Benalaxyl-M Human Health Risk Assessment DP No. D416044

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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF

CHEMICAL SAFETY AND

POLLUTION PREVENTION

MEMORANDUM

March 3, 2015

SUBJECT: Benalaxyl-M. Human-Health Risk Assessment for Tolerances in/on Imported 

Grape and Tomato. 

PC Code: 113510 DP Barcode: D416044

Decision No.: 476296 Registration No.: None

Petition No.: 3E8162 Regulatory Action: Tolerance, No U.S. 

Registration

Risk Assessment Type: Single Chemical Case No.: NA

TXR No.: NA CAS No.: 688046-61-9

MRID No.: NA 40 CFR: 180.TBD

FROM: William Donovan, Ph.D., Chemist/Risk Assessor

Linda Taylor, Ph.D., Senior Toxicologist

Risk Assessment Branch V/VII

Health Effects Division (HED) (7509P)

THROUGH: Michael Metzger, Branch Chief

Risk Assessment Branch V/VII, HED (7509P)

Anna Lowit, Senior Toxicologist

Immediate Office, HED (7509P)

Christine Olinger, Branch Chief

Risk Assessment Branch III, HED (7509P)

TO: Bewanda Alexander, Risk Manager

Rachel Holloman, Branch Chief

Fungicide/Herbicide Branch 

Registration Division (7505P)

The Health Effects Division (HED) of the Office of Pesticide Programs (OPP) is charged with 

estimating the risk to human health from exposure to pesticides. The Registration Division (RD) 

of OPP has requested that HED evaluate hazard and exposure data and conduct assessments, as 

needed, to estimate the risk to human health that will result from the proposed uses of the new 

fungicide active ingredient benalaxyl-M. 
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HED has evaluated the toxicity and exposure databases for the new active ingredient, benalaxylM, and has conducted a human health risk assessment in support of the proposed uses. Based on 

this assessment, HED has determined that there are no potential risk estimates of concern for the 

proposed uses of benalaxyl-M. HED recommends that the requested uses be granted.

A summary of the findings and an assessment of human health risk resulting from the proposed 

uses for benalaxyl-M are provided in this document. The HED team members contributing to 

this risk assessment include Linda Taylor (hazard evaluation) and William Donovan (chemistry, 

dietary, and risk assessments).
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Table of Contents

1.0 Executive Summary............................................................................................................. 5

2.0 HED Recommendations....................................................................................................... 6

2.1 Data Deficiencies ............................................................................................................. 6

2.1.1 Toxicity Studies............................................................................................................ 6

2.2 Tolerance Considerations................................................................................................. 7

2.2.1 Enforcement Analytical Method .................................................................................. 7

2.2.2 International Harmonization......................................................................................... 7

2.2.3 Recommended Tolerances............................................................................................ 7

2.2.4 Revisions to Petitioned-For Tolerances........................................................................ 8

3.0 Ingredient Profile ................................................................................................................. 8

3.1 Chemical Identity ............................................................................................................. 8

3.2 Physical/Chemical Characteristics................................................................................... 8

3.3 Pesticide Use Pattern........................................................................................................ 8

3.4 Anticipated Exposure Pathways..................................................................................... 10

3.5 Consideration of Environmental Justice ........................................................................ 10

4.0 Hazard Characterization and Dose-Response Assessment................................................ 10

4.1 Toxicology Studies Available for Analysis ................................................................... 10

4.2 Absorption, Distribution, Metabolism and Elimination (ADME) ................................. 11

4.2.1 Dermal Absorption ..................................................................................................... 11

4.3 Toxicological Effects..................................................................................................... 12

4.4.1 Completeness of the Toxicology Database................................................................. 13

4.4.2 Evidence of Neurotoxicity.......................................................................................... 13

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal ............. 13

4.4.4 Residual Uncertainty in the Exposure Database......................................................... 13

4.5 Toxicity Endpoints and Points of Departure Selections................................................. 14

4.5.1 Dose-Response Assessment......................................................................................... 14

4.5.2 Recommendation for Combining Routes of Exposure for Risk Assessment .............. 14

4.5.3 Cancer Classification and Risk Assessment Recommendations ................................. 14

4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk 

Assessment................................................................................................................................ 15

5.0 Dietary Exposure and Risk Assessment ............................................................................ 16

5.1 Metabolite/Degradate Residue Profile ........................................................................... 16

5.1.1 Summary of Plant and Animal Metabolism Studies .................................................. 16

5.1.2 Comparison of Metabolic Pathways........................................................................... 16

5.1.3 Residues of Concern Summary and Rationale ........................................................... 16

5.2 Food Residue Profile...................................................................................................... 17

5.3 Water Residue Profile .................................................................................................... 18

5.4 Dietary Risk Assessment................................................................................................ 18

5.4.1 Description of Residue Data Used in Dietary Assessment......................................... 18

5.4.2 Percent Crop Treated Used in Dietary Assessment.................................................... 18

5.4.3 Acute Dietary Risk Assessment ................................................................................. 18

5.4.4 Chronic Dietary Risk Assessment .............................................................................. 18

5.4.5 Cancer Dietary Risk Assessment................................................................................ 18

5.4.6 Summary Table........................................................................................................... 19

6.0 Residential (Non-Occupational) Exposure/Risk Characterization .................................... 19
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7.0 Aggregate Exposure/Risk Characterization....................................................................... 19

8.0 Cumulative Exposure/Risk Characterization..................................................................... 19

9.0 Occupational Exposure/Risk Characterization .................................................................. 20

10.0 References.......................................................................................................................... 20

Appendix A. Toxicology Profile and Executive Summaries....................................................... 21

A.1 Toxicology Data Requirements...................................................................................... 21

A.2 Toxicity Profiles............................................................................................................. 22

A.3 Hazard Identification & Endpoint Selection .................................................................. 28

A.4 Executive Summaries..................................................................................................... 28

Appendix B. Metabolism............................................................................................................ 47

B.1 Metabolism Summary Table .......................................................................................... 47

B.2 Metabolic Pathways ....................................................................................................... 50

Appendix C. Physical/Chemical Properties................................................................................. 52

Appendix D. Review of Human Research................................................................................... 52
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1.0 Executive Summary

Benalaxyl-M is a new fungicide that is proposed for tolerances without U.S. registrations on 

grapes and tomatoes, with foliar application use proposed for these commodities. The end-use 

products include water-dispersible granules and wettable powders, where benalaxyl-M is coformulated with another active ingredient (mancozeb, folpet, or chlorothalonil). These fungicide 

formulations are intended to control downy mildew (Plasmopara vitacola) in grapes and late 

blight (Phytophthora infestans) in tomato.

The database of experimental toxicology studies available for benalaxyl-M provides a robust 

characterization of the hazard potential of this pesticide. A comparative thyroid assay in rats is 

required to assess the potential impact of benalaxyl-M exposure on thyroid function in the young 

given the pivotal role of thyroid hormones in brain development.

The liver and thyroid are the primary target organs for benalaxyl-M. In rats, increased liver 

weights, clinical chemistry changes indicative of liver toxicity, hepatocellular hypertrophy, and 

thyroid follicular cell hypertrophy were seen following subchronic and chronic exposure. In 

mice, increased liver weight and microscopic lesions in the liver (hepatocellular hypertrophy, 

necrosis, eosinophilic foci) were observed following subchronic and chronic exposure. 

Additionally, chronic exposure in rats and mice led to increases in the incidence of liver (rat, 

mouse) and thyroid (rat) tumors. In dogs, increased liver weight, changes in clinical chemistry

indicative of liver toxicity, and hepatocellular hypertrophy were observed following subchronic 

exposure via the diet, whereas clinical chemistry changes indicative of liver toxicity, fat 

vacuolation in the liver, and thyroid follicular cell hypertrophy were observed following chronic 

exposure via capsules. No evidence of increased quantitative or qualitative susceptibility was 

seen in the benalaxyl-M hazard database following in utero exposure with rats or rabbits in the 

prenatal developmental studies or in young rats in the 2–generation reproduction study. No 

evidence of maternal toxicity or developmental effects was observed in the developmental 

toxicity studies in rabbits or rats. There is no reproductive concern. No neurotoxic effects were 

observed in the acute and subchronic neurotoxicity studies in rats, and no immunotoxic effects 

were observed in the immunotoxicity study in rats. Benalaxyl-M was classified as “Likely to be 

Carcinogenic to Humans”. This determination was based on the treatment-related liver tumors 

observed in male mice, liver tumors observed in male and female rats; and thyroid follicular cell 

tumors observed in female rats. No treatment-related tumors were observed in female mice. A 

linear low-dose extrapolation model (Q*1) was used to estimate cancer risk, based on the male 

mouse liver tumor rates. There is no mutagenicity concern from the in vivo or in vitro genetic 

toxicity assays.

Toxicological endpoints were selected for dietary exposure scenarios only. An acute dietary

endpoint was not selected because toxicity from a single exposure was not identified in the 

benalaxyl-M database. The chronic population adjusted dose (cPAD) is derived from the chronic 

toxicity/carcinogenicity study in rats, which had a lowest observed adverse effect level (LOAEL)

of 135 mg/kg/day based on an increase in gamma glutamyl transferase (GGT) in males, 

increased triglycerides in males, increased cholesterol in both sexes, slight increases liver weight 

in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased 

incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell 
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hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and an 

increased incidence of ovarian stromal cell hyperplasia in females and a no observed adverse 

effect level (NOAEL) of 20 mg/kg/day. For the point of departure (POD) for the chronic dietary

risk assessment, a total uncertainty factor of 1000X was applied (10X for interspecies variability, 

10X for intraspecies variability, and 10X for database uncertainty to account for the lack of a 

comparative thyroid assay). A full 10X factor was retained for interspecies variability since 

other effects not linked to thyroid perturbations were also the basis of the POD. 

The residue chemistry database is complete for the proposed uses, supporting a parent only 

residue definition for tolerance enforcement and risk assessment. Based on the use of the 

Organization for Economic Cooperation and Development (OECD) Maximum Residue Limit 

(MRL) calculation procedures with residues reflecting critical Good Agricultural Practice 

(cGAP) use patterns, the recommended tolerance levels are 3.0 and 0.20 ppm for grape and 

tomato, respectively. 

A risk assessment was conducted for dietary exposure only based on the request for 

establishment of tolerances for import uses in/on grapes and tomatoes. There are no drinking 

water, occupational or residential exposure assessments completed for non-domestic uses. 

Chronic dietary analyses were performed using conservative assumptions (i.e., 100% crop 

treated and mean field trial residue levels). There is no U.S. drinking water exposure associated 

with the establishment of an import tolerance; therefore, the chronic dietary exposure and risk 

assessments include food residues only. The resulting chronic exposure estimates were less than 

HED’s level of concern (≤100% chronic PAD) for general U.S. population (1.4% cPAD) and all 

population sub-groups. The most highly exposed population subgroup was Children 1-2 years 

old with an estimated risk of 7.1% cPAD. The cancer risk estimate for the general U.S. 

population is 2 × 10-6.

This risk assessment does not rely on data from studies in which human subjects were 

intentionally exposed to a pesticide or other chemical. 

2.0 HED Recommendations

Provided that benalaxyl-M reference standard is submitted to the National Pesticides Standards 

Repository, there are no data deficiencies that would preclude establishing the recommended 

tolerances for benalaxyl-M in/on grape and tomato. The specific tolerance recommendations are 

provided in Section 2.2.3. 

2.1 Data Deficiencies

2.1.1 Toxicity Studies

HED has determined that a comparative thyroid assay in rats is required to generate specific data 

on the thyroid hormone levels to protect the developing nervous system from thyroid hormone 

disrupting chemicals. In the absence of these data, the 10X FQPA database uncertainty factor 

has been retained in this risk assessment for the uncertainty associated with the potential for 

enhanced sensitivity of the developing organism to prenatal or postnatal thyroid toxicity. This 
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uncertainty may be addressed by submission of a comparative thyroid assay or a proposal 

describing an alternative approach that adequately addresses the issue of disruption of thyroid 

hormone homeostasis during development.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

An adequate analytical method is available to enforce the proposed tolerances for residues of 

benalaxyl-M in plant commodities. Method RA.09.01, a high-performance liquid 

chromatography method with tandem mass spectrometry detection (HPLC/MS/MS), has been 

adequately validated and radiovalidated, and has undergone a successful ILV (independent 

laboratory validation). The method has limits of detection and quantitation of 0.005 and 0.010 

ppm, respectively. 

Benalaxyl-M has been tested using the Food and Drug Administration (FDA) multi-residue 

method (MRM) protocols. Complete recovery of benalaxyl-M from tomato puree samples 

indicates that Protocol D of FDA Pesticide Analytical Methods (PAM) Vol. 1 may be applicable 

for determination of benalaxyl-M.

2.2.2 International Harmonization

Organization for Economic Cooperation and Development (OECD) Maximum Residue Limit 

(MRL) calculation procedures give rounded MRL estimates of 3 and 0.2 ppm for grape and 

tomato, respectively. Codex has set benalaxyl MRLs of 0.3 and 0.2 mg/kg for grape and tomato, 

respectively. Thus, the HED recommendations in this memo will result in harmonization of the 

U.S. tolerance with the Codex MRL for tomato, but not for grape since benalaxyl-M residues 

from the grape trials in Argentina were significantly higher than the Codex MRL. 

2.2.3 Recommended Tolerances

HED has reviewed the available residue data and determined the appropriate tolerance levels for 

residues of benalaxyl-M (Table 2.2.3). The recommended tolerance expression is as follows:

Tolerances are established for residues of the fungicide benalaxyl-M, including its 

metabolites and degradates, in or on the commodities in the table below. Compliance 

with the tolerance levels specified below is to be determined by measuring only

benalaxyl [methyl N-(2,6-dimethylphenyl)-N-(phenylacetyl)-DL-alaninate] in or on the 

commodity.

Table 2.2.3. Tolerance Summary for Benalaxyl-M.

Commodity Proposed Tolerance 

(ppm)

HED-Recommended 

Tolerance (ppm)

Comments;

Correct Commodity Definition

Grape 1.1 3.0 Grape

Grape, raisin 2.2 None Covered by grape tolerance

Tomato 0.25 0.20 Tomato
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The tolerance regulation should include a footnote to the tolerance stating:

“There is no U.S. registration for use of benalaxyl-M on grape or tomato.”

2.2.4 Revisions to Petitioned-For Tolerances

The proposed tolerance levels differ from those being recommended by HED. The petitioner 

used the NAFTA calculator to determine proposed tolerance levels while HED used OECD 

MRL calculation procedures. Further, for determination of the grape and tomato tolerance 

levels, the petitioner included the results from all trials. In contrast, HED included only those 

data which matched the critical Good Agricultural Practice (cGAP). 

 

3.0 Ingredient Profile

3.1 Chemical Identity

TABLE 3.1. Test Compound Nomenclature.

Compound

Common name Benalaxyl-M

Company experimental name IR6141 and IR6141 M

IUPAC name methyl N-(phenylacetyl)-N-(2,6-xylyl)-D-alaninate

CAS name methyl N-(2,6-dimethylphenyl)-N-(phenylacetyl)-D-alaninate

CAS # 98243-83-5

End-use product/EP Fantic, Sidecar

3.2 Physical/Chemical Characteristics

The physical and chemical properties of benalaxyl-M are provided in Appendix C. Technical 

grade benalaxyl-M is a solid at room and biologically-relevant temperatures. The compound has 

low volatility with little likelihood for exposure in the vapor phase. The technical-grade material 

has low aqueous solubility and a log Kow of 3.5, indicative of potential for being a fat-soluble 

compound. 

3.3 Pesticide Use Pattern

The petitioner provided translations of the benalaxyl-M labels from Europe, Mexico, and 

Argentina. Table 3.3 summarizes the key use parameters for benalaxyl-M application to grape 

and tomato. 
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Table 3. 3. Summary of Directions for Use of Benalaxyl-M.

Country

Formulation

Application 

Type

Application 

Rate 

(kg ai/ha)

Max. No. 

Applic. 

per 

Season

RTI1

(days)

Max. 

Seasonal 

Applic. Rate

(kg ai/ha)

PHI1

(days) Limitations

Grape

Italy

Fantic M NC WG;

Fantic M blu; [WP]

Fantic F WG 

Foliar

0.10

0.10

0.075

3

3

3

10-14

10-14

10-14

0.30

0.30

0.225

42

42

42

Apply the first treatment at 

the beginning of flowering 

(BBCH 60). Apply each 

subsequent treatment at an 

interval roughly coincident 

with the phenological stages 

of fully flowering (BBCH 

65) and early development 

of the bunches (BBCH 73), 

when the berries reach the 

pea size.

France

Fantic F WG

Sidecar [WP]

0.10

0.10

3

3

12-14

10-14

0.30

0.30

42

42

For table grapes, do not treat

after flowering (Stage

BBCH69)

Spain

Fantic M [WP]

Fantic F [WG]

0.10

0.075

2

3

10-14

10-14

0.20

0.225

40

42

This product cannot be used

in vineyards intended for

multiplication or on wine

trellises.

Argentina

Fantic M2 [WG] 0.10 4 7-10 0.40 21

For sensitive varieties, it is

advised to preventively

apply the product pre

and post-flowering until

the clusters close.

Tomato

Italy

Fantic M NC WG;

Fantic M blu [WP]

Foliar

0.10

0.10

3

3

7-10

10

0.30

0.30

14

14

Begin treatment when there 

are favorable conditions for 

the disease or when the main 

bud has developed (BBCH 

61).

Spain

Fantic M [WP] 0.10 3 10 0.30 3

Begin application when 

environmental conditions 

favor disease development.

Mexico

Sidecar M; [WG]

Fantic Star [WG]

0.12

0.12

3

3

7

7

0.36

0.36

14

14

Begin application when 

environmental conditions 

favor disease development 

(temperatures between 18

and 22oC and relative

humidity above 90%).

Do not apply when

temperatures are very high,

when winds are stronger

than 10 km/hour, or while it

is raining.

1 RTI = Retreatment interval. PHI = Preharvest interval.

2 Fantic M use in/on grape is pending in Argentina.

The use directions are adequate to allow evaluation of the residue data relative to the proposed 

uses. The cGAP for grape and tomato were identified as the GAPs from Argentina and Spain, 

respectively. 
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3.4 Anticipated Exposure Pathways

The Registration Division requested that HED perform an assessment of human health risk to 

support the petitioner’s request for benalaxyl-M tolerances on imported grapes and tomatoes. No 

previous Agency risk assessments have been conducted for benalaxyl-M since there are currently 

no U.S. registrations for benalaxyl-M-based products. Humans may be exposed to benalaxyl-M

through ingestion of imported grapes, tomatoes, and their processed commodities (i.e., grape 

juice, raisins, wine, dried tomato, tomato paste, puree, etc.). Dietary exposures are expected 

based on detectable residues observed. There are no U.S. drinking water, occupational or 

residential exposures associated with the requested tolerance. 

3.5 Consideration of Environmental Justice

Potential areas of environmental justice concerns, to the extent possible, were considered in this 

human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions 

to Address Environmental Justice in Minority Populations and Low-Income Populations," 

(http://epa.gov/compliance/ej/resources/policy/exec_order_12898.pdf). As a part of every

pesticide risk assessment, OPP considers a large variety of consumer subgroups according to 

well-established procedures. In line with OPP policy, HED estimates risks to population 

subgroups from pesticide exposures that are based on patterns of that subgroup’s food and water 

consumption, and activities in and around the home that involve pesticide use in a residential 

setting. Extensive data on food consumption patterns are compiled by the USDA under the 

National Health and Nutrition Survey/What We Eat in America (NHANES/WWEIA) and are 

used in pesticide risk assessments for all registered food uses of a pesticide. These data are 

analyzed and categorized by subgroups based on age and ethnic group. Additionally, OPP is 

able to assess dietary exposure to smaller, specialized subgroups and exposure assessments are 

performed when conditions or circumstances warrant. Whenever appropriate, non-dietary

exposures based on home use of pesticide products and associated risks for adult applicators and 

for toddlers, youths, and adults entering or playing on treated areas postapplication are 

evaluated. Further considerations are currently in development as OPP has committed resources 

and expertise to the development of specialized software and models that consider exposure to 

bystanders and farm workers as well as lifestyle and traditional dietary patterns among specific 

subgroups.

4.0 Hazard Characterization and Dose-Response Assessment

4.1 Toxicology Studies Available for Analysis

The database of experimental toxicology studies available for benalaxyl-M provides a robust 

characterization of the hazard potential of this pesticide. HED has determined that a comparative 

thyroid assay in rats is required to assess the potential impact of benalaxyl-M exposure on 

thyroid function in the young (Dellarco et al., 2005). The agency is requiring these data given 

the pivotal role of thyroid hormones in brain development. In the absence of these data, the 10X 

FQPA database uncertainty factor has been retained.

The data from the following studies were used to evaluate the hazard potential of benalaxyl-M:
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 subchronic oral toxicity studies in rats, mice, and dogs

 chronic oral toxicity studies in rats and dogs 

 carcinogenicity studies in rats and mice 

 developmental toxicity studies in rats and rabbits 

 reproduction toxicity study in rats 

 acute and subchronic neurotoxicity studies in rats

 subchronic dermal toxicity study in rats 

 immunotoxicity study in rats 

 complete mutagenicity study battery 

 metabolism study in rats 

4.2 Absorption, Distribution, Metabolism and Elimination (ADME)

Benalaxyl-M was well absorbed and rapidly excreted. Absorption was rapid and the peak 

plasma concentrations (Tmax) were reached within 2-6 hours and the terminal half-lives T1⁄2

ranged from 59 to 102 hrs. There were no major sex differences in the maximum concentration 

(Cmax) or in the time taken to reach Cmax (Tmax), and the estimated total absorption was similar 

between the sexes (~89% for males and ~82% for females). Radiolabel was excreted mainly via

the feces within 72 hours (84% of administered dose). Only 7% of the administered dose (AD) 

was excreted via the urine, and tissue radioactivity levels were low. The highest tissue 

concentrations (as percent of administered dose) 0.5 hours after treatment were found in the liver 

and intestinal tract, which is indicative of active absorption. Total radioactive dose recovered in 

the tissues 0.5 hours after treatment was 23%. Tissue concentrations as percent of administered 

dose were <1% for both sexes by 72 hours post dose. Metabolism of benalaxyl-M was extensive 

as at least 18 compounds were found. Benalaxyl-M was a minor component excreted in the 

feces, and it was not found in the urine. Feces contained a higher number of degradation 

products, but the main metabolites were identical both in urine and feces. The metabolic profile 

was the same in male and female rats. Benalaxyl-M was extensively metabolized principally by

oxidation of the methyl group on the aniline ring to the hydroxymethyl and finally to the 

carboxylic acid. Another metabolic pathway involved hydroxylation of the phenyl ring and 

hydrolysis of the carboxymethyl group. Following single low and high doses, the main route of 

elimination was fecal via the bile (62-81% AD), while urinary excretion (8-19% AD) played a 

minor role. There was evidence of extensive enterohepatic recirculation. Bile was an important 

route of excretion; a substantial portion of the administered dose was re-absorbed; biliary

excretion was substantial for both sexes (80.5% of dose male; 62% of dose females), indicating 

most of the radiolabeled test material was absorbed and then excreted via the bile into the feces. 

Excretion in the expired air was negligible (<0.1%). Residual radioactivity was < 1% AD in the 

carcasses 96 hours after treatment. 

4.2.1 Dermal Absorption

Dermal exposure is not expected in the U.S. with an import tolerance. There is no dermal 

absorption study on benalaxyl-M, but there is a subchronic dermal toxicity study available, 

which shows no dermal hazard at dose levels up to the limit dose.
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4.3 Toxicological Effects

Benalaxyl-M is a new fungicide and a member of the N-acylalanine fungicides, which act by 

inhibiting RNA polymerase. There are no data on whether this pesticidal mode of action is 

relevant to mammalian toxicology.

The liver and thyroid are the primary target organs for benalaxyl-M. In rats, increased liver 

weights, clinical chemistry changes indicative of liver toxicity, hepatocellular hypertrophy, and 

thyroid follicular cell hypertrophy were seen following subchronic and chronic exposure. In 

mice, increased liver weight and microscopic lesions in the liver (hepatocellular hypertrophy, 

necrosis, eosinophilic foci) were observed following subchronic and chronic exposure. 

Additionally, chronic exposure in rats and mice led to increases in the incidence of liver (rat, 

mouse) and thyroid (rat) tumors. In dogs, increased liver weight, changes in clinical chemistry

indicative of liver toxicity, and hepatocellular hypertrophy were observed following subchronic 

exposure via the diet, whereas clinical chemistry changes indicative of liver toxicity, fat 

vacuolation in the liver, and thyroid follicular cell hypertrophy were observed following chronic 

exposure via capsules.

Benalaxyl-M was classified as “Likely to be Carcinogenic to Humans”. This determination was 

based on the treatment-related liver tumors observed in male mice, liver tumors observed in male 

and female rats; and thyroid follicular cell tumors observed in female rats. No treatment-related 

tumors were observed in female mice. There is no mutagenicity concern from the in vivo or in 

vitro genetic toxicity assays. The negative data rule out mutagenicity as a possible mode of 

action (MOA) for the induction of tumors in the rat and mouse carcinogenicity studies. A 

plausible non-genotoxic mode of action involving mitogenesis was not established for the 

development of liver tumors in the male mouse bioassay with benalaxyl-M. MOA data for the 

liver tumors in male and female rats and the thyroid tumors in female rats were not provided. 

No evidence of increased quantitative or qualitative susceptibility was seen following in utero

exposure to benalaxyl-M with rats or rabbits in the prenatal developmental studies or in young 

rats in the 2-generation reproduction study. The 2-generation rat reproduction study resulted in 

no effects on fertility, although decreased primordial follicles/growing follicles/corpora lutea

were observed at the high dose. However, there is a clear NOAEL for these findings, and there 

is no reproductive concern. The offspring effects (decreased F2 pup body weight and increased 

incidence of hepatocellular hypertrophy in both sexes of F2 pups) occurred at the same dose that 

caused parental effects (increased liver weight and liver hypertrophy). No evidence of maternal 

toxicity and no developmental effects were observed in developmental toxicity studies in rabbits 

or rats. The rabbit was tested at the limit dose (1000 mg/kg/day), but the rat was not tested up to 

the limit dose. Since the point of departure is 10-fold lower than the NOAEL in the rat study, a 

repeat rat study at the limit dose would not impact the risk assessment and is not needed. 

No neurotoxic effects were seen in the acute or the subchronic neurotoxicity study. Benalaxyl-M 

was not found to be immunotoxic in the male rat.
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4.4 FQPA Safety Factor for Infants and Children

No evidence of increased quantitative or qualitative susceptibility was seen following in utero

exposure to benalaxyl-M with rats or rabbits in the prenatal developmental studies or in young 

rats in the 2-generation reproduction study. There is no evidence for neurotoxicity following 

oral exposures to benalaxyl-M. Thyroid toxicity was seen following subchronic and chronic 

exposures to adult rats. There are, however, no data regarding the potential effects of benalaxylM on thyroid homeostasis in the young animals. This lack of characterization creates uncertainty

with regards to potential life stage sensitivities due to exposure to benalaxyl-M, and raises the 

Agency’s level of concern. Therefore, the Agency is requiring a comparative thyroid assay in 

rats with benalaxyl-M. This study will better address the concern for potential thyroid toxicity in 

the young. There are no residual uncertainties in the benalaxyl-M residue database with regards 

to dietary exposure. The FQPA Safety Factor is retained at 10X in the form of a database 

uncertainty factor (UFDB).

4.4.1 Completeness of the Toxicology Database 

There are no outstanding studies according to Part 158 data requirements for the current request 

for an import tolerance on grapes and tomatoes; however, HED has determined that a 

comparative thyroid assay in rats is required, as discussed above. The uncertainty for the lack of 

this study is addressed by retaining the FQPA Safety Factor of 10X as the UFDB. 

4.4.2 Evidence of Neurotoxicity

There is no evidence for neurotoxicity following oral exposures to benalaxyl-M. Following a 

single exposure, no evidence for neurotoxicity was observed at the limit dose and twice the limit 

dose. Following repeated (dietary) exposures, there were no treatment-related clinical signs of 

neurotoxicity, behavioral changes, or neuropathology. 

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal

No evidence of increased quantitative or qualitative susceptibility was seen following in utero

exposure to benalaxyl-M with rats or rabbits in the prenatal developmental toxicity studies or in 

young rats in the 2-generation reproduction study. The 2-generation reproduction study resulted 

in no effects on reproductive function or fertility. The offspring effects occurred at the same 

dose that caused parental effects. No evidence of developmental delay or developmental toxicity

was observed in developmental toxicity studies in rabbits or in rats. The rabbit was tested at the 

limit dose (1000 mg/kg/day), but the rat was not tested up to the limit dose. Since the point of 

departure is 10-fold lower than the NOAEL in the rat study, a repeat rat study at the limit dose 

would not impact the risk assessment and is not needed.

4.4.4 Residual Uncertainty in the Exposure Database

There are no residential uses of benalaxyl-M in the U.S. and adequate food residue data are 

available for human health risk assessment. Residue values used in the dietary risk assessments 

are unlikely to underestimate risk. 
Benalaxyl-M Human Health Risk Assessment DP No. D416044

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Dietary exposure assessments were conducted using anticipated residues and assumed 100% 

crop treated. Therefore, the chronic dietary (food only) exposure is considered a conservative 

estimate. Drinking water exposure in the U.S. is not expected since there are no domestic 

registrations for benalaxyl-M. 

4.5 Toxicity Endpoints and Points of Departure Selections

4.5.1 Dose-Response Assessment

The details for the toxicity endpoint selection are presented in Appendix A2. In risk assessments 

for import commodities, endpoints are typically selected for dietary exposure only. Endpoints

for incidental oral, dermal and inhalation exposures are not selected for import tolerances due to 

lack of potential U.S. occupational or residential exposure. 

An acute dietary endpoint for the general population, including infants and children and females 

13+ was not selected for benalaxyl-M, based on the lack of toxicity resulting from a single 

exposure in the database. 

For chronic dietary risk assessment, a NOAEL of 20 mg/kg/day was selected based on an 

increase in gamma glutamyl transferase (GGT) in males, increased cholesterol in both sexes, 

increased triglycerides in males, slight increases liver weight in both sexes, increased incidence 

of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell 

hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased 

incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal 

cell hyperplasia in females at a LOAEL of 135 mg/kg/day in the chronic toxicity/carcinogenicity 

study in rats. For the POD for the chronic dietary risk assessment, a total UF of 1000X was 

applied (10X for interspecies variability, 10X for intraspecies variability, and 10X for database 

uncertainty) to account for the lack of a comparative thyroid assay. When the POD is based on 

thyroid effects, the interspecies extrapolation UF is typically reduced to 3X due to the 

pharmacodynamic differences between rats and humans pertaining to thyroid 

perturbations. However, in the case of benalaxyl-M, the full 10X factor was retained for 

interspecies UF since effects not related to thyroid function disruption (i.e., liver, ovarian stromal 

cell hyperplasia) were also the basis of the POD.

4.5.2 Recommendation for Combining Routes of Exposure for Risk Assessment

Since only oral exposure is anticipated for benalaxyl-M, it is not relevant to this action to discuss 

combining routes of exposure at this time. 

4.5.3 Cancer Classification and Risk Assessment Recommendations

In accordance with the EPA’s Final Guidelines for Carcinogenic Risk Assessment (March, 

2005): the Cancer Assessment Review Committee (CARC) classified benalaxyl-M as "Likely to 

be Carcinogenic to Humans”, based on the following weight of evidence considerations:
Benalaxyl-M Human Health Risk Assessment DP No. D416044

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(i) Treatment-related hepatocellular adenomas and hepatocellular adenomas and/or 

carcinomas combined in male rats; 

(ii) Treatment-related hepatocellular adenomas in female rats; 

(iii) Treatment-related thyroid follicular cell adenomas and adenomas and/or carcinomas 

in female rats; 

(iv) Treatment-related hepatocellular adenomas and hepatocellular adenomas and/or 

carcinomas combined in male mice;

(v) Lack of mutagenic concern. Benalaxyl-M does not act through a mutagenic mode of 

action; and 

(vi) The overall weight-of-evidence was not sufficient to indicate that benalaxyl-M

induces liver tumors via a mitogenic mode of action.

The CARC recommended that a low dose extrapolation model (Q1*) be applied to the 

experimental animal tumor data for quantification of cancer risk in humans from exposure to 

benalaxyl-M. The most potent unit risk, Q1

*

, is that for male mouse liver tumor rates at 5.90 x 

10-3(mg/kg/day)

-1

in human equivalents. The available data are inadequate to establish a mode 

of action (MOA) for the male mouse liver tumors.

4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk 

Assessment

Table 4.5.4. Summary of Toxicological Doses and Endpoints for Benalaxyl-M for Use in Dietary Human 

Health Risk Assessments

Exposure/ Scenario Point of 

Departure

Uncertainty/

FQPA 

Safety 

Factors

RfD, PAD, 

Level of 

Concern for 

Risk 

Assessment

Study and Toxicological Effects

Acute Dietary 

(General Population, 

including Infants,

Children, and 

females 13+)

No appropriate acute endpoint was identified.

Chronic Dietary

(All Populations)

NOAEL= 

20 

mg/kg/day

UFA= 10x

UFH= 10x

FQPA UFDB

= 10x

Chronic RfD = 

cPAD = 0.02 

mg/kg/day

Chronic Toxicity/Carcinogenicity Study -rat 

(49040634)

LOAEL = 135 mg/kg/day based on based 

on an increase in γ-glutamyl transferase

(GGT) in males, slight increases liver 

weight in both sexes, increased incidence of 

hepatocellular hypertrophy in both sexes, 

increased incidence of thyroid follicular cell 

hypertrophy in both sexes, increased 

incidence of thyroid cell hyperplasia in 

females, increased incidence of thyroid 

follicular ectasia in females, and an 

increased incidence of ovarian stromal cell 

hyperplasia in females.

Cancer (oral) Classification: “Likely to be Carcinogenic to Humans”. Based on male mouse liver tumors, 

Q1*= 5.90 × 10-3 (mg/kg/day)-1

Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and used to 

mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures. NOAEL 
Benalaxyl-M Human Health Risk Assessment DP No. D416044

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= no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UFA = 

extrapolation from animal to human (interspecies). UFH = potential variation in sensitivity among members of the human 

population (intraspecies). UFDB = to account for the absence of a comparative thyroid study. FQPA SF = FQPA Safety Factor. 

PAD = population adjusted dose (c = chronic). RfD = reference dose.

5.0 Dietary Exposure and Risk Assessment

5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Animal Metabolism Studies

The nature of the residue in grape, lettuce, and tomato is adequately understood. The parent 

compound comprised 26-44% of the total residues found in the submitted grape, tomato, and 

lettuce metabolism studies. The only metabolites found at more than 10% of the total residues

were benzyl alcohol glucoside and malonylglucoside conjugates of the parent molecule, present 

at summed levels comparable to the parent molecule. 

There are no significant livestock feedstuffs associated with grape or tomato. Therefore, no 

livestock metabolism data are relevant to the present tolerance petition.

5.1.2 Comparison of Metabolic Pathways

The proposed metabolic pathways for benalaxyl-M in grape and rats are provided in Appendix 

B. Metabolism in lettuce, grape, and tomato shared a common metabolic pathway with parent 

benalaxyl-M being the common and dominant residue. Other major metabolites (>10% TRR) 

were identified as glucoside or malonylglucoside conjugates of the parent compound after 

oxidation. The primary difference noted in the plant metabolism studies was the extent of 

benalaxyl-M metabolism, which corresponded with the PHIs of the metabolism studies. Thus, 

relatively more parent was found in the short-PHI studies (lettuce and tomato) and relatively less 

in the longer PHI study (grape). 

In the rat metabolism study, benalaxyl-M was extensively metabolized to at least 18 compounds 

with parent present at only low levels. The primary metabolic pathway involved oxidation of the 

methyl group on the aniline ring to the hydroxymethyl and then to the carboxylic acid. Another 

pathway involved hydroxylation of the phenyl ring and hydrolysis of the carboxymethyl group. 

Low levels of glucuronic acid conjugates were identified. 

Metabolism of benalaxyl-M is more extensive in the rat than in plants. The metabolic pathway

observed in plants also occurs in the rat, along with other pathways not found in plants. 

5.1.3 Residues of Concern Summary and Rationale

Major residue in the plant metabolism studies were the parent compound and its glucoside and 

malonylglucoside conjugates. These conjugates are expected to be stable in the gastrointestinal 

tract. Conjugation of the parent molecule is likely to decrease absorption and increase 

elimination, thereby reducing the toxicological concern of these residues. Therefore, the residue 

of concern in plants is parent benalaxyl-M for tolerance enforcement and risk assessment.
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Given that the only pathway for exposure at this time is via imported grapes and tomatoes, 

residue definitions for rotational crops, livestock, and water are not needed. Table 5.1.3 

summarizes the residues of concern for benalaxyl-M.

Table 5.1.3. Summary of Metabolites and Degradates to be included in the Risk 

Assessment and Tolerance Expression

Matrix Residues Included In Risk 

Assessment

Residues Included In 

Tolerance Expression

Plants Primary Crop Benalaxyl-M Benalaxyl-M

Rotational Crop

Not Applicable Not Applicable

Livestock Ruminant

Poultry

Drinking Water

5.2 Food Residue Profile

The submitted residue chemistry studies are adequate for supporting regulatory conclusions, 

establishing appropriate tolerances levels for enforcement, and for purposes of risk assessment. 

Analysis of residues can be accomplished through standard analytical techniques, and residues 

do not dissipate during frozen storage. Following foliar spray applications to grape and tomato, 

benalaxyl-M residues translocate through the plant. Processing studies demonstrate no residue 

concentration in any grape or tomato processed commodities except for raisin, where an average 

concentration factor of 2.3× was determined. Multiplying the concentration factor of 2.3× by the 

grape highest average field trial (HAFT) value of 0.98 ppm gives 2.3 ppm, which is less than the 

recommended grape tolerance of 3.0 ppm. Thus, there is no need for a separate raisin tolerance 

since residue concentration in this processed commodity will be covered by the grape raw 

agricultural commodity (RAC) tolerance. No confined rotational crop, livestock metabolism, or 

livestock feeding studies were submitted or required. 

At this time there are no U.S. registrations for benalaxyl-M. Establishment of U.S. tolerances for 

grape and tomato was requested to support the import of treated crops from Europe, Central 

America, and South America. For grape and tomato the maximum application rates are similar 

across countries; thus, critical Good Agriculture Practice (cGAP) selection was based on the 

shortest PHI, since residue decline trials showed a gradual decrease in residues with increasing 

PHI. The Argentina use pattern constitutes the cGAP for grape, and the recommended grape 

tolerance of 0.20 ppm was based on trials reflecting the cGAP. Three trials from Argentina 

conducted in 2011 gave residues approximately ten times higher than residues reported in any of 

the other grape trials and prevent harmonization with the Codex MRL for benalaxyl. For tomato, 

the Spain use pattern, with a preharvest interval (PHI) of 3 days, was identified as the cGAP and 

formed the basis for the recommended tomato tolerance, which is harmonized with the Codex 

MRL for benalaxyl. 
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5.3 Water Residue Profile

An assessment of residues in drinking water is not required for this assessment because there is 

no drinking water exposure in the U.S. associated with the establishment of an import tolerance.

5.4 Dietary Risk Assessment

5.4.1 Description of Residue Data Used in Dietary Assessment

For the chronic and cancer dietary analyses, anticipated residues (i.e., mean field trial residues) 

were used. The residue chemistry database for benalaxyl-M included processing studies for all 

relevant processed commodities from grape and tomato except for dried tomato. Thus, the 

default processing factor of 14.3× was used for dried tomato. The only processed commodity

showing residue concentration was for raisin, with an average concentration factor of 2.3×. All 

other processed commodities showed residue reduction upon processing; for these commodities 

a processing factor of 1.0 was used in the analysis. The Agency considers these to be 

conservative assessments. 

5.4.2 Percent Crop Treated Used in Dietary Assessment

For the chronic and cancer dietary analyses, a 100% crop treated assumption was used.

5.4.3 Acute Dietary Risk Assessment

Because no acute endpoint was identified in the toxicity database, no acute dietary analysis was 

conducted.

5.4.4 Chronic Dietary Risk Assessment

The results of the chronic dietary exposure analyses are reported in Table 5.4.6. The resulting 

chronic risk estimates were less than HED’s level of concern (≤100% cPAD) for the general U.S. 

population (1.4% cPAD) and all population sub-groups. The most highly exposed population 

subgroup was Children 1-2 years old with an estimated risk of 7.1% cPAD.

5.4.5 Cancer Dietary Risk Assessment

The results of the cancer dietary exposure analyses are reported in Table 5.4.6. The cancer 

dietary assessment made use of the same input assumptions as the chronic analysis. BenalaxylM has been classified as “Likely to be Carcinogenic to Humans” (TXR No. 0057093, L. 

Brunsman, 04-DEC-2014). A linear low-dose extrapolation model (Q1*) was used to estimate 

cancer risk, with a Q1* = 5.90 × 10-3(mg/kg/day)

-1

. The cancer risk estimate to the U.S. 

population is 1.7 x 10-6.
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5.4.6 Summary Table

Table 5.4.6. Summary of Dietary (Food Only) Exposure and Risk for Benalaxyl-M.1

Population Subgroup

Acute Dietary Chronic Dietary Cancer

Dietary 

Exposure 

(mg/kg/day)

% aPAD

Dietary 

Exposure

(mg/kg/day)

% cPAD

Dietary 

Exposure

(mg/kg/day)

Risk

General U.S. Population

NA NA

0.000287 1.4 0.000287 1.7 × 10-6

All Infants (< 1 year old) 0.000477 2.4

NA NA

Children 1-2 years old 0.001416 7.1

Children 3-5 years old 0.000901 4.5

Children 6-12 years old 0.000381 1.9

Youth 13-19 years old 0.000148 < 1

Adults 20-49 years old 0.000201 1.0

Adults 50-99 years old 0.000225 1.1

Females 13-49 years old 0.000206 1.0

1 Highest exposure identified in bold.

6.0 Residential (Non-Occupational) Exposure/Risk Characterization

There are no registered residential uses for benalaxyl-M.

7.0 Aggregate Exposure/Risk Characterization

As stated previously, there are no existing or proposed US registrations of benalaxyl-M and the 

only route of exposure is via dietary ingestion from imported grape and tomato commodities. 

Therefore, aggregate exposure and risk estimates are equivalent to the dietary exposures and risk 

estimates discussed in Section 5.

8.0 Cumulative Exposure/Risk Characterization

Unlike other pesticides for which EPA has followed a cumulative risk approach based on a 

common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as 

to benalaxyl-M and any other substances and benalaxyl-M does not appear to produce a toxic 

metabolite produced by other substances. For the purposes of this tolerance action, therefore, 

EPA has not assumed that benalaxyl-M has a common mechanism of toxicity with other 

substances. For information regarding EPA’s efforts to determine which chemicals have a 

common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the 

policy statements released by EPA’s Office of Pesticide Programs concerning common 

mechanism determinations and procedures for cumulating effects from substances found to have 
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a common mechanism on EPA’s website at http://www.epa.gov/pesticides/cumulative/.

9.0 Occupational Exposure/Risk Characterization

An occupational assessment is not relevant for uses on imported commodities.

10.0 References

Dellarco, V. et al. 11/01/2005. Interim Guidance: Thyroid Disrupting Pesticides: Use of Rat 

Thyroid Data and Application of Uncertainty Factors for RfD Derivation. Prepared and 

Reviewed by the Hazard Science Policy Council.

OPP Guidance. 10/24/2005. Guidance for Thyroid Assays in Pregnant Animals, Fetuses and 

Postnatal Animals, and Adult Animals. Office of Pesticide Programs, Health Effects Division, 

Washington, DC. Cited in Dellarco, V. et al 2005

Benalaxyl-M. Report of the Residues of Concern Knowledgebase Subcommittee (ROCKS). –

E. Holman, D421423, 09/09/14.

Benalaxyl-M. Report of the Cancer Assessment Review Committee. J. Rowland and K. 

Middleton, TXR 0057035, 12/02/14.

Benalaxyl-M. Quantitative Risk Assessment Based on Rat and Mouse Dietary Studies. L. 

Brunsman, TXR 0057093, 12/04/14.

Residue Chemistry Assessment – W. Donovan, D416583, 12/02/14.

Dietary Exposure Assessment – W. Donovan, D416584, 01/13/15.
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Appendix A. Toxicology Profile and Executive Summaries

A.1 Toxicology Data Requirements

The requirements (40 CFR 158.340) for food (import tolerance) for benalaxyl-M are in Table 1. Use of the new 

guideline numbers does not imply that the new (1998) guideline protocols were used. 

Study

Technical

Required Satisfied

870.1100 Acute Oral Toxicity.......................................................

870.1200 Acute Dermal Toxicity ..................................................

870.1300 Acute Inhalation Toxicity..............................................

870.2400 Primary Eye Irritation....................................................

870.2500 Primary Dermal Irritation ..............................................

870.2600 Dermal Sensitization......................................................

yes

no

no

no

no

no

yes

---

---

---

---

---

870.3100 Oral Subchronic (rodent)...............................................

870.3150 Oral Subchronic (nonrodent).........................................

870.3200 21-Day Dermal ..............................................................

870.3250 90-Day Dermal ..............................................................

870.3465 90-Day Inhalation..........................................................

yes

no

no

no

no

yes

yes

---

---

---

870.3700a Developmental Toxicity (rodent)...................................

870.3700b Developmental Toxicity (nonrodent).............................

870.3800 Reproduction .................................................................

yes

yes

yes

yes

yes

yes

870.4100a Chronic Toxicity (rodent)..............................................

870.4100b Chronic Toxicity (nonrodent)........................................

870.4200a Oncogenicity (rat)..........................................................

870.4200b Oncogenicity (mouse)....................................................

870.4300 Chronic/Oncogenicity....................................................

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.5100 Mutagenicity—Gene Mutation - bacterial.....................

870.5300 Mutagenicity—Gene Mutation - mammalian................

870.5375 Mutagenicity—Structural Chromosomal Aberrations...

870.5xxx Mutagenicity—Other Genotoxic Effects.......................

yes

yes

yes

no

yes

yes

yes

---

870.6100a Acute Delayed Neurotoxicity (hen)...............................

870.6100b 90-Day Neurotoxicity (hen)...........................................

870.6200a Acute Neurotoxicity Screening Battery (rat) .................

870.6200b 90-Day Neurotoxicity Screening Battery (rat)...............

870.6300 Develop. Neurotoxicity .................................................

no

no

yes

yes

no

---

---

yes

yes

---

870.7485 General Metabolism ......................................................

870.7600 Dermal Penetration........................................................

870.7800 Immunotoxicity .............................................................

yes

no

yes

yes

---

yes
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A.2 Toxicity Profiles

A.2.1 Acute Toxicity

Table A.2.1 Acute Toxicity Profile – Benalaxyl-M (technical)

Guideline No. Study Type MRID(s) Results Toxicity Category

870.1100 Acute oral [rat] 49040619 LD50 = >2000 mg/kg

No deaths or clinical 

signs

III

870.1200 Acute dermal [rat] 49040620 LD50 = >2000 mg/kg III

870.1300 Acute inhalation [rat] 49040621

49040622

LC50 = mg/L

Aerosol could not be 

generated (waiver granted)

-

870.2400 Acute eye irritation [rabbit] 49040623 No corneal opacity or 

iritis; + score of 2 for 

conjunctival redness 

at 24 hrs; no + scores 

at 48 hrs; all eyes free 

of irritation at 72 hrs

III

870.2500 Acute dermal irritation [rabbit] 49040624 Not an irritant IV

870.2600 Skin sensitization [Guinea pig] 49040625 Not a sensitizer -

A.2.2 Subchronic, Chronic, Other Toxicity

Table A.2.2 Subchronic, Chronic and Other Toxicity Profile – Benalaxyl-M

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3100

90-Day Oral Toxicity 

(Crl:CD BR rat)

49040627 (2000)

Acceptable/guideline

0, 80, 400, 2000, or 

10000 ppm (diet)

Males: 0, 6.2, 30.1, 154, 

or 861 mg/kg/day 

Females: 0, 6.7, 32.8, 

167, or 892 mg/kg/day

NOAEL = 2000 ppm (males 154 mg/kg/day; females 

167 mg/kg/day). 

LOAEL = 10000 ppm (males 861/females 892 

mg/kg/day), based on elevated GGT values in both 

sexes, increased cholesterol and phospholipids in both 

sexes, increased liver weights in both sexes, increased 

thymus weight in females, increased incidence of 

hepatocellular hypertrophy in both sexes, and increased 

incidence of thyroid follicular cell hypertrophy in both 

sexes.

Findings at 2000 ppm are considered to be minor 

[increased GGT (both sexes); increased phospholipids 

(males), adaptive hepatocellular hypertrophy (both 

sexes), and not biologically adverse due to lack of 

severity (thyroid follicular cell hypertrophy]
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Table A.2.2 Subchronic, Chronic and Other Toxicity Profile – Benalaxyl-M

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3100

90-Day Oral Toxicity 

(CD-1 mouse)

49040628 (2005)

Acceptable/guideline

0, 600, 2000, or 7000 

ppm (diet)

Males: 0, 93.2, 296.6, or 

1053.8 mg/kg/day 

Females: 0, 111.8, 348.1, 

or 1253.3 mg/kg/day

Systemic NOAEL = 2000 ppm (296.6 mg/kg/day in 

males and 348.1 mg/kg/day in females).

Systemic LOAEL = 7000 ppm (1054 mg/kg/day in 

males and 1253 mg/kg/day in females), based on 

increased liver weights, increased incidence and 

severity of periportal hepatocellular hypertrophy in 

females, increased glucose in the urine in both sexes, 

focal necrosis in the liver in males, and depleted 

glycogen in the liver in females.

870.3150

90-Day Oral Toxicity 

(Beagle dog)

49040629 (2000)

Acceptable/guideline

0, 150, 450, 900, or 3000 

ppm (diet)

Males: 0, 5.6, 16, 32, or

112 mg/kg bw/day 

Females: 0, 5.85, 17, 35, 

or 110 mg/kg bw/day 

Systemic NOAEL = 900 ppm (32 mg/kg/day in males 

and 35 mg/kg/day in females). 

Systemic LOAEL = 3000 ppm (112 mg/kg/day in 

males and 110 mg/kg/day in females), based on 

elevated alkaline phosphatase activity in both sexes, 

elevated triglyceride levels in males, increased liver 

weight in both sexes, and an increased incidence/

severity of hepatocyte hypertrophy in the centrilobular, 

midzonal, and periportal areas in males and females.

870.3200

28-Day Dermal Toxicity 

(Wistar rat)

49040631 (2013)

Acceptable/guideline

0, 62.5, 250, or 1000 

mg/kg/day 

6 hours/day, 7 days/week 

for 4 weeks 

NOAEL = 1000 mg/kg/day

LOAEL = not identified

Endpoints of concern (liver, thyroid) were assessed; no 

developmental, neurotoxicity, immunotoxicity, or 

reproductive concern.

870.3465

28/90-Day Inhalation 

Toxicity

49066308

Waiver request

49040621/49040622

See MRID49040621-49040622 (acute inhalation)

870.1300 waived. Aerosol could not be generated.

870.3700a

Prenatal developmental 

(Crl: CD (SD) BR rat)

49049637 (2001)

Acceptable/nonguideline

0, 10, 50, or 250 

mg/kg/day (dose volume 

10 mL/kg/day)

(0.5% methylcellulose 

aqueous solution)

GD 6-15

Maternal NOAEL = 250 mg/kg/day (HDT)

Maternal LOAEL = not identified

Developmental NOAEL =250 mg/kg/day

Developmental LOAEL = not identified

Non-guideline since it did not test to limit dose and no 

adverse effect was observed in the maternal animals.

A new study is not required since an endpoint more 

sensitive than the one used for risk assessment would 

not be identified by a study at dose levels >250 

mg/kg/day

870.3700b

Prenatal developmental in 

(Himalayan rabbit)

49040638 (2003)

Acceptable/guideline

0, 50, 250, or 1000 

mg/kg/day (0.5% 

methylcellulose)

GD 6-27

(dose volume 4 

mL/kg/day)

Maternal NOAEL = 1000 mg/kg/day (HDT)

Maternal LOAEL = not identified

Developmental NOAEL = 1000 mg/kg/day

Developmental LOAEL = not identified
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Table A.2.2 Subchronic, Chronic and Other Toxicity Profile – Benalaxyl-M

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3800

Reproduction and Fertility 

Effects (Han Wistar rats)

49040639 (2008)

Acceptable/guideline

0, 120/80, 360/240, 

1080/725, or 3240/2170 

ppm

Males: 0, 7.9, 23.5, 71.1, 

or 221.3 mg/kg/day

Females 0, 8.9, 26.7, 

77.5, or 241.8 mg/kg/day

70-day pre-mating

Parental NOAEL = 1080 ppm (males 71/females 78 

mg/kg/day)

Parental LOAEL = 3240 ppm (males 221/females 242 

mg/kg/day), based on decreased # primordial follicles 

and corpora lutea in F1 females, decreased uterus 

weight in F0 females, and a slight increase in 

hepatocellular hypertrophy in both sexes and 

generations (males P 16/24, F1 12/24; females P 21/24; 

F1 23/24),.

Offspring NOAEL = 1080 ppm (males 71/females 78 

mg/kg/day)

Offspring LOAEL = 3240 ppm (males 221/females 

242 mg/kg/day), based on decreased pup body weight 

in F2 pups of both sexes and increased incidence of 

hepatocellular hypertrophy in F2 pups of both sexes. 

Reproductive NOAEL = 1080 ppm (males 71/females 

78 mg/kg/day)

Reproductive LOAEL = 3240 ppm (males 221/ 

females 242 mg/kg/day), based on a decrease in 

ovarian follicle counts (primordial follicles and corpora 

lutea) in F1 females.

outside historical control and concurrent control values 

were generally low compared to the historical control 

values.

870.4100a

Chronic Toxicity

(Sprague Dawley CD 

Crl:CD(SD)BR rat)

49040634 (2013) See under 870.4200a

870.4100b

Chronic toxicity 

(Beagle dog)

49040630 (2006)

Acceptable/guideline

0, 10/300A, 30, or 100 

mg/kg/day (52 weeks)

capsule 

A

low dose ↑to 300 mkg 

(week 27)

NOAEL = not selected.

LOAEL = not selected.
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Table A.2.2 Subchronic, Chronic and Other Toxicity Profile – Benalaxyl-M

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.4200a

Carcinogenicity 

(Sprague Dawley CD 

Crl:CD(SD)BR rat)

49040634 (2013)

Acceptable/guideline

0, 60, 390, or 2535 ppm

Main study: Males 0, 

2.4, 15.66, 104.27 

mg/kg/day

Females 0, 3.13, 20.04, 

or 135.28 mg/kg/day

52-week chronic study: 

Males: 0, 2.92, 19, 126 

mg/kg/day

Females: 0, 3.82, 24.6, or 

167 mg/kg/day

NOAEL = 390 ppm (males 15.7 mg/kg/day/females 20 

mg/kg/day.

LOAEL = 2535 ppm (males 104/females 135 

mg/kg/day), based on increase in GGT in males,

increased cholesterol in both sexes, increased 

triglycerides in males, slight increases liver weight in 

both sexes, increased incidence of hepatocellular 

hypertrophy in both sexes, increased incidence of 

thyroid follicular cell hypertrophy in both sexes, 

increased incidence of thyroid cell hyperplasia in 

females, increased incidence of thyroid follicular 

ectasia in females, and an increased incidence of 

ovarian stromal cell hyperplasia in females.

evidence of carcinogenicity: increase in liver tumors 

(adenomas) at the high dose in both sexes; increase in 

thyroid follicular cell adenomas and follicular cell 

adenomas/carcinomas combined in high dose females

(CARC 8/13/2014; Likely/Q*1).

870.4200b

Carcinogenicity (78 weeks)

(CD-1 mouse)

49040635 (2013)

Acceptable/guideline

0, 200, 1200, or 7000 

ppm

Males: 0, 24.86, 156.1, 

or 962 mg/kg/day

Females: 0, 31.36, 196.7, 

or 1180 mg/kg/day

Systemic NOAEL = 1200 ppm (156 mg/kg bw/day for 

males and 197 mg/kg/day for females).

Systemic LOAEL = 7000 ppm (962 mg/kg bw/day for 

males and 1180 mg/kg bw/day for females), based on

increased liver weight in both sexes and an increased 

incidence of liver lesions (diffuse hepatocellular 

hypertrophy in both sexes, single cell necrosis in both 

sexes, focal/ multifocal necrosis in males, and 

eosinophilic foci in males). 

evidence of carcinogenicity: increased incidence of 

liver adenomas in males and liver adenomas/ 

carcinomas in both sexes at HDT; (CARC 8/13/2014; 

Likely/Q*1).

870.4300

Combined Chronic 

Toxicity/Carcinogenicity

(Sprague Dawley CD 

Crl:CD(SD)BR rat) 

49040634 (2013) See under 870.4200a

870.5100

Bacterial reverse gene 

mutation (Ames Assay)

49040640 (2002)

Acceptable/guideline

5- 5000 μg/plate +/ –S9 

(plate incorporation)

5- 1500 μg/plate +/ –S9 

(preincubation) 

Negative up to the limit dose; compound precipitation 

at ≥1500 μg/plate

870.5300

In vitro mammalian 

forward cell gene mutation 

(mouse lymphoma cells)

49040642 (1999)

Acceptable/guideline

5- 75 μg/mL –S9 

15- 100 μg/mL +S9 

Negative up to cytotoxic concentration 
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Table A.2.2 Subchronic, Chronic and Other Toxicity Profile – Benalaxyl-M

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.5375

In vitro mammalian 

chromosome aberration test

[Chinese Hamster Ovary 

(CHO cells)]

49040641 (1999)

Acceptable/guideline

Assay 1 5- 3000 

μg/mL+/ –S9 (3 hrs.)

Assay 2 5- 150 μg/mL –

S9 (18 hrs.)

15- 150 μg/mL +S9 (3 

hrs.)

Negative up to cytotoxic & insoluble concentration

870.5395

Mammalian erythrocyte 

micronucleus assay 

(mouse)

49040643 (2000)

Acceptable/guideline

0, 100, 200, 400 mg/kg 

M

0, 75, 150, 300 mg/kg F

Single i.p. injections

Negative up to doses (≥300 mg/kg) causing overt 

toxicity in preliminary study (deaths and/or 

piloerection)

870.6200a

Acute Neurotoxicity 

Screening Battery

(CRL Wistar rat)

49040626 (2013)

Acceptable/guideline

0, 500, 1000, or 2000 

mg/kg

10 mL/kg dose volume

0.5% aqueous CMC 

(peak effect time 30 

minutes)

NOAEL =2000 mg/kg

LOAEL = not identified.

870.6200b

Subchronic Neurotoxicity 

Screening Battery

(CRL Wistar rat)

49143501 (2013)

Acceptable/guideline

0, 1000, 3000, or 10000 

ppm

Males: 0, 67, 206, or 784 

mg/kg/day

Females: 0, 78, 325, or 

1139 mg/kg/day

Systemic NOAEL = 3000 ppm (males 206/females 325

mg/kg/day.

Systemic LOAEL = 10000 ppm (males 784/females 

1139 mg/kg/day, based on increased serum GGT 

activity in both sexes and increased absolute and 

relative liver weights. 

No neurobehavioral or neuropathological effects were 

observed in either sex.

870.6300

Developmental 

Neurotoxicity

No study available or required.

870.7485

Metabolism and 

Pharmacokinetics

(Sprague-Dawley rat)

49040644

Acceptable/guideline

Radiolabel readily absorbed and excreted mainly via 

the feces (≈85%) and to a smaller extent via urine 

(≈8%). Biliary excretion explains high elimination of 

absorbed radiolabel via fecal route. By 72 hours, all 

tissue concentration were <1%; highest tissue 

concentrations in intestinal wall (8%), liver (8%), and 

stomach (6%); PK profile characterized by a peak level 

between 10 minutes and 1 hour post dose; elimination 

half-life of ≈18 hours; IR6141 was extensively 

metabolized, principally by oxidation of methyl group 

on aniline ring to the hydroxymethyl and finally to the 

carboxylic acid; minor pathways were hydroxylation of 

phenyl ring and hydrolysis of carboxymethyl group. 

shows urinary metabolic pathway identical to fecal 

metabolic pathway.

870.7485

Metabolism and 

Pharmacokinetics

(Sprague-Dawley rat)

49040645 (1999)

Acceptable/guideline
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Table A.2.2 Subchronic, Chronic and Other Toxicity Profile – Benalaxyl-M

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.7485

Metabolism and 

Pharmacokinetics

(Sprague-Dawley rat)

4904046 (2008)

Acceptable/guideline

Total absorption similar to that in studies above, 

although females excreted more in urine than males 

(Females 17% vs males 8%); however, biliary 

excretion was substantial in both sexes (males 81%; 

females 62%), indicating most of radiolabel was 

absorbed and then excreted via the bile into feces; 

estimated total absorption was similar (males 89%; 

females 82%); recovery 97%-99%. Highest tissue 

concentrations in liver and GI tract in both sexes; Tmax 

was 1.8 hours post dose; terminal half-life of 62.4 

hours; data suggest that absorption and elimination 

were not saturated since there was not a 10-fold 

increase in Cmax and tmax between the 10 mkd and 

100 mkd doses.

870.7485

Metabolism and 

Pharmacokinetics

(Sprague-Dawley rat)

49040647 (2008)

Acceptable/guideline

870.7600

Dermal penetration 

No study available

870.7800

Immunotoxicity

(Wistar rat, male)

49040648 (2013)

Acceptable/guideline

0, 1000, 3000, or 10000 

ppm (28 days) 

0, 80.5, 250.4, or 924.6 

mg/kg/day

Systemic NOAEL = 1000 ppm (80.5 mg/kg/day)

Systemic LOAEL = 3000 ppm (250.4 mg/kg/day), 

based on increased absolute and relative-to-body liver 

weights.

Immunotoxicity NOAEL = 10000 ppm (924.6 

mg/kg/day)

Immunotoxicity LOAEL = not identified.

Non-guideline 

(mechanistic)

(Crl:CD-1(Icr) BR mouse)

49040636 (2013)

Acceptable/nonguideline

0, 200, 1200, or 7000 

ppm (diet; 3 or 14 day)

0, 30.9, 184.4, or 1094.1 

mg/kg/day (males)

Assessment of cell proliferation not adequate; 

microsomal liver enzymes EROD, MROD, and 

LA12OH activities: slightly increased at 1200 and 7000 

ppm; PROD, 6β-OH, and LA11OH activities: 

moderately increased at 7000 ppm; PROD activity:

moderately increased at 1200 ppm; and 6β-OH and 

LA11OH activities: slightly increased at 1200 ppm.

Expression of the Cyp1A-1, Cyp1A-2, Cyp2B10, and 

Cyp3A11 genes were increased at 7000 ppm;

expression of Cyp1A-1, CypB10, and Cyp3A11 were 

increased at 1200 ppm; expression of Cyp2B10 was 

increased 45.8- and 195.7-fold at 1200 and 7000 ppm, 

respectively; expression of Cyp3A11 was increased 

15.58-fold. Examination of the parameters of oxidative 

stress, reduced glutathione (GSH) and oxidized 

glutathione (GSSH) in liver, and thiobarbituric acid 

reactive substance (TBARS) in liver and serum, 

showed no effect of treatment with IR6141 for 14 days.
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A.3 Hazard Identification & Endpoint Selection 

A.3.1 Acute Reference Dose (aRfD) – All Populations

An endpoint for an aRfD for either females age 13-49 or the general population was not 

identified in the toxicity database, including acute neurotoxicity or developmental toxicity

studies. 

A.3.2. Chronic Reference Dose (cRfD)

Study Selected: 2-year carcinogenicity study in rats

MRID No.: 

Executive Summary: See Appendix A.4.5, Guideline § 870.4200.a 

Dose and Endpoint for Risk Assessment: NOAEL = 20 mg/kg/day based 

Comments about Study/Endpoint/Uncertainty Factors: This study was the most sensitive

endpoint and is the appropriate duration and route of exposure. The LOAEL of 135 mg/kg/day

is based on an increase in GGT in males, increased triglycerides in males, increased cholesterol 

in both sexes, slight increases liver weight in both sexes, increased incidence of hepatocellular 

hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both 

sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid 

follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in 

females. A 10X interspecies uncertainty factor to extrapolate from animals to humans; a 10X 

uncertainty factor to account for potential variation in sensitivity among the human population; 

and a 10X database uncertainty factor to account for the lack of a critical study (comparative 

thyroid assay (CTA)) were used. For the POD for the chronic dietary risk assessment, a total 

uncertainty factor of 1000X was applied (10X for interspecies variability, 10X for intraspecies 

variability, and 10X for database uncertainty to account for the lack of a CTA study). When the 

basis of the POD is based on thyroid effects, the interspecies extrapolation UF is typically

reduced to 3X due to the pharmacodynamic differences between rats and humans pertaining to 

thyroid perturbations. However, in the case of benalaxyl-M, the full 10X factor was retained for 

interspecies UF since effects not related to thyroid function disruption are also the basis of the 

POD. 

A.3.3 Incidental Oral Exposure, Dermal Exposure, Inhalation Exposure 

Endpoints for these exposure routes are not needed to assess non-domestic uses. 

A.4 Executive Summaries 

A.4.1 Subchronic Toxicity

870.3100 90-Day Oral Toxicity - Rat
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In a subchronic oral toxicity study (MRID 49040627), five groups of Crl:CD BR rats 

(10/sex/group; ~5 weeks of age) were administered 0, 80, 400, 2000, or 10,000 ppm of 

benalaxyl-M [97.27% (Batch No. FCT/T/156-99 (20398/15)] in the diet and observed for 90 

days. The respective dose levels based on food consumption were 0, 6.2, 30.1, 154, and 861 

mg/kg bw/day for males and 0, 6.7, 32.8, 167, and 892 mg/kg bw/day for females. The animals 

in each test group were subjected to neurobehavioral testing, clinical pathology, and gross and 

microscopic examinations.

There was no adverse effect on survival for either sex, and no clinical signs attributed to the test 

substance were observed at any dietary concentration. Both sexes showed body weight deficits of 

12%-14% throughout the 13-week exposure period at 10000 ppm, which may be attributed to an 

initial aversion to the diet. Decreased food consumption was observed in both sexes during the 

first week of the study but was comparable to the control thereafter 

No statistically significant differences were observed between any treatment group and the 

controls during the functional operational battery (FOB) and locomotor testing. The minor 

changes in hematology parameters were not considered adverse. The most notable changes in the 

clinical chemistry data were dose-related elevations in GGT activity in both sexes at the two 

highest dietary concentrations, elevations in cholesterol at the highest dietary concentration in 

both sexes, and elevations in phospholipids at the mid-high and high concentration in males and 

at the high concentration in females. There were no treatment-related or toxicologically

significant findings in the urinalysis or gross examination findings.

Males and females in the high dose group had statistically higher absolute and relative liver 

weights compared to controls. Females in the mid-high dose group had statistically higher 

relative liver weights. Males in the high dose group had statistically higher relative brain, 

thyroid, testes, and epididymides weights compared to controls, which were likely related to the 

lower body weight at this dietary concentration. Females in the high dose group had statistically

higher absolute and relative thymus weights, and lower absolute and relative heart weights. 

Generalized hepatocyte hypertrophy was observed in all males and females in the high dose 

group and in 8/10 males and 2/10 females in the mid-high dose group. Increased thyroid 

follicular cell hypertrophy was observed in five males and three females at the high dose and in 

three males at the mid-high dose compared to none in the control, low- and low-mid dose groups. 

There was an increase in the incidence and prominence of vacuolated cells in the pituitary pars 

distalis in six males at the high dose. There was an increased incidence of kidney corticomedullary mineralization in females at the high dose (10/10) compared to the control (6/10) and 

other dose groups (5/10).

The NOAEL was 2000 ppm (males 154 mg/kg/day; females 167 mg/kg/day), based on 

elevated GGT values in both sexes, increased cholesterol and phospholipids in both sexes, 

increased liver weight in both sexes, increased thymus weight in females, increased 

incidence of hepatocellular hypertrophy (both sexes), and increased thyroid follicular cell 

hypertrophy (in both sexes) at the LOAEL of 10000 ppm (males 861 mg/kg/day; females 

892 mg/kg/day.
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This subchronic neurotoxicity study is classified as Acceptable/Guideline and satisfies the 

guideline requirement for a 13-week toxicity study in rats (OCSPP 870.3100; OECD 408). 

870.3100 90-Day Oral Toxicity - Mouse

In a subchronic oral toxicity study (MRID 49040628), four groups of CD-1 mice (10/sex/group; 

6 weeks of age) were administered 0, 600, 2000, or 7,000 ppm of benalaxyl-M [96.33% (Batch 

No. G018/04)] in the diet and observed for 90 days. The respective dose levels based on food 

consumption were 0, 93.2, 296.6, and 1053.8 mg/kg bw/day for males and 0, 111.8, 348.1, and 

1253.3 mg/kg bw/day for females. The animals in each test group were subjected to clinical 

pathology testing, and gross and microscopic examinations.

All animals survived to scheduled sacrifice. No treatment-related clinical signs or body weight 

effects were observed. The most notable statistically significant clinical chemistry finding was a 

test material-related elevation in total bilirubin (p<0.01) in females at the high dietary

concentration. Urinalysis data showed a treatment-related elevation in glucose in males and 

females at the mid and high dose levels. Males and females in the high dose group had 

statistically higher absolute and relative liver weights compared to controls (p≤0.01). Females in 

the mid-high dose group had statistically higher relative liver weights (p≤0.01). Minimal to slight 

periportal hepatocellular hypertrophy (without signs of necrosis) was observed in both sexes in 

all treated dose groups (none in controls), but only the females showed a dose-response. The 

average grade of hepatocellular glycogen deposits decreased in both sexes in all treated dose 

groups, but only the females showed a dose-response. 

The LOAEL in mice was 7000 ppm (1054 mg/kg/day in males and 1253 mg/kg/day in 

females), based on increased liver weights, increased incidence and severity of periportal 

hepatocellular hypertrophy in females, increased glucose in the urine in both sexes, focal 

necrosis in the liver in males, and depleted glycogen in the liver in females. The NOAEL is 

2000 ppm (296.6 mg/kg/day in males and 348.1 mg/kg/day in females).

This subchronic oral toxicity study is classified as Acceptable/Guideline and satisfies the 

guideline requirement for a 13-week toxicity study in a rodent (OCSPP 870.3100; OECD 408).

870.3150 90-Day Oral Toxicity – Dog

In a 13-week oral toxicity study (MRID 49040629), five groups of beagle dogs (4/sex/group; 23-

36 weeks of age) were administered 0, 150, 450, 900, or 3000 ppm of benalaxyl-M [97.27% 

(Batch No. FCT/T/156-99 (20398/15))] via the diet. The respective dose levels based on food 

consumption were 0, 5.58, 16.24, 32.02, and 112 mg/kg bw/day for males and 0, 5.85, 17.34, 

34.97, and 110 mg/kg bw/day for females. The animals in each test group were subjected to 

clinical pathology, and gross and microscopic examinations.

All animals survived to the end of the study. No clinical signs attributed to the test substance 

were observed at any dietary concentration. Body weights were slightly lower at the 3000 ppm 

dose level in both sexes at termination (males ↓4%/females ↓8%) compared to the control 

groups. Food consumption was not adversely affected in either sex. The most notable changes in
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clinical chemistry data were statistically elevated alkaline phosphatase activity (ALK) in both 

sexes (males ↑353%-494%/females ↑287%-374%) at the high dietary concentration, with the 

magnitude of the effect increasing with time. ALK was also elevated at the mid-high 

concentration in males (↑110%-141%) at both the 6- and 13-week sampling intervals and in 

females (↑85%) at the mid-high concentration at the 13-week interval. The historical control 

range for ALK activity was exceeded in males and females at the high dietary concentration. 

Triglycerides were significantly elevated at the high dose level in males (↑70%-96%) at 6 and 13 

weeks and elevated in high-dose females (↑40%-68%) at the same time points but statistical 

significance was not attained. There were no treatment-related or toxicologically significant 

findings in the hematology or urinalysis data. Enlarged liver was observed in 2/4 males and 1/4 

females at the high dietary concentration during gross examinations. Males and females in the 

high dose group had statistically higher absolute (↑19%) and relative (↑23%-29%) liver weights 

compared to controls (p≤0.01). Hepatocyte hypertrophy in centrilobular and midzonal areas were 

observed in all males and females in the mid-high and high dose groups. The change extended to 

the periportal area in two males and two females at the high dose and in one female from the 

mid-high dose group. No signs of liver necrosis were observed.

The no-observed-adverse effect level (NOAEL) for benalaxyl-M in beagle dogs was 900 

ppm (32 mg/kg/day in males and 35 mg/kg/day in females). A lowest-observed-adverse 

effect level (LOAEL) for IR 6141 in beagle dogs was 3000 ppm (112 mg/kg/day in males 

and 110 mg/kg/day in females), based on elevated alkaline phosphatase activity in both 

sexes, elevated triglyceride levels in males, increased liver weight in both sexes, and an 

increased incidence/severity of hepatocyte hypertrophy in the centrilobular, midzonal, and 

periportal areas in males and females.

This subchronic toxicity study in the dog is classified as Acceptable/Guideline and satisfies the 

guideline requirement for a 13-week toxicity study in non-rodents (OCSPP 870.3150; OECD 

409). 

870.3200 21/28-Day Dermal Toxicity – Rat

In a 28-day dermal toxicity study (MRID 49040631), benalaxyl-M technical (96.53% a.i., Lot 

No. P/10/106) was applied to the shaved skin of 10 Wistar rats/sex/group at concentrations of 0, 

62.5, 250, or 1000 mg/kg bw/day, 6 hours/day for 28 consecutive days. Additional groups of 10 

rats/sex were treated for the same time period at concentrations of 0 or 1000 mg/kg bw/day, 

followed by a two-week recovery period. 

No dermal irritation was observed during treatment, and no treatment-related moribundity or 

mortality were found. No neurological effects were observed. There were no treatment-related 

effects on body weight, food consumption, ophthalmoscopic parameters, the estrus cycle, or 

effects on hematology, clinical chemistry, or urinalysis parameters. There were no treatmentrelated effects noted at necropsy on absolute or relative to body or brain organ weights, or 

microscopic treatment-related effects. 

The dermal toxicity NOAEL for male and female Wistar rats was 1000 mg/kg bw/day. A 

LOAEL was not determined. 
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This 28-day dermal toxicity study in the rat is Acceptable/Guideline and satisfies the guideline 

requirement for a 28-day dermal toxicity study (OPPTS 870.3200; OECD 410) in rats.

870.3465 90-Day Inhalation – Rat

An inhalation study is not required for an import tolerance. 

A.4.2 Prenatal Developmental Toxicity

870.3700a Prenatal Developmental Toxicity Study - Rat

In a developmental toxicity study (MRID 49040637), benalaxyl-M [>99% a.i.; batch # 

FCF/T/173-00 (ex 20493/57/B lotto II)] was administered to 25 female Crl:CD (SD) BR 

rats/dose in 0.5% aqueous methylcellulose by oral gavage at dose levels of 0, 10, 50, or 250 

mg/kg bw/day on gestation days (GD) 6 through 15 (dose volume of 10 mL/kg bw/day). Dams 

were sacrificed and necropsied on GD 20. Pregnant uterus weight, numbers of corpora lutea, 

implantations, early and late resorptions, and live and dead fetuses were recorded. Individual 

fetal and placental weights were recorded. All fetuses were sexed and subjected to gross external 

evaluations. Approximately half of the fetuses per litter underwent skeletal evaluations while the 

other half underwent visceral examinations.

There was no evidence of maternal toxicity. No clinical signs, behavioral changes, body weight 

effects, or deaths were observed in dams during the study. An initial reduction in food 

consumption (GD 8-10) was observed in the dams at 250 mg/kg bw/day, but no differences were 

noted for absolute body weight or corrected body weight gain. No gross pathological effects 

were noted at sacrifice, and no treatment-related effects on cesarean parameters were observed at 

any dose level. 

The maternal lowest-observed-adverse-effect level (LOAEL) for benalaxyl-M in rats was 

not determined. The maternal NOAEL was greater than or equal to 250 mg/kg bw/day. 

There were no treatment-related effects on fetal sex ratio, fetal weight, post-implantation loss, 

resorptions per litter, or live litter size. Although the 10 and 250 mg/kg bw/day groups had 

increases in early resorptions, there was no dose response. Further, a slightly lower mean 

number of live fetuses per litter was noted in the 250 mg/kg bw/day group but was not 

considered treatment-related due to small magnitude (<1 fetus), a corresponding lower number 

of mean implantations per dam, and the absence of a correlated increase in post-implantation 

loss. One to two malformed fetuses were present in all groups, including the control group. 

Therefore, no developmental effects were observed at doses up to 250 mg/kg bw/day. 

The developmental LOAEL for benalaxyl-M in rats was not determined, and the 

developmental NOAEL was greater than or equal to 250 mg/kg bw/day.

This developmental toxicity study in the rat is classified Acceptable/Non-Guideline, but it does 

satisfy the guideline requirement for a developmental toxicity study (OSCPP 870.3700; OECD 
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414) in the rat. It is non-guideline because no significant developmental or maternal toxicity

occurred at the highest dose level tested. However, a new study is not required at this time since 

a study at higher dose levels would not identify a more sensitive endpoint for risk assessment 

than the endpoint selected for risk assessment. 

870.3700b Prenatal Developmental Toxicity Study - Rabbit

In a developmental toxicity study (MRID 49040638), benalaxyl-M [97.14% a.i., batch # 

FCF/T/173-00 (ex 20493/57/B lotto II)] was administered to 24 mated female Himalayan 

rabbits/dose in 0.5% aqueous methylcellulose by oral gavage (dose volume of 4 mL/kg bw/day) 

at dose levels of 0, 50, 250, or 1000 mg/kg bw/day on gestation days (GD) 6 through 27, 

inclusive. Dams were sacrificed on day 28 of gestation and underwent gross macroscopic 

examination. All fetuses were weighed, killed, sexed, examined for gross external abnormalities, 

and examined for both soft tissue and skeletal alterations. Heads from one-half of the fetuses 

were subjected to evaluation of the internal structures, including the eyes, brain, nasal passages, 

and tongue.

Treatment with benalaxyl-M did not cause any maternal deaths, clinical signs, or effects on food 

consumption or body weight/body weight gain at doses up to and including 1000 mg/kg bw/day. 

The maternal LOAEL for benalaxyl-M in rabbits was not determined. The maternal

NOAEL was greater than or equal to 1000 mg/kg bw/day, the highest dose tested.

No treatment-related fetal deaths or resorptions were noted, and fetal sex ratio was unaffected by 

treatment. At 1000 mg/kg bw/day, there was a slight decrease (6%) in fetal body weight, 

correlated with a slight delay in the stage of development in this dose group as indicated by

increased percentages of fetuses with incomplete chondrification of cartilaginous sternebra(e) 2, 

3, and/or 4 compared to controls. However, these findings were significant only when analyzed 

on an individual fetal basis, while the litter should be considered the statistical unit of measure. 

Skeletal abnormalities were limited to common sternebral abnormalities (including fused and 

abnormally shaped sternebrae) and abnormalities of cartilaginous sternebrae and/or costal 

cartilages (including bipartite and/or abnormally shaped cartilaginous sternebrae, fused or 

bifurcated costal cartilages). The combined incidence of these abnormalities was 2 (2), 3 (2), 3 

(3), and 6 (6) fetuses (litters) in the 0, 50, 250, and 1000 mg/kg bw/day groups, respectively; the 

slight increase in the high dose group was not statistically significant or considered adverse. 

External and visceral abnormalities were incidental and not related to treatment and included one 

fetus in the 50 mg/kg bw/day group with an encephalocele and an open skull and two fetuses in 

the 1000 mg/kg bw/day group: one with flexure of the right forelimb and one with minimal 

bilateral dilation of the lateral brain ventricles.

The fetal LOAEL for benalaxyl-M in rabbits was not determined; and the fetal NOAEL 

was greater than or equal to 1000 mg/kg bw/day. 

The developmental toxicity study in the rabbit is classified Acceptable/Guideline and satisfies 

the guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414) in 

rabbits. 
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A.4.3 Reproductive Toxicity

870.3800 Reproduction and Fertility Effects - Rat

In a two-generation reproduction study (MRID 49040639), benalaxyl-M (Benalaxyl-M; 96.33% 

a.i.; Batch No. G 018/04) was administered to groups of 24 HanRcc:WIST (SPF) rats/sex/dose in 

the diet at concentrations of 0, 120 [80], 360 [240], 1080 [725], or 3240 [2170] ppm, with 

lowered concentrations [those indicated in brackets] administered to lactating females and 

weaned pups to maintain a consistent intake of the test material in terms of mg/kg bw/day. 

Premating doses were respectively 0, 7.9, 23.5, 71.1, or 221.3 mg/kg bw/day for F0 males, 0, 8.9, 

26.7, 77.5, or 241.8 mg/kg bw/day for F0 females, 0, 8.9, 27.1, 81.0, or 257.0 mg/kg bw/day for 

F1 males, and 0, 9.9, 29.5, 85.8, or 280.2 mg/kg bw/day for F1 females. Except for one high-dose 

F0 “replacement” male, the F0 and F1 parental animals were administered the treated or control 

diets for at least 70 days (for F0) or 90 days (for F1) prior to mating, throughout mating, 

gestation, and lactation, and until necropsy after weaning of litters, and one litter was produced 

by each generation. The replacement animal was treated for only 55 days prior to mating. 

Additional groups of 24 F1 pups/sex/dose were selected for post-weaning functional evaluations, 

including a functional observational battery/modified Irwin screen (FOB/modified Irwin) and 

locomotor activity measurement.

No significant treatment-related effects on mortality, clinical signs, body weight, or food 

consumption were noted in either sex or generation. Treatment-related increased incidences of 

hepatocellular hypertrophy (mainly centrilobular) were observed in both sexes and generations at 

the high and high-mid doses and in low-mid-dose F0 males, F1 males, and F1 females. Other 

findings included increased liver weights in high-dose males of both generations (absolute and 

relative-to-brain weights 20% and 16-17% greater than controls for F0 and F1 high-dose males, 

respectively; p<0.01), and increased incidence of gross enlargement of the liver in high-dose F1

females (9/24 high-dose F1 females vs. 0 controls; p<0.01). The liver is a target organ following 

benalaxyl-M exposure, and these liver findings are consistent with other benalaxyl-M rat studies. 

Treatment-related decreased spleen weights were seen in high-dose females of both generations, 

with the absolute and relative (to brain) spleen weights 14% lower than controls for F1 and 16%-

17% lower than controls for F2. Since hematology parameters and spleen histopathology were 

not examined as part of the current study, the toxicological significance of the decreased spleen 

weights is not known. A similar decrease was not observed in the chronic rat study in either sex 

at comparable dose levels. No treatment-related effects were observed on fertility or 

reproductive performance in male or female rats in either generation. Estrous cycle length, 

sperm measures, reproductive indices, precoital interval, gestation length, and postimplantation 

loss were not affected by treatment. Significantly decreased numbers of primordial follicles and 

corpora lutea was observed in high-dose F1 females (respectively -26% and -29%, relative to 

controls); it is unknown whether this effect extended into the lower dose groups because the 

ovarian follicles were only counted in high-dose and control F1 females. However, the magnitude 

of the deficit at the high dose suggests that any treatment-related effects at lower dose levels 

would not be toxicologically significant.
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Under the conditions of this study, the parental systemic LOAEL for benalaxyl-M in 

female rats is 3240/2170 ppm (241.8 mg/kg bw/day), based on decreased numbers of 

primordial follicles and corpora lutea in the F1 females, decreased uterus weights in F0 

females, and a slight increase in the incidence of hepatocellular hypertrophy in both 

generations. The parental systemic NOAEL in females is 1080/725 ppm (77.5 mg/kg 

bw/day). The parental systemic LOAEL in males is 3240 ppm (221.3 mg/kg bw/day), based 

on increased liver weights and an increased incidence of hepatocellular hypertrophy in 

both generation. The parental systemic NOAEL in males is 1080 ppm (71.1 mg/kg bw/day).

During postnatal days (PNDs) 7-14 and 14-21, the body weight gain of the high-dose F2

offspring of both sexes was significantly decreased (-11% to -13%) with resultant significantly 

decreased absolute body weights on PND 14 (males: -8%, p<0.05; females: -10%, p<0.01) and 

PND 21 (males: -10%; females: -11%; p<0.01 for both). High-dose F1 females had a treatmentrelated 1.5-day delay in vaginal opening (p<0.05), with the group’s mean body weight at 

completion of vaginal opening comparable to that of the control group. Decreased absolute and 

relative (to brain) spleen weights were noted in high-dose males and females of the F1 generation 

(-16% for both sexes and both parameters) and the F2 generation (males: -21% to -22%; females: 

-17% to -19%). Since hematology parameters and spleen histopathology were not examined as 

part of the current study, the toxicological significance of the decreased spleen weights is not 

known. There were no effects on pup survival/mortality, litter size from birth to weaning, 

postimplantation loss, viability indices, sex ratio, or male sexual maturation. There were no 

treatment-related clinical observations or gross findings in pups or weanlings. There was an 

increased incidence of hepatocellular hypertrophy in both sexes of F2 pups at the high dose.

Under the conditions of this study, postweaning functional investigations (FOB and locomotor 

testing) did not reveal any treatment-related effects. However, due to the absence of positive 

control data demonstrating competence of the testing laboratory, the lack of provided 

information concerning the test methods and conditions, and the use of 15-minute subsessions 

for the locomotor testing, the reviewer does not consider the apparent absence of 

neurobehavioral effects to be definitive. Since these parameters are not typically assessed in a 

reproduction study, and there are acute and subchronic neurotoxicity studies available to assess 

these parameters, these deficiencies do not interfere with the interpretation of this study.

The offspring LOAEL for benalaxyl-M in rats is 221.3 mg/kg bw/day for males and 241.8 

mg/kg bw/day for females, based on decreased pup body weight in F2 pups of both sexes

and increased incidence of hepatocellular hypertrophy in F2 pups of both sexes. The 

offspring NOAEL is 1080/725 ppm (71.1 mg/kg bw/day) for males and 77.5 mg/kg bw/day 

for females. 

Findings potentially indicative of reproductive toxicity included decreased ovarian follicle counts 

(primordial follicles and corpora lutea). The decrease in ovarian follicle counts in the high-dose 

F1 females is considered treatment-related and adverse. The results in the treated group were 

outside the historical control range, and the concurrent control values were generally low 

compared to the historical control values. Although ovarian follicle counts were not assessed for 

the lower dose groups, it was concluded that the magnitude of the findings at the high dose are 

such that the results at lower doses would not be considered adverse. 
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The reproductive toxicity LOAEL for benalaxyl-M is 3240/2170 ppm (241.8 mg/kg 

bw/day), based on decreased numbers of primordial follicles and corpora lutea in the F1 

females. The reproductive toxicity NOAEL is 1080/725 ppm (77.5 mg/kg bw/day). 

This study is Acceptable/Guideline and satisfies the guideline requirement for a two-generation 

reproductive study [OCSPP 870.3800; OECD 416] in the rat. Despite deficiencies in the conduct 

and reporting of this study and the occurrence of only minimal parental toxicity, offspring 

toxicity and potential reproductive toxicity were seen. 

A.4.4 Chronic Toxicity

870.4100a (870.4300) Chronic Toxicity – Rat

See under 870.4200a

870.4100b Chronic Toxicity - Dog

In a 52-week oral toxicity study (MRID 49040630), four groups of beagle dogs (4/sex/group; 

5.5-7.5 months of age) were administered via capsule 0, 10 (26 weeks)/300 (26 weeks), 30, or 

100 mg/kg/day (mg/kg) of benalaxyl-M [96.33% (Batch No. G018/04] for 52 weeks. The 10 

mg/kg/day dose was increased to 300 mg/kg beginning week 27 to increase the chance of 

detecting toxic effects. The animals in each test group were subjected to clinical pathology, and 

gross and microscopic examinations.

All animals survived to the end of the study. No clinical signs attributed to the test substance 

were observed at any dose. There were no treatment-related or toxicologically significant 

findings observed in the body weight data. No treatment-related effects were observed on 

hematology values, clinical chemistry values, urinalysis values, organ weight data, or during 

gross examination of tissues. Minimal or slight periportal fat vacuolation in the liver was 

observed in 3/4 males and 2/4 females from the 10/300 mg/kg/day dose group. All females from 

the 10/300 mg/kg/day dose group had a minimal diffuse hypertrophy of the thyroid gland. 

The lowest observed-adverse effect level (LOAEL) for benalaxyl-M in beagle dogs was not 

identified.

This chronic toxicity study in the dog is classified as Acceptable/Guideline. 

A.4.5 Carcinogenicity

870.4200a Carcinogenicity Study - rat

In a combined chronic toxicity/carcinogenicity study (MRID 49040634), benalaxyl-M (96.33% 

a.i., Lot/Batch # G018/04) was administered in the diet to 20 male and 20 female Sprague 

Dawley CD Crl:CD(SD)BR rats at concentrations of 0, 60, 390, or 2535 ppm (0, 2.92, 19.0, and 

126 mg/kg bw/day, respectively, for male rats and 0, 3.82, 24.6, and 167 mg/kg bw/day, 
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respectively, for female rats) for 52 weeks (chronic study) and groups of 65 male and 65 female 

rats were administered the same dietary concentrations for 104 weeks (main carcinogenicity

study). The equivalent doses for the main study were 0, 2.40, 15.7, and 104 mg/kg bw/day, 

respectively, for male rats and 0, 3.13, 20.0, and 135 mg/kg bw/day, respectively, for female rats. 

No treatment-related, adverse effects were observed on mortality, clinical signs, detailed clinical 

observations, FOB parameters, body weight and body weight gain, hematology, nodules/masses, 

water consumption, food consumption, or eyes in either sex. Increased γ-glutamyl transferase

(GGT) levels were observed in both sexes at 7000 ppm, with males displaying the greater effect. 

Both sexes showed slightly increased cholesterol values also. Absolute liver weight was not 

significantly increased (11%-12%) by treatment with the test substance, but the liver/body 

weight ratio was significantly increased by 10% to 18% in high-dose male and female rats after 

52 and 104 weeks of treatment. The liver/brain weight ratio was not significantly affected by

treatment with the test substance. 

Gross and microscopic examination showed treatment-related changes primarily in the liver and 

thyroid gland. Gross examination showed accentuated lobulated pattern in the liver of 4/20 highdose male rats compared with none of the controls. In the chronic study (52 weeks), treatmentrelated microscopic lesions in the liver included fatty change and hepatocellular hypertrophy

(primarily centrilobular) at the mid- and high-dose level in male rats, hepatocellular hypertrophy 

at the mid- and high-dose level in female rats, and fatty change and infiltrating inflammatory

cells at the high-dose level in female rats. The incidences of bile duct hyperplasia and peribiliary

fibrosis were decreased in high-dose male and female rats compared with that of controls. The 

incidence of thyroid follicular cell hypertrophy was significantly increased in male rats in the 

mid- and high-dose groups and in females in the high-dose group compared with that of controls. 

In the main study (104 weeks), the incidences of eosinophilic foci and spongiosis hepatis in the 

liver of male rats and biliary cysts in females showed statistically significant positive doserelated trends but did not reach statistical significance at any dose; the incidence of eosinophilic 

foci was significantly increased in females in the high-dose group compared with that of 

controls. The incidence of lesions in the thyroid gland was increased only in female rats in the 

high-dose group. These included follicular cell hypertrophy, follicular cell hyperplasia, and 

follicular ectasia. The incidence of ovarian stromal cell hyperplasia was increased in females at 

the high-dose level.

The lowest-observed-adverse-effect level (LOAEL) for benalaxyl-M in rats is 2535 ppm 

(males 104/females 135 mg/kg bw/day), based on an increase in GGT in males, increased 

triglycerides in males, increased cholesterol in both sexes, slight increases liver weight in 

both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased 

incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid 

cell hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and 

an increased incidence of ovarian stromal cell hyperplasia in females. The no-observedadverse-effect level (NOAEL) is 390 ppm (males 15.7 mg/kg bw/day and females 20 mg/kg 

bw/day).

At the doses tested, the incidences of thyroid follicular cell adenomas and follicular cell 

adenomas/carcinomas combined were marginally increased in females at the high dose compared 
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with that of controls. Additionally, there was an increased incidence of hepatocellular adenomas 

in both sexes at the high dose. 

This chronic/carcinogenicity study in the rat is Acceptable/Guideline and satisfies the guideline 

requirement for a chronic/carcinogenicity study (OCSPP 870.4300; OECD 453) in rats. 

870.4200b Carcinogenicity (feeding) - Mouse

In a carcinogenicity study (MRID 49040635), benalaxyl-M (96.33% a.i., Batch/Lot # G 018/04) 

was administered in the diet to 50 male and 50 female CD-1 (SPF) mice per dose at 

concentrations of 0, 200, 1200 or 7000 ppm for 78 weeks (equivalent to 0, 24.84, 156.05, and 

962.23 mg/kg bw/day for males and 0, 31.36, 196.72, and 1180.41 mg/kg bw/day for females).

No treatment-related effects were observed on the following parameters: mortality, clinical signs, 

body weight, food consumption, and hematology parameters. The liver was identified as the 

target organ with treatment-related effects observed at 1200 ppm in male mice and at 7000 ppm 

in both sexes. At 7000 ppm, absolute liver weight, liver to brain weight ratio, and liver to body

weight ratio were significant increased by 52%-63% in males and 52%-61% in females 

compared with that of controls. Macroscopic examination showed a significant increase in liver 

nodules in males (18/50 vs 8/50 in controls, p<0.01) and in females 4/50 vs 1/50, N.S.) at 7000 

ppm. At 1200 ppm, the only treatment-related finding in the liver was an increased incidence of 

diffuse hepatocellular hypertrophy in male mice (21/50 vs 8/50, p<0.01). Treatment-related 

microscopic liver lesions were observed in male mice at 7000 ppm were diffuse hepatocellular 

hypertrophy (49/50 vs 8/50 in controls, p<0.01), single cell necrosis (30/50 vs 10/50 in controls, 

p<0.01), focal/multifocal necrosis (12/50 vs 5/50 in controls, p<0.05), and eosinophilic focus 

(6/50 vs 1/50 in controls, p<0.01). Treatment-related liver lesions were observed in female mice 

at 7000 ppm were diffuse hepatocellular hypertrophy (44/50 vs 0/50 in controls, p<0.01) and 

single cell necrosis (21/50 vs 5/50 in controls, p<001). In addition, the severity of the liver 

lesions also increased in both sexes at 7000 ppm, as evidenced by the average severity grade and 

the incidence of mice with moderate and/or severe forms of the lesion. At 7000 ppm, one male 

and one female displayed thyroid follicular cell hyperplasia. 

The lowest-observed-adverse-effect level (LOAEL) for benalaxyl-M in male and female 

mice is 7000 ppm (962.23 mg/kg bw/day for males and 1180 mg/kg bw/day for females) 

based on increased liver weight in both sexes and an increased incidence of liver lesions 

(diffuse hepatocellular hypertrophy in both sexes, single cell necrosis in both sexes,

focal/multifocal necrosis in males, and eosinophilic foci in males). The NOAEL is 1200 

ppm (156 mg/kg bw/day for males and 197 mg/kg/day for females).

At the doses tested, there was a treatment related increase in the incidence of hepatocellular 

adenoma in male mice, and an increase in the incidence of hepatocellular adenoma/carcinoma 

combined in male mice and female mice. The increased incidences exceeded those of historical 

controls for hepatocellular adenoma in male mice and hepatocellular adenoma/carcinoma 

combined in both sexes. 
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This carcinogenicity study in mice is Acceptable/guideline and satisfies the guideline 

requirement for a carcinogenicity study [OPPTS 870.4200; OECD 451] in mice.

A.4.6 Mutagenicity

Guideline No./

Study Type

MRID No. (year)/ 

Classification/Doses

Results

Benalaxyl-M

870.5100

Bacterial reverse gene 

mutation (Ames Assay)

49040640 (2002)

Acceptable/guideline

5- 5000 μg/plate +/ –S9 (plate 

incorporation)

5- 1500 μg/plate +/ –S9 

(preincubation)

Negative up to the limit 

dose; compound 

precipitation at ≥1500 

μg/plate

870.5300

In vitro mammalian forward 

cell gene mutation (mouse 

lymphoma cells)

49040642 (1999)

Acceptable/guideline

5- 75 μg/mL –S9 

15- 100 μg/mL +S9 

Negative up to cytotoxic 

concentration 

870.5375

In vitro mammalian 

chromosome aberration test

[Chinese Hamster Ovary

(CHO cells)]

49040641 (1999)

Acceptable/guideline

Assay 1 5- 3000 μg/mL+/ –S9 (3

hrs.)

Assay 2 5- 150 μg/mL –S9 (18 

hrs.)

15- 150 μg/mL +S9 (3 hrs.)

Negative up to cytotoxic 

& insoluble concentration

870.5395

Mammalian erythrocyte 

micronucleus assay (mouse)

49040643 (2000)

Acceptable/guideline

0, 100, 200, 400 mg/kg M

0, 75, 150, 300 mg/kg F

Single i.p. injections

Negative up to doses 

(≥300 mg/kg) causing 

overt toxicity in 

preliminary study (deaths 

and/or piloerection)

N-malonyl-N-(2, 6-xylyl)-D, L-alanine

870.5100

Bacterial reverse gene 

mutation (Ames Assay)

49040606 (2002)

33- 5000 μg/plate +/ –S9 

Acceptable/guideline

Negative up to the limit 

dose; plate incorporation

& preincubation

870.5300

In vitro mammalian cell gene 

mutation (mouse lymphoma 

cells)

49040608 (2002)

168.8 – 2700 μg/mL –S9 (4 hrs.)

168.8 – 2565 μg/mL +S9 (4 hrs.)

168.8 – 2700 μg/mL –S9 (24 hrs.)

Acceptable/guideline

Negative up to insoluble 

concentrations

870.5375

In vitro mammalian 

chromosome aberration test

[Chinese Hamster Ovary

(CHO cells)]

49040609 (2002)

Assay 1 60.3 – 1930 μg/mL-/+S9 

(4 hrs.)

60.3 – 1930 μg/mL-S9 (24 hrs.)

Assay 2

Negative up to a 

cytotoxic concentration

(1930 μg/mL-S9, 24 & 

46

hrs.)
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Guideline No./

Study Type

MRID No. (year)/ 

Classification/Doses

Results

241.3 – 1930 μg/mL-S9 (46 hrs.)

241.3 – 1930 μg/mL+S9 (4 hrs.)

Acceptable/guideline

Methyl-N-malonyl-N-(2, 6-xylyl)-D, L-alaninate

870.5100

Bacterial reverse gene 

mutation (Ames Assay)

49040605 (2002)

33- 5000 μg/plate +/ –S9 

Acceptable/guideline

Negative up to the limit 

dose; plate incorporation 

& preincubation

870.5300

In vitro mammalian cell gene 

mutation (mouse lymphoma 

cells)

49040607(2002)

156.3 – 2500 μg/mL +/ –S9 (4 

hrs.)

156.3 – 2500 μg/mL +/ –S9 (24 

hrs.)

Acceptable/guideline

Negative up to insoluble 

concentrations

870.5375

In vitro mammalian 

chromosome aberration test

[Chinese Hamster Ovary

(CHO cells)]

49040610 (2002)

Assay 1 19.5 – 2500 μg/mL-/+S9 

(4 hrs.)

Assay 2 625 – 1875 μg/mL-S9 (24

& 46 hrs.)

625 – 2500 μg/mL +S9 (4 hrs.)

Assay 3 1000- 1500 μg/mL-S9 

(24& 46 hrs.)

Acceptable/guideline

Confirmed positive:

Assay 2 937.5, 1250 & 

1875 μg/mL-S9 (24 hrs.)

& 937.5 μg/mL-S9 (46

hrs.).

Assay 3 1500 μg/mL-S9 

(24 hrs.)

870.5375

In vitro mammalian 

chromosome aberration test

(Human lymphocytes)

49040611 (2002)

Assay 1 0.078 – 10mM -/+S9 (3

hrs.)

Assay 2 0.313 – 10 mM -S9 (20 

hrs.)

0.313 – 10 mM +S9 (3 hrs.)

Acceptable/guideline

Positive: at

10 mM -S9 (20 hrs.)

Limit dose

870.5395

Mammalian erythrocyte 

micronucleus assay (mouse)

49040612 (2002)

0, 500, 1000, 2000 mg/kg M & F

Single i.p. injections

Acceptable/guideline

Negative up to an overtly 

toxic dose (2000 mg/kg)
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A.4.7 Neurotoxicity

870.6100 Delayed Neurotoxicity Study - Hen

A delayed neurotoxicity is not required and is not available. 

870.6200 Acute Neurotoxicity Screening Battery

In an acute neurotoxicity study (MRID 49040626), groups of unfasted, 6- to 9-week-old CRL: 

Wistar rats (12/sex/dose) were given a single oral (gavage) dose of benalaxyl-M (96.53% w/w; 

Batch/Lot #P/10/106) in 0.5% aqueous carboxymethylcellulose (CMC) solution (dose volume of 

10 mL/kg) at doses of 0, 500, 1000, or 2000 mg/kg bw and observed over a 14-day postdose 

period. In addition to observations of all animals for clinical signs of overt toxicity conducted 

daily from the day of dosing to study termination on Day 15, weekly body weight measurements 

and neurobehavioral assessments (functional observational battery, landing foot splay testing, 

grip strength measurements, and motor activity testing) were conducted on all animals on predose Day -7, dose Day 0 (starting at approximately 30 minutes after dosing), and post-dose Days 

7 and 14. At study termination on Day 15, at least 6 animals/sex/group were perfusion-fixed. 

Other surviving animals were euthanized under pentobarbital anesthesia by exsanguination. 

Gross necropsy was performed on each animal. Brain weights of non-perfused animals were 

obtained on Day 15; brains of perfused animals were weighed on Day 17, after a 48 hour period 

of post-perfusion whole body fixation. Central nervous system and peripheral nervous system 

tissues from the control (0 mg/kg) and high dose (2000 mg/kg) groups of perfused animals (6 

animals/sex/group) were processed into slides (embedded in paraffin or plastic Epoxy resin and 

sectioned) for histopathological evaluation. 

There were no treatment related effects on mortality, clinical signs, body weight, brain weight or 

gross and histologic pathology or neuropathology. FOB, landing foot splay, fore- and hindlimb 

grip strength, and motor activity testing revealed no treatment related effects.

Based on the absence of treatment related effects in this study, a LOAEL was not 

identified. The NOAEL for benalaxyl-M technical product was 2000 mg/kg bw 

administered orally in adult male and female Wistar rats.

This neurotoxicity study is classified as Acceptable/Guideline and satisfies the guideline 

requirement for an acute neurotoxicity study in rats (OCSPP 870.6200a; OECD 424). 

870.6200 Subchronic Neurotoxicity Screening Battery

In a subchronic neurotoxicity study (MRID 49143501), benalaxyl-M technical product 

(Benalaxyl-M; 96.53% a.i.; Batch/Lot #P/10/106) was administered in the diet to 12 CRL:Wistar 

rats/sex/group at dietary levels of 0, 1000, 3000, or 10,000 ppm (resulting in calculated daily

dose levels of 0, 67, 206 and 784 mg/kg bw/day for males and 0, 78, 325, and 1139 mg/kg 

bw/day for females, respectively) for 90 days. Treatment was initiated on Day 0, and the 

neurobehavioral assessment (functional observational battery [FOB] and motor activity testing) 

was performed in all animals on Days -7, 14, 42, and 77 (Weeks -1, 3, 7 and 12). At study
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termination (Day 90), at least 6 animals/sex/group were euthanized via in situ perfusion fixation, 

and the central and peripheral nervous system tissues from 6 male and 6 female perfused control 

and high-dose animals were subjected to histopathological evaluation. Additional measures 

included ophthalmoscopic examination (all animals on Day -6 and control and high-dose at 

term), serum GGT activity (in all animals at term), gross pathology (of all surviving animals), 

and brain and liver weights (all surviving animals).

There were no treatment-related effects on mortality, general or detailed clinical signs, brain 

weight, or macroscopic and neurohistologic evaluations. Neurobehavioral test measures 

including FOB, landing foot splay, grip strength and motor activity testing also revealed no 

treatment-related effects for either males or females at any dietary level. Absolute body weights 

and food consumption were not affected by treatment in either sex. Liver weights (absolute and 

relative) were significantly increased in non-perfused males at the low-, mid-, and high-dose 

treatment levels (absolute weights: +29%. +42%, and +51%, respectively; p<0.05 or p<0.01) and 

in non-perfused mid- and high-dose females (absolute weights: +20% and +45%; p<0.01 for 

high-dose, only). In perfused animals, liver weights were increased only at the high-dose 

treatment level (both sexes), but it is possible that liver weights of these animals were influenced 

by the perfusion/fixation process. Increased serum GGT activity was seen in high-dose animals 

of both sexes (males: 14.0 U/L; females: 16.1 U/L; both vs. 5.0 U/L for controls and p<0.01), 

which correlated with the increased liver weights in the high-dose animals of both sexes. 

Based on increased serum GGT activity in both sexes and increased absolute and relative 

liver weights, the LOAEL for benalaxyl-M in rats is 10000 ppm (equivalent to 784 mg/kg 

bw/day for males and 1139 mg/kg bw/day for females). The NOAEL is 3000 ppm 

(equivalent to 206 mg/kg bw/day for males and 325 mg/kg bw/day for females).

The study is classified as Acceptable/Guideline and satisfies the guideline requirement for a 

subchronic neurotoxicity study in rats [OCSPP 870.6200b; OECD 424]. 

870.6300 Developmental Neurotoxicity Study

A developmental neurotoxicity study is not required and is not available. 
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A.4.8 Metabolism

870.7485 Metabolism - Rat

In four metabolism studies (MRIDs 49040644, 49040645 and 49040646, 49040647), [14C-Uaniline ring]-benalaxyl-M (>99% a.i., Batch/lot No. - MRID 49040644: FCF/T/155-98, MRID 

49040645: 20376/76, MRIDs 49040646 and 49040647: B01/L-4) was administered to groups of 

5 male Sprague Dawley rats at doses of 0 or 10 mg/kg bw or to groups of 2 – 4 male and female 

Sprague Dawley rats at doses of 10 or 100 mg/kg bw by oral gavage. 

(MRIDs 49040644 and 49060445). Following a single 10 mg of 14C [U-aniline ring] –

benalaxyl-M/kg bw oral dose to male rats, the test material was readily absorbed and excreted 

mainly through the fecal route (~85%) and to a smaller extent via the urine (~8%) within 48 

hours. Biliary excretion, as demonstrated in the subsequent studies, explains the high 

elimination of absorbed radioactivity via the fecal route. The highest tissue concentrations, as 

percent of administered dose 0.5 hours after treatment, were found in the intestinal wall (8.0%), 

liver (8.3%), and stomach wall (6.4%). Total radiolabel recovered in the tissues 0.5 hours after 

treatment was 23%. By 72 hours after treatment, all tissue concentrations were <1% of the 

administered dose. The pharmacokinetic profile was characterized by a peak level between 10 

minutes (first sampling time) and 1 hour after administration. After peaking, a gradual decline of

radioactivity was observed leading to an elimination half-life (t1⁄2) of ~18 hours. Cmax was 452 ± 

158 ng eq./mL while AUC0-∞ was 6593 ± 470 hour • ng eq./mL. The average MRT (mean 

residence time extrapolated to infinity) was 25 ± 4 hours. Excretion in the expired air was 

negligible (<0.1%). No residual radioactivity was found in the carcasses 168 hours after 

treatment. The total excretion accounted for 93.4% of the administered dose. Benalaxyl-M was 

extensively metabolized, principally by oxidation of the methyl group on the aniline ring to the 

hydroxymethyl and finally to the carboxylic acid. Minor metabolic pathways were the 

hydroxylation of the phenyl ring and the hydrolysis of the carboxymethyl group.

(MRIDs 49040646 and 49040647). The pharmacokinetics of total radioactivity in blood, 

plasma, tissue, urine, feces, bile and expired air following single oral dose of 10 mg/kg of 14C-

[U-aniline ring]-benalaxyl-M was investigated in male and female Sprague Dawley rats. 

Elimination of absorbed radioactivity was mostly through the feces. Biliary excretion was 

substantial for both sexes (80.5% of dose male; 62% of dose females within 72 hours), indicating

most of the radiolabeled was absorbed and then excreted via the bile into the feces. Female rats 

excreted approximately double the amount of radiolabel into the urine (7.61% of dose males; 

17.23% of dose females). The highest tissue concentrations were found in the liver and GI tract 

of both male and female rats. The amount of radiolabel recovered in the tissues was 

approximately double in males (17.29% of dose) relative to females (7.96% of dose) 1 hour after 

treatment. Thereafter, tissue concentrations were <1% for both male and female rats at 72 and 

168 hours. Total recovered radioactivity was high, 99% in the male and 97% in the female, 

being most of the radioactivity was eliminated within 48 hours post dosing. Negligible levels of 

radioactivity (<0.03%) were recovered in the expired air. After oral administration of 10 mg/kg 

bw 14C-benalaxyl-M to male rats, the blood Cmax was 1.22 ± 0.45 μg eq./g at tmax 1.8 hours postdose. The radioactivity was detectable up to 120-144 hours, resulting in a t1⁄2,z (apparent terminal 

half-life of total radioactivity) of 62.4 hours. The AUC0-∞ (area under the plasma concentration 
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versus time curve up to infinite time) to the total radioactivity was 17.8 ± 1.9 μg eq.∙hour/g. 

After oral administration of 10 mg/kg bw 14C-benalaxyl-M to female rats, Cmax was 1.93 ± 0.49 

μg eq./g at 1.8 hours post-dose. The levels of radioactivity declined with a t1⁄2,z of 80 hours. The 

AUC0-∞ was 34.3 ± 5.8 μg eq.∙hour/g. 

After oral administration of 14C-benalaxyl-M at a dose of 100 mg/kg bw to male rats, Cmax was 

5.44 ± 2.25 μg eq./g at 3.3 hours post-dose. Detectable levels of radioactivity were measured 

until 96-120 hours post-dose, with an average t1⁄2,z of 102 hours. The AUC0-∞ was 155.9 ±26 μg 

eq.∙hour/g. After oral administration of 100 mg/kg bw 14C-benalaxyl-M to female rats, total 

radioactivity in blood reached a Cmax of 8.5 ± 3.21 μg eq./g at 6 hours post-dose. The levels of 

radioactivity declined with a t1⁄2,z of 59 hours. The AUC0-∞was 212.3 ± 71.2 μg eq.∙hour/g. The 

data suggest that absorption and elimination were not saturated as there was not a 10-fold 

increase in Cmax or tmax. 

Parent 14C-benalaxyl-M was excreted exclusively in the feces as a minor component and 

accounted for 1.49% in females and 2.94% in males. No parent compound was found in the 

urine. 14C-benalaxyl-M was extensively metabolized by oxidation of the methyl group on the 

aniline ring to hydroxymethyl and finally to carboxylic acid. Other metabolic pathways were 

hydroxylation and di-hydroxylation (followed by methylation) of the phenyl ring. Conjugation of 

hydroxyl group with glucuronic acid was also found. Total identified radioactivity accounted for

82.11% and 87.69% of the administered dose in the excreta of male and female rats respectively.

The above studies are well conducted and complement each other. 

Together, these metabolism studies are classified Acceptable / Guideline and satisfy the 

guideline requirement for a metabolism study [OCSPP 870.7485, OECD 417] in the rat. 

870.7600 Dermal Absorption - Rat

A dermal absorption study is not required and is not available. 

A.4.9 Immunotoxicity

870.7800 Immunotoxicity

In an immunotoxicity study (MRID 49040648), benalaxyl-M technical product (purity 96.53%, 

Lot No. P/10/106), was administered in the diet for 28 consecutive days at concentrations of 0, 

1000, 3000, or 10,000 ppm (equivalent to 0.0, 80.5, 250.4, and 924.6 mg/kg bw/ day, 

respectively) to groups of 12 male Crl:WI rats/dose. A concurrent cyclophosphamide (CPS) 

positive control group of 12 male Wistar rats received 25 mg CPS in saline/kg bw/day by IP 

injection from days 22 – 27. The rats were inspected for signs of morbidity and mortality twice 

daily. General clinical observations were made at least once a day while detailed clinical observations were made weekly. Body weight and food consumption were determined twice weekly

while water consumption was measured daily. All rats received a single IV injection into the tail 

vein of sheep red blood cells (SRBCs, 2×108cells/rat) on Day 22. The rats were sacrificed 

following an overnight fast and blood collected for an enzyme-linked immunosorbent assay for 
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anti-SRBC IgM antibody and gamma glutamyl transferase (GGT) activity. All rats were 

necropsied and observed macroscopically. Histopathology was not done. 

No treatment-related effects were found on body weight, food consumption, food efficiency, or 

water consumption. No clinical signs of toxicity were observed, and all rats survived until 

scheduled sacrifice. There was a slight increase in GGT activity (6.5±1.7 U/L vs 5.0±0.0) in rats 

receiving 10,000 ppm, however, the significance of this result is obscured by the low sensitivity

of the assay at the low end of the linear range. No treatment-related effects were noted at 

necropsy. The absolute liver weights of mid- and high-dose rats were statistically increased 17% 

and 39%, respectively, while the liver weight relative to body weight of low-, mid-, and highdose rats were statistically increased 10%, 23%, and 48%, respectively. In addition, the relative 

to body kidney weight of high-dose rats was statistically increased 8%. While the increased 

absolute and relative to body liver weights were observed, no histopathology was done. The liver 

effect was a common observation of this chemical and was considered treatment-related.

The systemic LOAEL for male Wistar rats following dietary treatment for 28 days with 

benalaxyl-M was 3000 ppm (250.4 mg/kg bw/day) based on the increases in absolute and 

relative to body liver weights. The NOAEL was 1000 ppm (80.5 mg/kg bw/day). 

Dietary treatment of male rats with 1000, 3000, or 10,000 ppm benalaxyl-M technical product 

for 28 continuous days did not affect spleen, thymus, or mesenteric lymph node weights or 

decrease the anti-SRBC IgM immune response. There were no statistically significant 

differences in anti-SRBC IgM levels in treated groups when compare to the vehicle control 

group. High inter-individual variability was noted in all the treatment groups as well as in the 

control group. Evaluation of the individual animal data of this study did not show any trend or 

distribution that would demonstrate significant suppression of anti-SRBC antibody response. 

Positive control group had statistically significant decrease in the anti SRBC IgM levels. This 

confirmed the ability of the test system to detect immuno-suppressive effects and confirmed the 

validity of the study design.

The Natural Killer (NK) cells activity was not evaluated in this study. The toxicology database 

for benalaxyl-M does not reveal any evidence of immunotoxicity. The overall weight of 

evidence suggests that the chemical does not directly target the immune system. Under HED 

guidance a NK cell activity assay is not required at this time.

The immunotoxicity NOAEL for male Wistar rats treated for 28 consecutive days with 

benalaxyl-M was 10,000 ppm (924.6 mg/kg bw/day). A LOAEL was not established. 

This immunotoxicity study is classified Acceptable / Guideline and satisfies the guideline 

requirement for an immunotoxicity study (OCSPP 870.7800) in the rat. 

A.4.10 Other

Non-guideline Mechanistic

In a 14-day mechanistic study (MRID 49040636), benalaxyl-M (96.53% a.i.; Lot/Batch # 

P/10/106) was administered in feed to groups of 18 male Crl: CD-1(Icr) Br mice at dietary
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concentrations of 0, 200, 1200, or 7000 ppm (0, 30.9, 184.4, and 1094.1 mg/kg bw/day, 

respectively). Another group of 18 male mice were administered phenobarbitone (PB) in the diet 

at 850 ppm (139.6 mg/kg bw/day). Six mice/groups were sacrificed for evaluation of cell 

proliferation after treatment for 3 and 14 days (Allocation A and B animals, respectively). 

Allocation A and B animals were implanted subcutaneously with osmotic mini-pumps 

containing bromodeoxyuridine (BrdU) on days 6 and 8 of acclimatization, respectively. Six 

mice/group were sacrificed after treatment for 14 days for evaluation of liver microsomal 

enzyme activity, mRNA gene expression, and oxidative stress (Allocation C animals). 

No effects were observed on mortality, clinical signs, body weight, or body weight gain, in mice 

administered benalaxyl-M or PB. Absolute liver weight, liver/body weight ratio, and liver/brain

weight ratio were increased 16% to 17% and 15% to 20% in mice treated with benalaxyl-M for 3 

days or 14 days, respectively, and absolute and relative liver weights in mice treated with PB 

were increased by 32% to 36% and 33% to 41% after 3 and 14 days, respectively. No benalaxylM- or PB-related macroscopic lesions were observed in this study. Centrilobular to diffuse 

hepatocellular hypertrophy were observed in 0/6, 1/6, 2/6, and 4/6 mice treated with 0, 200, 

1200, or 7000 ppm of the test substance, respectively, for 3 days and 0/6, 2/6, 4/6, and 3/6 mice, 

respectively, after 14 days. All six mice treated with PB for 3 or 14 days had centrilobular to 

diffuse hypertrophy. There was a dose-related increased severity of the lesions in mice treated 

with benalaxyl-M, and the lesions in PB-treated mice was more severe than in those treated with 

IR6141. 

Analysis of the number of BrdU-positive nuclei in hepatocytes as an indicator of cell 

proliferation showed that cell proliferation was increased at all doses of IR6141 after 14 days; 

the data was equivocal after treatment for 3 days. PB treatment resulted in an increase in cell 

proliferation after 3 and 14 days. The effect of benalaxyl-M and PB treatment was analyzed on 

following microsomal liver enzymes: 7-ethoxyresorufin O-dealkylase (EROD, Cyp1a1

isoenzyme), 7-methoxyresorufin O-dealkylase (MROD, Cyp1a2 isoenzyme), 7-pentoxyresorufin O-dealkylase (PROD, Cyp2b1), testosterone 6β-hydroxylase (6β-TOH, Cyp3a), lauric 

acid 12-hydroxylase (LA12OH, Cyp4a1), and lauric acid 11-hydroxylase (LA11OH, Cyp2e1). 

EROD, MROD, and LA12OH activities were slightly increased at 1200 and 7000 ppm; PROD, 

6β-OH, and LA11OH activities were moderately increased at 7000 ppm; and PROD activity was 

moderately increased at 1200 ppm and 6β-OH and LA11OH activities were slightly increased at 

1200 ppm. Treatment with PB for 14 days caused moderate increases in EROD, MROD, 6-βTOH, and LA11OH activities, a pronounced increased in PROD activity and a slight increase in 

LA12OH activity. The expression of the Cyp1A-1, Cyp1A-2, Cyp2B10, and Cyp3A11 genes 

were increased at 7000 ppm and the expression of Cyp1A-1, CypB10, and Cyp3A11 were 

increased at 1200 ppm. The expression of Cyp2B10 was increased 45.8- and 195.7-fold at 1200 

and 7000 ppm, respectively, and the expression of Cyp3A11 was increased 15.58-fold. 

Treatment with PB for 14 days caused increased expression of all the genes tested. Examination 

of the parameters of oxidative stress, reduced glutathione (GSH) and oxidized glutathione 

(GSSH) in liver, and thiobarbituric acid reactive substance (TBARS) in liver and serum, showed 

no effect of treatment with benalaxyl-M or PB for 14 days.

This 14-day mechanistic study in the mouse is Acceptable/Non-guideline. The study provides 

an assessment of possible liver toxicity for use in the evaluation of the mode of action of 

hepatocellular tumor induction in mice by benalaxyl-M.
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Appendix B. Metabolism

B.1 Metabolism Summary Table

Table B.1. Tabular Summary of Metabolites and Degradates

Chemical Name (other names in 

parenthesis) and Structure

Matrix Percent TRR (PPM)

Matrices - Major 

Residue 

(>10%TRR)

Matrices -

Minor Residue 

(<10%TRR)

Benalaxyl-M Grape - Fruit 26.4 % (0.023) NA

Tomato - Fruit 43.5 % (0.025) NA

Lettuce 41.5 % (0.84) NA

Rat NA 3% feces ♂

2% feces ♀

R5

N-(hydroxyphenylacetyl)-N-(2-

hydroxymethyl-6-methylphenyl)-

alanine

Grapes - Fruit NA NA

Tomato - Fruit NA NA

Lettuce NA NA

Rat 22% feces ♀ 3% feces ♂

R6

methyl-N-(phenylacetyl)-N-(2-carboxy6-hydroxymethylphenyl)-alaninate

Grapes - Fruit NA NA

Tomato - Fruit NA NA

Lettuce NA NA

Rat 11% feces ♂ 4% feces ♀
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Table B.1. Tabular Summary of Metabolites and Degradates

Chemical Name (other names in 

parenthesis) and Structure

Matrix Percent TRR (PPM)

Matrices - Major 

Residue 

(>10%TRR)

Matrices -

Minor Residue 

(<10%TRR)

R7

(methyl-N-(hydroxyphenylacetyl)-N-

(2-carboxy-6-methylphenyl)-alaninate

Grapes - Fruit NA NA

Tomato - Fruit NA NA

Lettuce NA NA

Rat 24% feces ♀

8% urine ♀

38% feces ♂

1% urine ♂

G10, T4, L4 Grapes - Fruit 11% (0.0093) NA

Tomato - Fruit NA 3.2% (0.0018)

Lettuce 11% (0.23) NA

Rat NA NA
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Table B.1. Tabular Summary of Metabolites and Degradates

Chemical Name (other names in 

parenthesis) and Structure

Matrix Percent TRR (PPM)

Matrices - Major 

Residue 

(>10%TRR)

Matrices -

Minor Residue 

(<10%TRR)

G13, [T9, T11], [L3, L5-L9, L11] Grapes - Fruit 14% (0.012) NA

Tomato - Fruit NA 5.3% (0.0030)

Lettuce 10% (0.21) NA

Rat NA NA

G15, T10, L10 Grapes - Fruit 11% (0.0097) NA

Tomato - Fruit 32% (0.018) NA

Lettuce 30% (0.61) NA

Rat NA NA
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B.2 Metabolic Pathways

Proposed Metabolic Pathway of Benalaxyl-M for Grapes

The figure below was copied without alteration from MRID 49040680.
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Proposed Metabolic Pathway for the Rat.

The figure below was copied without alteration from MRID 49040647.
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Appendix C. Physical/Chemical Properties

TABLE C.1. Physicochemical Properties of the Technical Grade Test Compound: Benalaxyl-M. 

Parameter Value Reference

Melting point/range 74.86 °C

D421271, S. Mathur, 

22-JUL-2014

Boiling point 330 °C

pH 7.1

Density 1.19 ± 0.022 g/ml (19.5 °C)

Water solubility (at 25°C) 37.0 mg/L

Solvent solubility (g/L)

n-heptane - 17074 mg/L

xylene - >39 (%w/w)

acetone - >49 (%w/w)

ethyl acetate - >49 (%w/w)

1,2-dichloroethane - >50 (%w/w)

methanol - >50 (%w/w)

Vapor pressure at 25°C 6.7 x 10-4

Pa

Dissociation constant (pKa) Not applicable

Octanol/water partition coefficient Log(KOW) 3.54 at 20 °C

UV/visible absorption spectrum

202 nm - 3.4 x 104ξ (dm3/(mol x cm))

254 nm - 4.6 x 102 ”

259 nm - 4.6 x 102 ”

265 nm - 6.1 x 102 ”

275 nm - 4.4 x 102 ”

Appendix D. Review of Human Research

This risk assessment does not rely on data from studies in which human subjects were 

intentionally exposed to a pesticide or other chemicals.