.  It is further classified as GRAS (generally recognized as safe) by the FDA.  Following an exhaustive review, The Flavour and Extract Manufacturers' Association (FEMA) Expert Panel concluded that trans-anethole does not pose a risk to human health when used as a flavoring substance.  The use of trans-anethole as a fragrance in biopesticide formulations at 3% w/w is not expected to significantly increase the exposure to humans.]

	2. Infants and children. [The history of human use and consumption and evaluations of such use do not suggest there will be a safety concern for infants and children with the use of trans-anethole as a fragrance in biopesticide formulations at 3% w/w.]

F. International Tolerances

	[No known tolerances exist for trans-anethole.]




