Amitraz Human Health Risk Assessment DP No.D407721 

t lNITED STATES ENVIRONMRNTAL PROTECTION AGENCY 

WASHINGTON, I>.C. 204()(} 

MEMORANDUM 

Date: January 8, 2013 

OFFICE OF 

CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

SUBJECT: Amitraz. Aggregate Human Health Risk Assessment for Section 3 New Use in 

Beehives. 

PC Code: I 0620 I 

Decision No.: NA 

Petition No.: 9F7673 

DP Barcode: 0407721 

Registration No.: 87243- R 

Regulatory Action: Section 3 

Risk Assessment Type: Single Chemical Aggregate Case No.: NA 

TXRNo.: NA CAS No.: 33089-61-1 

MRIDNo.: NA 40 CFR: 180.287 

FROM: 

THROUGH: 

TO: 

and 

Danett

Wade Bri

e Drew

tton, 

, 

MPH, 

Chemist/Ri

Industria

sk Assessot_ 

l Hygienist 

'u4_ ~ 

/P' (l'fJ ___ -

Risk 

William 

Assessme

Irwin, 

n

PhD, 

t Branch 

DABT, 

V 

Toxicologist 

W~-7 

/, r ~ OrJ: _ Q... 

/ 

~ 

1 J/ r>-. 

Health Effects Division (7509P) ·' ~ v 

1 L 

Office of Pesticide Programs 

Michael Metzger, Branch Chief ~~~~ () k[ - Risk Assessment Branch V /VII / · 

Health Effects Division (7509P) 

Office of Pesticide Programs 

Autumn Metzger, CRM 

Insecticide Rodenticide Branch 

Stacey Groce, CRM 

Risk Integration, Minor Use & Emergency Response Branch 

Registration Division (7505P) 

Office of Pesticide Programs 

Attached please find the l-IED Human Health Risk Assessment for amitraz to support new use in 

beehives. 

Page 1 of 48 
Amitraz Human Health Risk Assessment DP No.D407721

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Table of Contents

1.0 Executive Summary ........................................................................................................... 4

2.0 HED Recommendations .................................................................................................... 5

2.1 Data Deficiencies ............................................................................................................ 5

2.2 Tolerance Considerations.............................................................................................. 6

2.2.1 Enforcement Analytical Method ........................................................................... 6

2.2.2 Recommended Tolerances...................................................................................... 6

2.2.3 Revisions to Petitioned-For Tolerances ................................................................ 7

2.2.4 International Harmonization................................................................................. 7

3.0 Introduction........................................................................................................................ 7

3.1 Chemical Identity ........................................................................................................... 7

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

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

3.4 Anticipated Exposure Pathways................................................................................... 9

3.5 Consideration of Environmental Justice...................................................................... 9

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

4.1 Summary of Toxicological Effects.............................................................................. 10

4.2 Safety Factor for Infants and Children (FQPA Safety Factor) ............................... 12

4.2.1 Completeness of the Toxicology Database.......................................................... 12

4.2.2 Evidence of Neurotoxicity .................................................................................... 13

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

4.2.4 Residual Uncertainty in the Exposure Database................................................ 14

4.3 Toxicity Endpoint and Point of Departure Selections .............................................. 14

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

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

5.2 Food Residue Profile .................................................................................................... 16

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

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

6.1 Residential Handler Exposure- Pet Collar................................................................. 21

6.2 Post-Application Exposure- Pet Collar ...................................................................... 22

6.3 Risk Characterization of Residential Assessment – Pet Collar................................ 25

6.4 Residential Bystander Post-application Inhalation Exposure ................................. 27

6.5 Spray Drift.................................................................................................................... 27

7.0 Aggregate Exposure/Risk Characterization.................................................................. 27

7.1 Aggregate Risk (All Durations)................................................................................... 28

8.0 Cumulative Exposure/Risk Characterization ............................................................... 28

9.0 Occupational Exposure/Risk Characterization ............................................................ 28

10.0 References......................................................................................................................... 29
Amitraz Human Health Risk Assessment DP No.D407721

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Appendix A. Toxicology Profile and Executive Summaries .................................................. 30

Appendix B. Physical/Chemical Properties............................................................................. 46

Appendix C. Review of Human Research................................................................................ 47

Appendix D. Summary of Amitraz US and International Tolerances and Maximum 

Residue Limits (MRLs) .............................................................................................................. 48
Amitraz Human Health Risk Assessment DP No.D407721

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1.0 Executive Summary

The Registration Division (RD) of the Office of Pesticide Programs (OPP) has requested that the 

Health Effects Division (HED) perform a human health risk assessment to evaluate the Section 3 

petition to register the insecticide amitraz (as an impregnated strip) to control varroa mites in 

beehives and to establish tolerances for residues of amitraz in honey. This assessment takes into 

account previously registered uses, including livestock dermal treatments and residential use on 

pets. The most recent human health risk assessment for amitraz was performed 10/18/12 in 

conjunction with a Section 18 emergency exemption request for the use of amitraz in beehives in 

South Dakota (D406097). The exposure assumptions and risk estimate conclusions have not 

changed since the 10/18/12 amitraz assessment.

The exposure database is complete for amitraz. The toxicology data base of amitraz, while 

incomplete, is adequate for purposes of risk assessment. An Extended One-Generation 

Reproductive Toxicity (EOGRT) study is being required for amitraz; the cohorts in this study 

will evaluate the reproductive, neuro- and immunotoxicity potential of amitraz. The 10X FQPA 

Safety Factor is retained in the form of a data base Uncertainty Factor (UFDB) to account for the 

lack of the EOGRT study.

Based on the studies available in human subjects and laboratory animals, human subjects appear 

to be the most sensitive species for amitraz-induced toxicity. In various species of laboratory 

animals, clinical signs of toxicity include central nervous system (CNS) depression, ataxia, 

ptosis, emesis, labored respiration, muscular weakness, tremors, hypothermia, dry mouth, 

hypertension and bradycardia. The current toxicity database for amitraz suggests that the CNS 

effects of amitraz are not cumulative (i.e., do not accumulate with increased duration, but are a 

response to each daily dose). Because of the reversibility of the CNS effects, exposures of all 

durations can be regarded as a series of repeating one-day (acute) exposures. Therefore, effects 

seen after a single exposure are appropriate for assessing exposures to amitraz regardless of 

exposure duration.

In the current risk assessment, the oral study in humans is used for establishing the point of 

departure (POD) for assessing risks following oral, dermal and inhalation exposures of all 

durations. An acute population adjusted dose (PAD) of 0.00125 mg/kg is derived from a 

NOAEL of 0.125 mg/kg and the application of a 100X uncertainty factor (UF) which includes 

10X for intra-species variation, 10X FQPA factor for the data gap (EOGRT study) and 1X for 

the inter-species extrapolation. Since a human study was selected, the inter-species factor is 

reduced to 1X. A margin of exposure (MOE) of 100X is adequate for oral, dermal and 

inhalation scenarios.

Amitraz is classified as showing “Suggestive Evidence of Carcinogenicity” based on the 

presence of commonly observed tumors (liver and lung) in a single species (mouse) and the lack 

of concern for mutagenicity. Quantification of risk using a non-linear approach (i.e., RfD/PAD) 

is adequate to account for all chronic toxicity, including carcinogenicity, which could result from 

exposure to amitraz. While the Agency would typically use a chronic population-adjusted dose 
Amitraz Human Health Risk Assessment DP No.D407721

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(cPAD) to protect for cancer concerns when using a non-linear approach, use of the acute 

population-adjusted dose (aPAD) is protective for amitraz because increasing toxicity with 

increasing duration of exposure is not seen for amitraz.

A partially refined acute probabilistic dietary exposure analysis for amitraz was conducted which 

included honey along with currently registered livestock uses. Drinking water was not included 

in the dietary assessment as it was determined that amitraz is not expected to enter water-bodies 

or drinking water through the current and proposed uses. The acute dietary (food only) 

exposures estimates are below HED’s level of concern. 

There are no residential (non-occupational) exposures expected from the beehive use so a nonoccupational risk assessment is not required for the proposed use. However, there is a potential 

for residential exposure to amitraz from existing pet uses (dog collars and spot-on treatments), 

either from applying (handling) the products or from post-application contact with the treated 

dog. An updated assessment for exposures to amitraz from dog collar uses was performed in the 

10/18/12 human health risk assessment for a requested Section 18 emergency exemption. The 

assumptions and conclusions of that assessment are presented in this document as well. The 

worst case residential handler and post-application exposure estimates (dog collar use) do not 

exceed the level of concern for adults and children.

Aggregate risk estimates combine exposures from dietary and residential (non-occupational) 

sources. The aggregate risk assessment for amitraz combines the dietary (food only; there are no 

drinking water exposures anticipated for amitraz uses) and residential exposures from amitraz pet 

collar uses. The aggregate assessment for amitraz combines the oral dietary and dermal 

exposures for adults and combines oral dietary, oral hand to mouth, and dermal exposures for 

children. The aggregate risk estimates (residential plus dietary) do not exceed the level of 

concern for adults and children. 

Occupational (dermal and inhalation) exposures are not anticipated for the proposed use of 

amitraz plastic strips in beehives; therefore, a quantitative occupational risk assessment is not 

performed.

This risk assessment relies in part on data from studies in which adult human subjects were 

intentionally exposed to a pesticide or other chemical. See Appendix C for more information.

2.0 HED Recommendations

HED recommends tolerances for total amitraz residues in honey at 0.2 ppm and in honeycomb at 

9 ppm for the proposed use of amitraz in beehives. The specific tolerance recommendations are 

discussed in section 2.2.

2.1 Data Deficiencies

The following data requirements were previously identified in D406097, 10/18/12 (ID#:10SD04 

Updated Section 18 Emergency Exemption for the Use of Amitraz in Beehives in South Dakota). 

The Agency has determined that an Extended One-Generation Reproduction Toxicity (EOGRT) 
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study is required to evaluate the reproductive, neuro- and immuno-toxic potential of amitraz.

The EOGRT study will provide comprehensive evaluation of three cohorts of F1 animals: 

Cohort 1: assesses reproductive/developmental endpoints; this cohort may be extended to include an F2 

generation. 

Cohort 2: assesses the potential impact of chemical exposure on the developing nervous system.

Cohort 3: assesses the potential impact of chemical exposure on the developing immune system. 

The 10X FQPA Safety Factor is retained in this assessment in the form of a data base 

Uncertainty Factor (UFDB) to account for the lack of the EOGRT study. 

The Agency has also determined that a “Triple pack” dermal absorption study is required for 

refinement of the dermal absorption factor. The Triple pack approach uses three types of dermal 

absorption data from: 1) in vivo animal; 2) in vitro animal; 3) and in vitro human skin. The 

Triple pack allows moderation of in vitro results using rat skin by correcting for any differences 

between in vitro and in vivo rates in that species as well as for species differences, which is 

important since absorption by human skin is usually lower than that by rat skin. Therefore, the 

combined use of data offers greater precision in estimating dermal absorption. The Agency 

recommends submission of a protocol for review prior to initiation of the studies.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

There are two adequate methods listed in FDA's Pesticide Analytical Manual (PAM Vol. II) for 

purposes of data collection and enforcement of tolerances for residues of amitraz and its 

metabolites containing the 2,4-dimethylaniline (2,4-DMA) moiety. Methods I (designed for 

animal tissues and milk) and II (designed for plant commodities) are both GLC methods with 

electron capture detection (ECD), and involve conversion of residues of amitraz and its 

metabolites containing the 2,4-dimethylaniline moiety to 2,4-DMA using acid and base 

hydrolysis, respectively. The data collection method for honey and honeycomb is similar to the 

PAM methods.

The FDA PESTDATA database dated 11/01 (PAM Volume I, Appendix I) indicates that amitraz 

has low recovery (<50%) using Multi-residue Method Section 302 (Luke Method; Protocol D) 

and that metabolite BTS-27919 is completely recovered using Section 302. The detection limits 

of the methods are 0.01 ppm for milk and 0.05 ppm for plant commodities and animal tissues. 

2.2.2 Recommended Tolerances

HED recommends tolerances for residues of total amitraz of 0.2 ppm in honey and 9 ppm in 

honeycomb for the proposed Section 3 use of amitraz in beehives. The tolerance definition in 40 

CFR 180.287 should read as:

“Tolerances are established for residues of the insecticide amitraz (N'-[2,4-dimethylphenyl]-N-

[[(2,4-dimethylphenyl)imino]methyl]]-N-methylmethanimidamide), 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 those amitraz residues convertible 
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to 2,4-dimethylaniline, expressed as the stoichiometric equivalent of amitraz, in or on the 

following raw agricultural commodities:” 

Table 2.2.3 Tolerance Summary for Amitraz

Commodity Proposed Tolerance 

(ppm)

Recommended 

Tolerance (ppm)

Comments

Honey 1.0 0.2

Honeycomb None 9

2.2.3 Revisions to Petitioned-For Tolerances

A tolerance of 1.0 ppm for amitraz in honey is proposed. The requested tolerance of 1.0 ppm 

was based upon a now-revoked tolerance for a different amitraz embedded strip (Miticur) 

designed for beehives for which uses have been cancelled. Subsequent to the Section 3 request, 

new field trial data (for honey and honeycomb) using the Apivar® strips in beehives were 

submitted (MRID 48341001). A screen of the Organization for the Economical Cooperation and 

Development (OECD) calculation procedure using the field trial data points from the 15 day preharvest interval (PHI) field trial honey samples resulted in a value of 0.15 ppm. A tolerance of 

0.2 ppm for amitraz in honey would be adequate to cover the proposed Section 3 use of Apivar 

strips in beehives and would harmonize with the European Union (EU) maximum residue level 

(MRL) for total amitraz in honey.

A tolerance for honeycomb was not proposed. The OECD calculation procedure of the 15 day 

PHI honeycomb field trial samples resulted in a value of 9 ppm. HED recommends a tolerance 

for amitraz in honeycomb be established based on the potential for residues in honeycomb as 

observed in the field trials.

2.2.4 International Harmonization

Canada, Codex, and the U.S. have essentially the same amitraz residue definition; metabolites 

containing the dimethylaniline moiety are regulated. There are currently no established or 

proposed Canadian, Mexican, or Codex maximum residue levels (MRLs) for amitraz in honey or 

honeycomb (see Appendix D). Presently, the European Union (EU) is the only authority that has 

established an MRL for amitraz residues in honey (at 0.2 mg/kg). 

3.0 Introduction

3.1 Chemical Identity

Table 3.1 Amitraz Nomenclature

Chemical structure

Common name Amitraz

N N N

H C3 CH3 H C3 CH3

CH3
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Table 3.1 Amitraz Nomenclature

Molecular Formula C19H23N3

Molecular Weight 293.42

IUPAC name N-methylbis(2,4-xylyliminomethyl)amine

CAS name N'-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]methyl]-Nmethylmethanimidamide

CAS # 33089-61-1

PC Code 106201

Current Food/Feed Site Registration [livestock dermal treatment for cattle and swine]

3.2 Physical/Chemical Characteristics

Amitraz is soluble in xylene, acetone, and methanol and insoluble in water. It has a low to 

moderate vapor pressure and exposure to the gaseous state should be negligible. Amitraz is 

highly lipophilic/hydrophobic. See Appendix B.

3.3 Pesticide Use Pattern

The petitioner is proposing the use of Apivar® strips (containing 3.33% amitraz; 0.5 g amitraz 

per strip) in beehives for the control of varroa mites. 

Directions for proposed beehive use: Apply 2 Apivar® strips per brood chamber. Separate the 

double strip and hang each strip between two comb frames inside the brood area or the bee 

cluster, with a minimum distance of 2 frames between strips. Suspend Apivar® strips in the 

brood chamber in such a way that the bees can walk on both sides of the strips. Leave strips 

inside the hive for 42 days, and then remove. In case of bee movement inside the beehive far 

from the strips, a repositioning of the strips should be done into the bee cluster, and the strips left 

in place for 14 more days before removal. Strips must be removed after a maximum of 56 days. 

All Apivar® strips should be removed 2 weeks before the honey flow starts. DO NOT USE 

APIVAR® STRIPS WHEN HONEY SUPERS ARE PRESENT. Wait 14 days after removing 

strips before placing honey supers on hives.

Table 3.3. Summary of Directions for Use of Amitraz

Applic. 

Timing, Type, 

and Equip.

Formulation 

[EPA Reg. 

No.]

Applic. 

Rate

Max. No. 

Applic. 

per Season

Max. 

Seasonal 

Applic. 

Rate 

PHI 

(days)

Use Directions and 

Limitations

Beehives

Two Strips (0.5 

g Amitraz each)

3.33% 

amitraz strip

[87243- R]

1.0 g 

a.i./hive 2

2.0 g 

a.i./hive 14

Do not use strips when 

honey supers are present. 

Wait 14 days after 

removing strips before 

replacing supers.
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3.4 Anticipated Exposure Pathways

Amitraz is currently registered for direct application on cattle and swine. Humans may 

potentially be exposed to amitraz in the diet from secondary residues in meat or in milk, or from 

consuming honey or honeycomb from treated beehives. Drinking water is not considered a 

source of dietary exposure as amitraz is not expected to enter water-bodies or drinking water 

through the current and proposed uses. The amitraz dietary risk assessment includes food only 

and considers existing and proposed uses. 

The proposed new use pattern does not indicate a significant potential for applicator or postapplication dermal or inhalation exposure. Dermal exposure resulting from use of these 

prepackaged impregnated strips is not likely to occur since there is no mixing or loading and 

latex gloves are prescribed to be worn when applying the strips. Since beehives are located 

outdoors, and amitraz has low volatility, there is negligible potential for inhalation exposure, at 

any time, to the applicator or during post-application activities. Because of the extremely low 

potential for dermal or inhalation exposure, quantitative occupational risk assessments are not 

performed for this proposed new use.

There are no residential (non-occupational) exposures expected from the proposed beehive use 

so a non-occupational risk assessment is not required for the proposed use. However, there is a 

potential for residential exposure to amitraz from existing pet uses, either from applying 

(handling) the products or from post-application contact with the treated dog. A dermal 

exposure assessment was performed for adults applying the amitraz pet products. For postapplication exposure to treated dogs, a dermal assessment was performed for adults and a dermal 

and oral (hand to mouth) assessment was performed for children 1-2 years of age. Handler and 

post-application inhalation exposure is expected to be negligible and was not quantitatively 

assessed. 

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://www.eh.doe.gov/oepa/guidance/justice/eo12898.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 Continuing 

Survey of Food Intake by Individuals (CSFII) 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, season of the year, ethnic group, and region of the country. 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 
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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

No new toxicity or metabolism data have been received since the last risk assessment on October 18, 

2012. This assessment includes summaries of prior assessments (D. Drew, Amitraz. ID#:10SD04 

Updated Section 18 Emergency Exemption for the Use of Amitraz in Beehives in South Dakota, 

10/18/2012, DP406097).

4.1 Summary of Toxicological Effects

Multiple studies in various mammalian species support that the common effects of amitraz are 

those of neurotoxicity, with signs such as sedation, hypothermia, drowsiness, etc. In insects, the 

mechanism of action is also neurotoxicity via interaction with the octopamine receptor, causing 

paralysis and death. Acute toxicity studies in various laboratory animals indicate that amitraz is 

moderately toxic (Toxicity Category II) via the dermal route, and it is slightly toxic (Category 

III) via the oral and not acutely toxic (Category IV) via inhalation routes of exposure. Further, it 

is not a skin or eye irritant, nor is it a skin sensitizer. 

Based on available human and animal studies, human subjects were shown to be more sensitive 

than any other species tested, followed by the dog. Multiple species display evidence of 

neurotoxicity following exposure to amitraz. Clinical signs of neurotoxicity were seen across 

species, sexes, and routes of administration. In both the oral subchronic and chronic studies in 

dogs, signs of central nervous system (CNS) depression were observed along with a decrease in 

pulse rate and hypothermia noted in the subchronic study. In both the oral subchronic and 

chronic studies and in the 21-day inhalation study in the rat, irritability, nervousness and/or 

excitability were observed. In the rabbit developmental toxicity study, clinical signs that were 

considered to be related to treatment included languor and polypnea. Sedation was also observed 

in rabbits in the repeated dose dermal study. In the single dose human metabolism study, 

neurotoxic effects such as dry mouth, drowsiness, decreased temperature, and bradycardia were 

seen within 90 to 160 minutes after ingestion and persisted for up to 12 hours at the lowest dose 

tested (0.25 mg/kg/day). 

No developmental toxicity was seen at the highest dose tested in two pre-natal developmental 

toxicity studies in rats. Two independent developmental toxicity studies were available in 

rabbits. Although technical deficiencies were encountered in the conduct of these studies, no 

developmental effects were seen either at the highest dose tested (in one study) or in the presence 

of maternal toxicity (second study). When taken together, these studies do provide data that 

shows 1) amitraz does not cause developmental toxicity in this species and 2) rabbits are not 

more sensitive than rats since the doses tested in the rabbits were higher than the doses tested in

the rat developmental study where no developmental toxicity was seen at any dose level. The 

database contains 1-generation and 3-generation reproduction studies in rats. In the 1-generation 

study, no reproductive toxicity was seen at the highest dose tested and offspring toxicity was 
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seen in the presence of parental/systemic toxicity. In the 3-generation reproduction study, no 

reproductive toxicity was seen at the highest dose tested, however, offspring toxicity was seen at 

a lower dose than the dose that caused parental/systemic toxicity. 

Amitraz is classified as showing “Suggestive Evidence of Carcinogenic Potential” based on the 

occurrence of common tumors (liver and lung) in a single species (mouse). Amitraz is not 

mutagenic, however, it forms a metabolite, 2,4-dimethylaniline, which has shown evidence of 

mutagenicity (in vitro only) and an equivocal carcinogenic response at high dose levels. EPA 

has determined that quantification of risk using a non-linear approach (i.e., RfD/PAD) for 

amitraz will adequately account for all chronic toxicity, including carcinogenicity, which could 

result from exposure to amitraz and its metabolites (TXR No. 0054272). That conclusion is 

based on the following considerations: (1) no carcinogenic response was seen in an acceptable 

rat cancer study; (2) the tumors found in the mouse are commonly seen in the mouse and were 

only found at a dose that appears to have been excessive given the other adverse effects seen in 

the animals; (3) amitraz is not mutagenic; and (4) although there is limited positive mutagenicity 

data and equivocal evidence of cancer for a minor amitraz metabolite, that equivocal cancer 

evidence was present only at high doses and was not consistent with the tumors seen in the 

amitraz study. 

While the Agency would typically use a chronic population-adjusted dose (cPAD) to protect for 

cancer concerns when using a non-linear approach, use of the acute population-adjusted dose 

(aPAD) is protective for amitraz because increasing toxicity with increasing duration of exposure 

is not seen for amitraz. The dog was the second most sensitive species after humans and the 90-

day dog study had identical NOAELs and LOAELs to the chronic dog study (i.e. 0.25 mg/kg/day 

and 1.0 mg/kg/day, respectively). In fact, the chronic data suggested that the dog was less 

susceptible to amitraz with increasing time, since the lowest POD in that study was based on 

toxicity observed during the first 2 days of exposure in the 2-year study. The NOAEL observed 

in the chronic rat study is 0.97 mg/kg/bw/day, 7-fold higher than the POD used for acute risk 

assessment and tumors were not observed at doses up to 12.59 mg/kg/day during a duration of 2 

years. Thus, the acute assessment is protective for exposure of longer durations for amitraz.

The amitraz data base contained studies conducted in human subjects following oral and dermal 

applications which were reviewed by the Human Studies Review Board (HSRB). The HSRB 

found these studies to be ethically sound, and concluded that “the results from the single oral 

dose study are informed by the human metabolism study, such that the single oral dose study is 

appropriate for developing a point of departure for acute dietary risk [...]”. The majority of the 

Board concluded that the human dermal study was not appropriate for developing a point of 

departure for dermal exposure of various durations since no effects were seen at the highest dose 

tested and the dermal loadings may have been too high.

In a human double-blind randomized crossover study (MRID 43283101), 6 male volunteers were 

given sequential oral doses of amitraz by capsule, at 0.0625 or 0.125 mg/kg with a placebo 

control. There were at least 14 days between treatments. Vital signs (pulse, respiration rate, 

blood pressure, and body temperature) and ECGs were taken. Pupil responsiveness and 

psychomotor performance were evaluated. Urine was collected for testing. Minimal and 

transient changes in blood pressure, temperature, ECG rate, and psychomotor performance were 
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observed at 0.125 mg/kg. In a human metabolism study, 2 male volunteers given 0.250 mg/kg 

by oral route experienced sedation, disorientation, and hypothermia. Based on the combined 

results of the human oral toxicity and metabolism studies, the acute oral NOAEL and LOAEL 

for humans are 0.125 mg/kg and 0.25 mg/kg, respectively, based on clinical signs in two human 

subjects in the metabolism study.

No gender differences were observed in the proportion of various metabolites recovered in 24-

hour urine samples. Metabolic process saturated at the 100 mg/kg level. No unchanged parent 

material was found in the urine. Major metabolites were N-(2,4-dimethylphenyl)-N-methyl 

formamidine, 4-formamido-3-methyl benzoic acid, 4-acetamido-3-methyl benzoic acid and a 

polar fraction. The polar fraction was labile to acid hydrolysis, yielding conjugates of 4-amino3-methylbenzoic acid, N-(2,4-dimethylphenyl)-N-methyl formamidine, 4-formamido-3-methyl 

benzoic acid and 4-acetamido-3-methyl benzoic acid. 

A dermal absorption study in rats resulted in a value of approximately 4%. A human dermal 

absorption value for amitraz can be extrapolated to 1.6% (4% DAF ÷ 2.5 permeability 

differences between rat and human skin). The full discussion of the dermal absorption studies is 

listed in Section 4.3.

A summary of existing toxicity studies can be found in Appendix A2 of this risk assessment 

document.

4.2 Safety Factor for Infants and Children (FQPA Safety Factor)

The proposed and existing use patterns for amitraz may result in dietary and residential exposure 

to infants and children. Thus, FQPA hazard and exposure considerations were addressed in 

HED’s evaluation of the database. HED recommends that the 10x FQPA Safety Factor (for the 

protection of infants and children) be retained in the form of a data base uncertainty factor 

(UFDB), due to multiple toxicology data deficiencies for amitraz (i.e. reproduction, 

immunotoxicity, and DNT studies). An Extended One-Generation Reproduction Toxicity 

(EOGRT) study is being required to evaluate the reproductive, neuro- and immuno-toxic 

potential of amitraz. A 10X FQPA safety factor is considered protective for the following 

reasons: (1) a clear NOAEL was used as the point of departure for risk assessment; (2) the 

NOAEL was from the most sensitive species; (3) the NOAEL is from an adequate study in 

humans that examined the most sensitive endpoint (neurotoxicity) seen in the animal data; (4) 

given the existing animal data, EPA expects that the most sensitive effect found in the EOGRT 

study will be a neurotoxic one; (5) EPA is applying a 10X intra-species safety factor to account 

for potential variability in the sensitivity in humans (including potentially greater sensitivity in 

infants and children than in the adults tested in the human study); and (6) in the 3-generation 

reproduction study, the only study showing the potential for increased susceptibility in offspring, 

offspring were <4X more sensitive than adult animals; retention of the additional default 10X 

safety factor for the protection of infants and children means that there will be a 100X factor to 

account primarily for potential sensitivity in the young even though the available (though 

incomplete) data show sensitivity in the young of no greater than 4X. 

4.2.1 Completeness of the Toxicology Database
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The toxicology data base is sufficient for endpoint selection, but not complete to assess 

susceptibility following pre-and/or post natal exposure to amitraz. The Agency has determined 

that an Extended One-Generation Reproduction Toxicity (EOGRT) study is required to evaluate 

the reproductive, neuro- and immuno-toxic potential of amitraz. Since the EOGRTS will contain 

a DNT component and other studies have demonstrated neurotoxicity, the ACN and SCN studies 

are not being requested. The Agency is requiring a “Triple pack” dermal absorption study to 

refine the dermal absorption factor. Therefore, the Agency has retained the 10X FQPA Safety 

Factor in the form a data base uncertainty factor (UFDB) for the lack of the EOGRT study. 

4.2.2 Evidence of Neurotoxicity

Various mammalian species in multiple studies have demonstrated the signs of neurotoxicity for 

amitraz (i.e. sedation, hypothermia, drowsiness, etc). The DNT study will be a component of the 

EOGRT study, thus will specifically monitor the potential neurotoxicity of amitraz in targeted 

testing. 

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

The toxicology data base is not complete to assess susceptibility following pre-and/or post natal 

exposure to amitraz. There was no evidence of increased susceptibility in rats since no 

developmental toxicity was seen at the highest dose tested in two independent pre-natal 

developmental toxicity studies in rats. Evidence for susceptibility in rabbits could not be 

ascertained due to technical deficiencies in the conduct of two independent developmental 

studies. However, the concern for the lack of susceptibility assessment is lessened because 1) in 

both studies, developmental effects occurred in the presence of maternal toxicity (one study) or 

at a dose higher than the dose that caused maternal toxicity (second study); 2) the doses tested in 

the rabbit studies were higher than the doses tested in developmental study in rats showing that 

rabbits are not more sensitive than rats. Two reproductive toxicity studies (1-generation and a 3-

generation) are available; in the 1-generation study, no reproductive toxicity was seen at the 

highest dose tested and offspring toxicity was seen in the presence of parental/systemic toxicity. 

In the 3-generation reproduction study, no reproductive toxicity was seen at the highest dose 

tested, however, offspring toxicity was seen at a lower dose than the dose that caused 

parental/systemic toxicity. Both studies were deemed to be unacceptable due to technical 

deficiencies in the conduct of these studies. Neurotoxicity was seen in a variety of animal 

studies and in human subjects and the data base does not contain specific neurotoxicity studies. 

The dietary and residential assessments are based on reliable exposure data and will not underestimate exposure.

The Agency has determined that an Extended One-Generation Reproduction Toxicity (EOGRT) 

study is required to evaluate the reproductive, neuro- and immuno-toxic potential of amitraz.

The EOGRT study will provide comprehensive evaluation of three cohorts of F1 animals: 

Cohort 1: assesses reproductive/developmental endpoints; this cohort may be extended to include 

an F2 generation. 

Cohort 2: assesses the potential impact of chemical exposure on the developing nervous system. 

Cohort 3: assesses the potential impact of chemical exposure on the developing immune system. 
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The Agency has retained the 10X FQPA Safety Factor in the form a data base uncertainty factor 

(UFDB) for the lack of the EOGRT study. 

4.2.4 Residual Uncertainty in the Exposure Database

There are no residual uncertainties with regard to dietary or residential exposure and no 

outstanding exposure data gaps. The dietary assessments are based on conservative, health 

protective assumptions regarding exposure from food and are designed not to underestimate 

exposures. Residential exposures resulting from contact with dogs wearing amitraz pet collars is 

conservative. The residential risk estimates are based upon protective assumptions of application 

rate, duration of exposure, and contact with the treated animal. The fraction of application rate 

transferred, while non-chemical specific, represents the best data available to assess risk from 

exposures to the amitraz collar and will not underestimate risk.

4.3 Toxicity Endpoint and Point of Departure Selections

There have been no changes in the endpoint selections since the prior assessment nor have there 

been changes to the prior recommendations for combining routes of exposure and/or cancer 

classification. The points of departure and endpoint selections are listed below.

Oral Exposure: The POD and the endpoint of concern are selected from the combined result of 

two studies conducted in human subjects; an oral toxicity and a metabolism study. Following a 

single oral dose, a NOAEL of 0.125 mg/kg was established based on dry mouth, drowsiness, 

decreased temperature, and decreased blood pressure and heart rate at 0.25 mg/kg (LOAEL). 

For acute dietary risk assessment, an acute PAD of 0.00125 mg/kg/day was derived from the 

NOAEL of 0.125 mg/kg and the application of 100X Uncertainty Factor which includes 1X for 

inter-species extrapolation, 10X for intra-species variation and 10X for the data gap for the 

EOGRT study. This POD was also selected for assessing risk via incidental oral exposure for 

infants and children. An MOE of 100 is adequate for this exposure scenario.

Dermal Exposure: The oral POD of 0.125 mg/kg established in the humans study (LOAEL = 

0.25 mg/kg) was also selected for dermal risk assessment. Since an oral dose was selected a 

dermal absorption factor (DAF) of 1.6% is used for route-to-route extrapolation. An MOE of 

100X is adequate for this exposure scenario.

The 1.6% human dermal absorption factor is derived from data obtained in a dermal absorption 

study in rats with amitraz as well as the comparative dermal absorption data available in rats and 

humans with other compounds which provide data to show that absorption by human skin is 

usually lower than that by rat skin. In the in vivo study (MRID No.42133501) the dermal 

absorption value was 3.69% at 24 hours following washing at of the skin application site at 10 

hours post dermal application. Another group of rats were continued for 120 hours following 

skin washing at 10 hours. The dermal absorption was estimated to be 6.56% at 120 hours. In this 

risk assessment the dermal absorption factor of 4% (rounded from 3.69% at 24 hours) was 

considered appropriate considering the short half-life of amitraz based on the rat metabolism 

study. A number of comparative studies available in open literature suggest that the rat skin is 

approximately 3 to 10 fold more permeable than the human skin (Bartek et al., 1972). OPP has 

reviewed a number of studies comparing dermal absorption in rats (in vivo and in vitro methods) 
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and in vitro studies with human skin. These studies suggest that rat skin is typically at least 2.5 

times more permeable than the human skin (2.3 to 9.2). Based on this information, a human 

dermal absorption value for amitraz can be extrapolated to 1.6% (4% DAF ÷ 2.5 permeability 

differences between rat and human skin). The 1.6% value used in this risk assessment differs 

from the 8% value used in the previous risk assessment because the 8% value was obtained from 

total absorption plus remaining chemical in skin seen at 120 hours compared to the 4% absorbed 

residue seen at 24 hours which is the appropriate time period based on the half-life of amitraz 

and toxicity following exposure to amitraz appearing early after dosing and reversing rapidly; in 

humans the effects were reversed within 12 hours (acute bolus dose). The Agency is requiring a 

“Triple pack” dermal absorption study to refine the dermal absorption factor.

Inhalation Exposure: A 21-day inhalation toxicity study in rats is available with a NOAEL of 1.8 

mg/kg/day and a LOAEL of 18 mg/kg/day; however, these values are based on the conversion of 

nominal values, since analytical concentrations were not measured. The lack of analytical data 

reduced the confidence in this study. Therefore, the oral NOAEL of 0.125 mg/kg established in 

human subjects was selected for assessing inhalation risk. Since an oral study was used, the 

Agency assumed equivalent oral and inhalation toxicity for route-to-route extrapolation. An 

MOE of 100 is adequate for this exposure scenario. 

Table 4.3.1 Summary of Toxicological Dose and Endpoints for Amitraz Risk Assessment

Exposure

Scenario

Dose Used in Risk Assessment, 

UF

Study and Toxicological Effects

Acute Dietary

(General population 

including infants and 

children)

NOAEL = 0.125 mg/kg

UF = 1001

Acute RfD = 0.0125 mg/kg

Acute PAD = 0.00125 mg/kg

A double-blind randomized crossover study in 

human subjects.

LOAEL = 0.25 mg/kg based on dry mouth, 

drowsiness, decreased temperature, decreased 

blood pressure and decreased heart rate.

Incidental Oral 

Short- and Intermediate 

Term

a

NOAEL= 0.125 mg/kg

Residential

LOC = MOE of 100

b

A double-blind randomized crossover study in 

human subjects.

LOAEL = 0.25 mg/kg based on dry mouth, 

drowsiness, decreased temperature, decreased 

blood pressure and decreased heart rate.

Dermal (All durations)

a Oral NOAEL = 0.125 mg/kg

Residential

LOC = MOE of 100 b 

Dermal absorption factor: 1.6%

A double-blind randomized crossover study in 

human subjects.

LOAEL = 0.25 mg/kg based on dry mouth, 

drowsiness, decreased temperature, decreased 

blood pressure and decreased heart rate.

Inhalation (All duration)

a Oral NOAEL = 0.125 mg/kg

(Inhalation toxicity assumed to be 

equivalent to oral route)

Residential

LOC= MOE of 100 b 

A double-blind randomized crossover study in 

human subjects.

LOAEL = 0.25 mg/kg based on dry mouth, 

drowsiness, decreased temperature, decreased 

blood pressure and decreased heart rate.
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Table 4.3.1 Summary of Toxicological Dose and Endpoints for Amitraz Risk Assessment

Exposure

Scenario

Dose Used in Risk Assessment, 

UF

Study and Toxicological Effects

Cancer “Suggestive Evidence of Carcinogenicity” 

UF = uncertainty factor, LOC = level of concern

a = Because of the reversibility of the CNS effects, exposures of all durations can be regarded as repeating one-day 

exposures. Therefore, the single-day human oral endpoint is appropriate for assessing exposures to amitraz 

regardless of exposure duration.

b= UF of 100 (10x for intra-species variation, and 10x FQPA factor for data gap for EOGRT study and 1x for interspecies extrapolation). Since the PODs used are from studies conducted in human subjects, the inter-species 

extrapolation factor is reduced to 1X.

5.0 Dietary Exposure and Risk Assessment 

5.1 Residues of Concern Summary and Rationale

The qualitative nature of the amitraz residue in plants and animals is adequately understood 

based on plant metabolism studies with apples, beans, lemons, citrus, cotton, and pears, and 

animal metabolism studies with cattle and swine (dermal application) as well as cattle, goats and 

hens (oral dosing). Tolerances for residues of amitraz in/on plant and animal commodities are 

currently expressed in terms of the combined residues of amitraz (N'-[2,4-dimethylphenyl]-N-

[[(2,4-dimethylphenyl)imino]methyl]]-N-methylmethanimidamide) and its metabolites 

containing the 2,4-dimethylaniline moiety, both calculated as the parent compound [40 CFR 

180.287]. 

Literature studies indicate that parent amitraz degrades quickly in honey with complete 

breakdown occurring within 10-15 days at ambient temperatures (Martel et al, 2002; Jimenez et 

al, 1997; Korta et al, 2001). In beeswax, amitraz degraded in one day. The degradation products 

in honey and beeswax were identified primarily as 2,4-dimethylphenyl formamide (referred to 

here as DMF but also known as DMPF) and N-(2,4-dimethylphenyl)-N-methyl formamidine 

(DPMF), along with small amounts of 2,4-dimethylaniline (DMA). DMF and DPMF were found 

in about equal proportions in honey in these studies. These metabolites contain the 

dimethylaniline moiety and are covered by the current tolerance expression. These are also the 

residues of concern for dietary risk assessment.

5.2 Food Residue Profile

The amitraz risk assessment in 2006 determined that the residue chemistry database was 

complete and that no additional residue studies are needed. Since all U.S. registrations on crops 

used as foods/feeds have since been cancelled, the only current source of amitraz residues in 

foods arise from dermal uses on livestock, and from imported treated cotton seed. 

Magnitude of the Residue (Beehive Use)

To support the Section 3 request Veto-Pharma and Arysta Lifescience America, Inc. submitted 

data (MRID 48341001) from a single field trial conducted in France during 1998 that examined 

amitraz residues in honey and honeycomb (beeswax) following treatment of eight beehives with 
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amitraz-impregnated strips (Apivar®). The field trial was performed following three 

consecutive years of Apivar® treatment of the beehives (two applications per year in the spring 

and autumn). In the submitted study two Apivar® strips, each containing 500 mg of the active 

ingredient (ai) amitraz, were hung between the frames in the brood chambers of the hives for 42 

days (total applied = 1 g amitraz). At the end of the 42-day treatment period, the strips were 

removed from each hive and honey supers were placed on top of five of the hives. Single 

samples of honey and beeswax were collected on Study Days 21, 42, 43, 44, 45, 46, 48, 52 and 

57, corresponding to a mid-treatment sampling and sampling at 0, 1, 2, 3, 4, 6, 10, and 15 days 

after removal of the strips (days after treatment; DAT) /addition of the honey supers. Samples 

were collected from the supers; for the three hives without supers, samples were collected around 

the brood chambers. 

Based on residue data from samples collected from the honey supers, residues of amitraz in fresh 

honey and beeswax were 0.059-0.092 ppm and <0.050-7 ppm, respectively, 10 DAT (Study Day 

52). Residues at 15 DAT (Study Day 57) were <0.050-0.084 ppm and 1-4 ppm in fresh honey 

and beeswax, respectively. 

[Note: The Study Report noted that the study was not designed to support the currently proposed 

label for Apivar® strips but the results can be used to ascertain maximum residue levels to be 

expected in honey from hives treated with Apivar strips. In the study, supers were installed on 

Day 42 (0 DAT), the same day that strips were removed. According to the Study Report, the 

proposed label for Apivar® strips requires a 14-day interval before installation of supers; 

therefore, residues in the supers from this study will reflect maximum expected residues. To 

more closely represent the use pattern for the proposed label, the Study Report disregarded 

residue data from samples collected from the brood chambers since honey collected from the 

brood chamber is not representative of commercially-gathered honey, which is always collected 

from supers. Data collected prior to Study day 52 (10 DAT) was also disregarded because, 

according to the proposed label, honey can only be collected on Day 56 (14 DAT) or later.]

Table 5.2 Summary of Residue Data for Honey and Beeswax from Hives Treated with Amitraz.

Treatment 

Rate

Commodity Study Day PHI/ 

DAT

(Days)

Combined Amitraz Residues (ppm)1

n Min. Max. Median Mean Std. Dev.

Apivar Strips Proposed Label Data2

Two 500 mg 

Strips

Honey 52 10 4 0.0586 0.0922 0.0735 0.0745 0.0172

57 15 4 <0.050 0.0839 0.0559 0.0614 0.0160

Beeswax 52 10 4 <0.050 7 2 3 3

57 15 4 1 4 2 2 2

1 The method LOQ for each matrix is 0.050 ppm. For samples with residues <LOQ, the LOQ was used for calculating the 

median, mean and standard deviation.

2

Includes only samples collected from the hives with supers. 

Storage Stability 

Storage stability data are available indicating that total residues of amitraz (which include any 

metabolites containing the 2,4-dimethylaniline moiety) are stable in honey stored at -20°C for up 

to 4 months and in honeycomb stored ambient for up to 1 month. Total residues of amitraz are 

stable in cottonseed for 13.5 months, citrus for 18 months and in animal muscle, fat, and milk for 
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12-15 months. 

5.3 Water Residue Profile

No residues of amitraz are expected in drinking water from the current uses or the proposed use 

in bee hives.

5.4 Dietary Risk Assessment

5.4.1 Description of Residue Data Used in Dietary Assessment

The partially refined acute dietary analysis was conducted using the Dietary Exposure Evaluation 

Model DEEM-FCIDTM, Version 2.03 and is based upon potential exposure through honey, 

imported cottonseed oil, and meat and milk from dermal treatments of livestock (D. Soderberg, 

4/12/12, D383216). The residue values used for livestock products, except for milk, are based 

upon tolerance level residues. Percents of livestock treated were incorporated as estimated by 

the Biological and Economic Analysis Division (BEAD, 10/27/09, D367939). For livestock 

commodities 0.3% head treated was used for cattle and 1.2% head treated was used for swine. 

Milk residues were based upon the high end result from the original cattle dosing study (as cited 

in D300301, J. Morales, 4/30/04). A tolerance remains in effect for amitraz in cottonseed 

although there are no registered uses on cotton in the U.S. To account for potential amitraz 

residues in imported cottonseed oil (Foreign Agriculture Service data show that about 2% of 

cottonseed oil is imported), residues in cottonseed oil were estimated at the cottonseed tolerance 

and 2% crop treated (imported) was incorporated.

For honey the residues at study day 57, i.e. 15 days after treatment (DAT), from MRID 

48341001 were used and it was assumed that 100% of honey is treated with amitraz. 

Honeycomb is not reported as being consumed in the DEEM-FCID.

Drinking water was not included in the dietary assessment as it was determined that amitraz is 

not expected to enter water-bodies or drinking water through the current and proposed uses. 

HED had determined that the acute assessment would be protective of any chronic dietary 

exposures to amitraz; therefore, a separate chronic dietary exposure assessment was not 

conducted.

5.4.2 Acute Dietary Risk Assessment

Acute dietary risk estimates were not of concern based on current amitraz uses and the proposed 

beehive use. The most highly exposed sub-population is children 1-2 years of age at 0.000956 

mgs/kg/day, or 76% of the aPAD, at the 99.9th percentile of exposure. The general population is 

estimated to be exposed at 0.000199 mgs/kg/day, or 16% of the aPAD. 
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Population Subgroup

Acute Dietary

(99.9th Percentile)

Dietary 

Exposure 

(mg/kg/day)

% aPAD

General U.S. Population 0.000199 16

All Infants (< 1 year old) 0.000286 23

Children 1-2 years old 0.000956 76

Children 3-5 years old 0.000606 48

Children 6-12 years old 0.000367 29

Youth 13-19 years old 0.000160 13

Adults 20-49 years old 0.000117 9.4

Adults 50+ years old 0.000118 9.4

Females 13-49 years old 0.000117 9.3

6.0 Residential (Non-Occupational) Exposure/Risk Characterization

There are no residential (non-occupational) exposures expected from the proposed beehive use 

so a non-occupational risk assessment is not required for the use in beehives. However, there is 

a potential for exposure in residential settings for homeowners who purchase and apply the 

amitraz spot-on product or the amitraz pet collar registered for use on dogs. There is also 

potential for post-application exposure (dermal and oral) from contacting a companion animal 

previously treated with these products. Handler and post-application inhalation exposure is 

expected to be negligible and was not quantitatively assessed. The current toxicity database for 

amitraz suggests that the CNS effects of amitraz are not cumulative, but are a response to each 

daily dose. Residential exposures of all durations can be regarded as a series of repeating oneday exposures.

An assessment of the amitraz-impregnated pet collar residential use was included as a part of the 

2006 HED amitraz human health risk assessment for the TRED. That assessment has been 

updated and includes the following revisions: 

1) Residential SOP: The current assessment uses the updated 2012 Residential SOPs, Health 

Effects Division’s (HED) 2012 Standard Operating Procedures for Residential Pesticide 

Exposure Assessment: Treated Pets.

2) Dermal Absorption Factor: The 1.6% DAF used in this risk assessment differs from the 8% 

value used in the previous risk assessment because the 8% value was obtained at absorption 

seen at 120 hours and the 1.6% value was derived from absorption seen at 24 hours which is 

the appropriate time period based on the half-life of amitraz and based on toxicity following 

exposure to amitraz which appears early after dosing and reverses rapidly; in humans the 
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effects were reversed within 12 hours (acute bolus dose). (See detailed discussion in Section 

3.0 of this document.)

3) Fur Residue Transfer Data: In the 2006 assessment a carbaryl pet collar fur residue study 

was used (MRID 45792201). HED reevaluated this data for use in the residential assessment 

to support the Section 18 bee hive use. The carbaryl pet collar residue transfer was 

conducted using a fur clipping methodology. While consistent with Agency policy at the 

time of the 2006 assessment, HED currently recommends a methodology which measures 

residue transfer by means of a scripted petting routine with the use of a gloved mannequin 

hand. This petting methodology is thought to be more representative of actual contact with a 

treated animal and, historically, it has resulted in more consistent outcomes than that 

observed from fur clipping studies (based on comparison of studies for non-collar pet 

formulations). Therefore, the carbaryl pet collar study was not recommended for use. HED 

considered all available residue transfer data for the updated amitraz pet collar assessment. 

There are no chemical-specific pet collar data for amitraz. The current assessment utilizes a 

recently submitted residue transfer study for a different chemical to refine residential postapplication exposure/risk estimates. This exposure study, which was done for a registered 

propoxur pet collar (MRID 48589901), uses the approved residue transfer methodology for 

the pet collar formulation. While not specific to amitraz (propoxur study), the propoxur pet 

collar study is the best available surrogate for amitraz collars and offers a realistic refinement 

from the high-end default fraction application rate (FAR) transferred recommended (2%) in 

the updated 2012 Residential SOPs when chemical-specific data are unavailable. The FAR

from the propoxur study is 0.1%. The default FAR relies on exposure data from multiple, 

liquid pet product formulations (e.g., aerosol and pump sprays, and spot-ons) which differ 

greatly from pet collars. It is well understood that the active ingredient (ai) in liquid 

formulated products are more readily available for transfer to the exposed individual than 

that of pet collars which are designed for slow release over time; as a result, the use of the 

default FAR is expected to result in an overestimate of the risk from pet collar exposures.

It is unclear, however, exactly what effect differing chemicals and the collar medium itself 

participate in the fraction transfer. HED expects that the amitraz and propoxur pet collars 

function in a similar manner; that is, through “rub off” or transfer from the collar to the 

animal’s hair coat via embedded lubricants which function like transfer agents at the surface 

of the collar. Diffusion of the ai through the collar matrix controls the amount of the active 

ingredient at the collar’s surface, which is modulated by the physical chemical properties of 

the compounds. Based on a comparison of differing physical chemical properties of the two 

chemicals, amitraz is much more hydrophobic and much less water soluble than propoxur. A 

study by Wang et al (2012) concluded that chemicals with higher hydrophobicity bind more 

to hair than chemicals with a lower log Kow value. Therefore, it is predicted that once 

transferred from the collar to the animal’s fur, amitraz will bind more strongly to the animal’s 

hydrophobic fur than propoxur and, as a consequence, less will be transferred to the exposed 

person from fur contact. Furthermore, hydrophobic chemicals can penetrate into the hair, 

thus not available for transfer. This principle is supported by dermal toxicology data in which 

chemicals with high hydrophobicity bind tightly to skin keratin (also present in hair), and are 

thus not available for dermal transfer. The propoxur collar data is predicted to be a 

conservative estimate for amitraz collar transfer.
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6.1 Residential Handler Exposure- Pet Collar

Residential handler exposures to amitraz from the pet collar use may occur via the dermal route 

while the collars are placed on a dog. Residential handler inhalation exposure is expected to be 

negligible from amitraz pet collars due to the chemical’s low volatility; thus, a quantitative 

assessment was not performed. A dermal residential handler exposure assessment for all 

durations was performed for homeowners applying the collar to a dog. 

No chemical-specific unit exposure data were provided in support of this submission and there 

are currently no pet collar handler exposure data available for other chemicals to use as a 

surrogate. As a result, HED used exposure data described in the 2012 Treated Pet SOP for spoton applications (MRID 44433303) as a surrogate to estimate handler exposures. The use of 

handler exposure data are not expected to underestimate exposures from the pet collar 

formulation. Unit exposures are expressed as mass active ingredient exposure, per mass active 

ingredient handled (e.g., mg/lb ai). This means that an individual’s exposure will increase by a 

given (and constant) amount for every “unit” increase in the amount of active ingredient handled. 

Therefore, the increased application rate of the pet collars relative to the typical spot-on product 

is accounted for due to the assumption of proportionality. These unit exposures were based on 

residential handlers wearing short pants, short-sleeved shirt, and no gloves. 

Dermal risk estimates for residential handlers (applicators) of pet collars are not of concern 

MOEs>100) for all exposure durations.
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1. No exposure data available for this application scenario. Exposure data for spot-on applications recommended as 

surrogate data.

2. Based on proposed label and percent ai in product.

3. Based on HED’s 2012 Standard Operating Procedures for Residential Pesticide Exposure Assessment; Treated Pets.

4. Dermal Dose = Dermal Unit Exposure (mg/lb ai) x Application Rate (lb ai/collar) x Amount Handled (collars/day) x DAF 

(1.6%)/BW (80 kg).

5. Dermal MOE = Oral NOAEL (0.125 mg/kg/day)/Dermal Dose (mg/kg/day).

6.2 Post-Application Exposure- Pet Collar

Residential post-application exposures to amitraz can occur after collars are placed on a dog via 

dermal and oral exposure routes. Residential post-application inhalation exposure is expected to 

be negligible from pet collars due to the chemical’s low volatility; thus, a quantitative assessment 

was not performed. Dermal post-application risks (all durations) were assessed for adults and 

children 1 to < 2 years old while oral post-application risks were only assessed for children 1 to 

< 2 years old. Children ages 1 to <2 years are chosen as a sentinel population for this assessment 

because their exposures are expected to be greater than those for other age groups, and therefore 

the assessment will be protective for all age groups.

No exposure data have been submitted in support of the amitraz pet collar use. In the absence of 

chemical-specific data, HED used data from a residue transfer study (MRID 48589901) 

conducted for another chemical, propoxur, to refine residential post-application exposure/risk 

estimates. This exposure study is the only study available to the Agency which was conducted 

using pet collars and using the approved residue transfer methodology (i.e., a scripted petting 

routine via gloved mannequin hand). Therefore, while not chemical-specific, the propoxur pet 

collar study is the best available surrogate for amitraz pet collar exposure assessment and offers a 

considerable refinement from the high-end default FAR recommendation; the default FAR would 

result in a significant overestimation of exposure from amitraz pet collar use. 

The adult dermal post-application risks for amitraz dog collars are not of concern (MOEs >100). 

The combined dermal and oral (hand to mouth) exposures for children 1 to < 2 years old are also 

not of concern (see Tables 6.2.1- 6.2.3 below).

Table 6.1 Residential Handler Exposure and Risk Estimates for Use of Pet Collars Containing 9% 

Amitraz

Exposure 

Scenario1

Type of 

collar

Application 

Rate (lb 

ai/collar)2

Amount 

Handled 

(collar/day)3

Baseline Unit Exposures 

(mg/lb ai)3 Dermal Dose 

(mg/kg/day)4

Baseline 

Dermal 

MOE5 Dermal Inhalation 

Applying 

a pet 

collar 

Up to 60 lb 

Collar

(18.5 g)

0.0037 2 120 Negligible 0.00018 700

Above 60 lb 

Collar

(27.5 g)

0.0055 2 120 Negligible 0.00026 480
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Table 6.2.1 Amitraz Collar Dermal Adult and Child 1 < 2 yrs Exposure/Risk Estimates

Lifestage AR 

(mg ai) Far SA of pet (cm2/animal)

Transferable 

residue 

(mg/cm2)

Transfer 

coefficient 

(cm2/hr)

Exposure 

time (hr) DAF BW Exposure (mg/day)

Dose 

(mg/kg/day) MOE

Small to Medium Dog

Adult

1,700

0.001 5,000

0.00033

5200 0.77

1.6%

80 1.3 0.00027 470

Child 1 < 2 0.001 5,000 1400 1 11 0.47 0.00065 190

Large Dog

Adult

2,500

0.001 9,400

0.00026

5200 0.77

1.6%

80 1.1 0.00021 590

Child 1 < 2 0.001 9,400 1400 1 11 0.37 0.00052 240

Table 6.2.2 Amitraz Collar Incidental Oral (Hand-to-Mouth) Child 1 < 2 yrs Exposure/Risk Estimates

Faihands

DE SAH HR Fm ET 

Replenishment 

interval 

(min)

N_Replen SE Freq_Replen

Body 

weight 

(kg)

Average

Daily 

Dose 

(mg/kg/day)

MOE Dermal 

Exposure 

(mg) 

Surface 

area of 

1 hand 

(cm2)

Hand 

residue 

loading 

(mg/cm2)

Fraction 

of hand 

mouthed

Exposure 

Time 

(hours/

day)

# Replenishment 

intervals per 

hour 

(intervals/hr)

Fraction 

Saliva 

Extractio

n

Number of 

hand-to-mouth 

contacts events 

per hour 

(events/hr)

Small to Medium Dog

0.040 0.47 150 0.000062 0.13 1 15 4 0.50 20 11 0.00041 310

Large Dog

0.040 0.37 150 0.000049 0.13 1 15 4 0.50 20 11 0.00032 390
Amitraz Human Health Risk Assessment DP No.D407721

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Table 6.2.3. Combined Residential Exposure and Risk Estimates from the Amitraz Pet Collar Use 

Lifestage Post-application 

Scenarios

Daily Dose

(mg/kg/day) MOE Combined MOE

Small to Medium Dog

Adult Dermal 0.00027 470 470

Child 1 < 2

Dermal 0.00065 190

120 Hand-to-Mouth 0.00041 310

Large Dog

Adult Dermal 0.00021 590 590

Child 1 < 2

Dermal 0.00052 240

150 Hand-to-Mouth 0.00032 390
Amitraz Human Health Risk Assessment DP No.D407721

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6.3 Risk Characterization of Residential Assessment – Pet Collar

The evaluation of the potential health risks from exposures to dogs treated with amitraz pet 

collars is considered conservative. The risk estimates calculated are based upon protective 

assumptions of application rate, duration of exposure, and contact with the treated animal. The 

fraction of application rate transferred, while non-chemical specific, represents the best data 

available to assess risk from exposures to the amitraz collar and is not expected to underestimate 

risk.

Application Rate: The application rates used in the assessment of the amitraz pet collar represent 

the amount of ai contained in the entire small to medium, or large size dog collars. The amitraz 

pet collar label directs the user to cut off and dispose of any excess length once the product is fit 

and buckled into place. As described in the HED Treated Pet SOP, the removal of excess length 

and corresponding ai loss cannot be determined and, therefore, the maximum application rate 

(total ai) of the collar is assumed for assessment of applicator risk. While this approach may 

result in an overestimate of collar application rate, it ensures protection of users who may contact 

dogs wearing an uncut collar. Because it is more likely that the removal of excess length will be 

required to fit small dogs wearing the “up to 60 lbs” size collar, the surface area of small and 

medium dogs (as recommended in the Treated SOP) were averaged to result in a more realistic 

application rate estimate for both sizes. Further, the recommended surface area of a large size 

dog corresponds to an animal weighing 50 lb. Because the larger size collar is for dogs over 60 

lbs, the surface area recommended for exposure assessment was adjusted to reflect the larger 

size. 

Fraction of Application Rate Transferred: As described previously, no chemical-specific 

transferable residue data has been submitted for the amitraz pet collar product. In the absence of 

such data, HED used transferable residue exposure data from a pet collar study for another 

chemical, propoxur, to refine residential post-application exposure/risk estimates. Typically, 

when data is lacking a default assumption of the fraction application rate (FAR) is used in 

conjunction with the application rate to estimate TR. Based on recommendation of the Treated 

Pet SOP, a 2% screening level FAR point estimate is used to assess liquid formulated products, as 

well as pet collars. The screening level assumption was determined from the review of 8 pet 

residue transfer, or “petting,” studies conducted with liquid formulations (i.e., aerosol and pump 

sprays, and spot-ons). Measurements of residue transfer were derived by taking the ratio of the 

amount of ai on a bare or gloved hand (on the day of highest observed transfer) to the amount of 

ai applied. Five residue transfer studies were performed by means of volunteers “petting” or 

“stroking” animals treated with a known amount of ai. Three additional residue transfer studies 

were conducted using a gloved mannequin hand. For each study the amount of residue 

transferred to the hands was determined. Of the 8 pet residue transfer studies used to establish 

FAR, none were conducted using a pet collar formulation. 

In comparison to other formulations used for pet pest control (e.g., dust, shampoos, spot-ons), the 

pet collar formulation is unique. When other pet product formulations are applied to a treated 

pet, it can be reasonably assumed that the entire amount of that applied (i.e., the application rate) 

is available on the hair coat of the animal for transfer to the exposed individual. For example, 

the labeled amount of a spot-on product can be used directly in the assessment of exposure/risk 
Amitraz Human Health Risk Assessment DP No.D407721

Page 26 of 48

from its use because it is assumed this amount is available on the surface to be contacted. Pet 

collar formulations operate by means of the slow release of ai from the collar medium, making 

them efficacious for pest treatment over longer periods of time. Due to this behavior, it can be 

difficult for the risk assessor to determine the amount which is initially available on the animal 

because the majority is retained within the collar. Accordingly, the propoxur pet collar study 

results in residue transfer much less than that from other, liquid formulated products. 

Some uncertainty exists regarding what effect differing ais and the collar medium itself play in 

the fraction transfer. HED expects that the amitraz and propoxur pet collars work in a similar 

manner; that is, through “rub off” or transfer from the collar to the animal’s hair coat via 

embedded transfer agents which transport the active ingredient to the collar’s surface. Amitraz is 

much more lipophilic than propoxur; therefore, HED predicts that once transferred from the 

collar the animal’s fur, amitraz will bind more strongly to the animal’s hydrophobic fur and, as a 

consequence, less will be transferred to the exposed person from fur contact. For all the reasons 

described, HED anticipates the use of the surrogate, propoxur pet collar data to be protective of 

individuals exposed to a dog treated with the amitraz collar product.

Exposure Time: The exposure time (ET) assumption used to assess residential post-application 

exposure to the amitraz pet collar is derived from a study which sought to evaluate the times that 

individuals spent performing different activities around the home. Based upon the Treated Pet 

SOP, the point estimates recommended for adult and child ET with pets are 0.75 and 1 hour, 

respectively. In the study, animal care is defined as “care of household pets including activities 

with pets, playing with the dog, walking the dog and caring for pets of relatives, and friends.” 

The data identified the time spent with an animal while performing household activities as 

recorded in 24 hour diaries by study volunteers. While the activities defined do not necessarily 

represent the time volunteers were actively engaged in constant contact with the animal as is 

implicit in the post-application dermal and incidental oral algorithms, the data are the most 

accurate representation of time spent with pets available and, therefore, it is assumed that contact 

is continual throughout the timed activity. HED assumes the ET value reflects a reasonable high 

end estimate of time spent in contact with a dog treated with an amitraz pet collar. 

Pet Contact: For the purpose of determining exposure to treated pets, transfer coefficients (TCs) 

can be defined as animal surface area contact per unit time (cm2/hr). The transfer coefficient 

used to assess exposure to a dog treated with the amitraz collar was derived from a study 

representing application and grooming activities with dogs using a shampoo product. These data 

were gathered while human volunteers applied a shampoo product to various dogs of differing 

sizes and fur lengths. Since these individuals extensively handled the dogs while shampooing 

and grooming, it is expected that their resulting exposures are a reasonable approximation of 

upper bound estimates of contact with an amitraz-treated dog. In the absence of direct exposure 

data for the residential activity with a treated pet scenario, the Agency concludes that the 

grooming activities are likely to result in a highly protective estimate of pet contact relative to 

contact associated with petting, hugging or sleeping with an amitraz-treated dog. 
Amitraz Human Health Risk Assessment DP No.D407721

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6.4 Residential Bystander Post-application Inhalation Exposure

Residential bystander post-application inhalation exposures are not expected to occur from the 

current uses (livestock dermal treatments, pet products) and proposed use (beehive strips) of 

amitraz. 

6.5 Spray Drift

There is no potential for spray drift for the current (livestock dermal treatments, pet products) or 

proposed uses (beehive strips) of amitraz. 

7.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and 

risks from three major sources: food, drinking water, and residential exposures. In an aggregate 

assessment, exposures from relevant sources are added together and compared to quantitative 

estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated. When 

aggregating exposures and risks from various sources, HED considers both the route and 

duration of exposure. The current toxicity database for amitraz suggests that the CNS effects of 

amitraz are not cumulative, but are a response to each daily dose. Residential exposures of all 

durations can be regarded as a series of repeating one-day exposures and the single-day human 

oral endpoint is appropriate for assessing exposures to amitraz regardless of exposure duration.

There are no residential exposures expected from the proposed amitraz beehive use. However, 

residential exposure to amitraz from existing pet uses (pet collars and spot-on treatments for 

dogs) is expected. The aggregate risk assessment for amitraz combines the dietary (food only; 

there are no drinking water exposures anticipated for amitraz uses) and residential exposures 

from amitraz pet collar uses. The use of pet collar exposure in the aggregate is protective of 

spot-on treatments. The aggregate assessment for amitraz combines the oral dietary and dermal 

exposures for adults and combines oral dietary, oral hand to mouth, and dermal exposures for 

children. For amitraz, an aggregate MOE of 100 or greater does not exceed HED’s level of 

concern.

In general aggregate assessments combine average (chronic) dietary exposures with conservative 

residential exposures. However, in the case of amitraz, a chronic dietary assessment was not 

performed since the acute dietary assessment will result in higher estimated exposure levels and 

will therefore be protective of any chronic exposures. As a screening level aggregate 

assessment, residential post-application exposures from the small to medium dog collar uses (the 

residential scenario resulting in the highest estimated exposures) were combined with acute 

dietary exposures at the 95th percentile of exposure. While aggregation using an average 

background exposure would more appropriately reflect expected exposures, in the absence of a 

chronic dietary assessment, use of acute exposures at the 95th percentile of exposure provides a 

high-end aggregate risk screen, and since risks are acceptable using this screen, a more refined 

assessment is not required. 
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7.1 Aggregate Risk (All Durations)

For adults, combining the acute dietary exposure with post-application dermal exposure from 

amitraz dog collar use results in an aggregate MOE of 450 which does not exceed HED’s level 

of concern. 

For children (1to <2 yrs), combining dermal and incidental oral exposures from amitraz dog 

collar use with acute dietary exposure results in an aggregate MOE of 120 which does not exceed

HED’s level of concern. 

Table 7.1 AMITRAZ Aggregate Risk Calculations –All Durations

(Small to Medium Dog Collar plus Dietary)

Population

All Durations

NOAEL 

mg/kg/day LOC1

Max 

Allowable 

Exposure2

mg/kg/day

95th

Percentile 

Food 

Exposure 

mg/kg/day

Residential 

Exposure 

mg/kg/day3

Total 

Exposure 

mg/kg/day4

Aggregate 

MOE (food, 

and 

residential)5

Adult 0.125 100 0.00125 0.000006 0.00027 0.000276 450

Child (1-2 yrs) 0.125 100 0.00125 0.000017 0.00106 0.00108 120

1 LOC of 100 includes 10x intraspecies uncertainty factor, 1x interspecies factor and 10x FQPA safety factor.

2 Maximum Allowable Exposure (mg/kg/day) = NOAEL/LOC

3 Residential Exposure = [Oral exposure + Dermal exposure]. (Table 6.2.3).

4 Total Exposure = 95th Percentile Food Exposure + Residential Exposure. (Soderberg, D383216)

5 Exposure + Residential Exposure)]

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 amitraz and any other substances and amitraz 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 amitraz 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 a common mechanism on 

EPA’s website at http://www.epa.gov/pesticides/cumulative/.

9.0 Occupational Exposure/Risk Characterization

The proposed new use pattern does not indicate a significant potential for applicator or postapplication dermal or inhalation exposure. The Apivar beehive strips come prepackaged and do 

not involve any mixing or loading of active ingredient. Occupational exposure resulting from 

use of these strips is not likely to occur, as the only potential times for worker contact are during 

application, when the beehive keeper removes the strips directly from the packaging and places 

them in the hives, and during removal and disposal of the strips six weeks later. Also the 

proposed label requires the use of latex gloves by applicators which will further reduce the 

potential for exposure. After six weeks, at the time of disposal, residues have significantly
Amitraz Human Health Risk Assessment DP No.D407721

Page 29 of 48

decreased, so post-application exposure is minimal. Since most beehives are located outdoors, 

there is negligible potential for inhalation exposure, at any time, to the applicator or during postapplication activities. Because of the very low potential for dermal or inhalation exposure, 

quantitative occupational risk assessments are not required for this proposed new use even 

though one-day dermal and inhalation endpoints for amitraz have been previously identified.

10.0 References

D. Drew et al., 10/18/12, Amitraz. ID#:10SD04 Updated Section 18 Emergency Exemption for 

the Use of Amitraz in Beehives in South Dakota, D406097.

W. Britton, 10/11/12, Amitraz: Occupational and Residential Exposure and Risk Assessment for 

the Pet Collar Use (2382-104) in Support of the Section 18 Use in Beehives, D405728.

D. Soderberg, 4/16/12, Amitraz: Revised Acute Probabilistic (Food Only) Dietary Exposure 

Assessment for the Section 3 Registration Action That Was Requested for Use of Amitraz in 

Apivar Strips in Bee Hives, D383216.

W. Irwin et al., 3/10/10, Amitraz: Human Health Assessment Scoping Document in Support of 

Registration Review, D367925.

D. Drew et al., 7/26/06, Amitraz. Revised Human Health Risk Assessment for the Tolerance 

Reassessment Eligibility Decision based on discussions of the Human Studies Review Board,

D310227. 

USEPA, 6/26/06, HSRB Final Report: April 4-6, 2006 Meeting EPA Human Studies Review Board 

Report. http://www.epa.gov/hsrb/apr-4-6-2006-public-meeting.html

J. Carley, 3/16/06, Initial Ethics Review of Amitraz Human Study.

Martel A.-C. and S. Zeggane, 2002. "Determination of acaricides in honey by high 

performance liquid chromatography with photodiode array detection." Journal of 

Chromatography A, 954, pp 173 to 180.

Korta, E., A. Bakkali, L.A. Berrueta, B. Gallo, F. Vicente, V. Kilchenmann, and S.

Bogdanov, 2001. "Study of Acaracide Stability in Honey. Characterization of

Amitraz Degradation Products in Honey and Beeswax. Journal of Agricultural and

Food Chemistry, 49, pp 5835 to 5842.

Jimenez, J.J., J. L Bernal, M.J. del Nozal, L. Toribo, and J. Atienza, 1997. "Characterization 

and Monitoring of Amitraz Degradation Products in Honey." Journal of High Resolution 

Chromatography, 20, pp 81 to 84.

Wang, L et al, "Kinetics and equilibrium of solute diffusion into human hair", Ann Biomed Eng,

July 2012 (Epub ahead of print)
Amitraz Human Health Risk Assessment DP No.D407721

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Appendix A. Toxicology Profile and Executive Summaries

A.1 Toxicology Data Requirements

The requirements (40 CFR 158.340) for [Type of Use (e.g., food vs. non food)] for [CHEMICAL NAME] are in 

Table 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

Table A1.1 Toxicology Requirements for Amitraz 

Guideline Number and Toxicity Study 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

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.3100 Oral Sub-chronic (Rodent)...........................................

870.3150 Oral Sub-chronic (Non-Rodent)...................................

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

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

870.3465 90/28-Day Inhalation ...................................................

yes

yes

yes

CR

CR

no

e

yes a 

yes b

no

Yes c

870.3700 Developmental Toxicity (Rodent)...............................

870.3700 Developmental Toxicity (Non-rodent).........................

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

yes

yes

yes

yes

yesd

no 

e

870.4100 Chronic Toxicity (Rodent) ...........................................

870.4100 Chronic Toxicity (Non-rodent) ....................................

870.4200 Oncogenicity (Rat).......................................................

870.4200 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 ...

870.5550 Mutagenicity: UDS Assay............................................

yes

yes

yes

yes

yes

yes

yes

yes

870.6100 Acute Delayed Neurotoxicity (Hen).............................

870.6100 90-Day Neurotoxicity (Hen) ........................................

870.6200 Acute Neurotoxicity Screening Battery (Rat) ..............

870.6200 90-Day Neurotoxicity. Screening Battery (Rat)...........

870.6300 Developmental Neurotoxicity ......................................

no

no

yes

yes

CR

-

-

no 

e

no 

e

no

e

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

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

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

yes

CR

yes

yes

yes

no

e

a= Satisfied by the availability of a chronic toxicity study in this species

b= Satisfied by the availability of an oral study in human subjects and used for risk assessment.

c = Satisfied by the availability of an oral study in human subjects and used for risk assessment.

d= Available unacceptable studies in non-rodents does not indicate this species to be more sensitive than rats.

 e = Will be satisfied by the Extended One-Generation Reproduction Toxicity (EOGRT) study
Amitraz Human Health Risk Assessment DP No.D407721

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A.2 Toxicity Profiles

Table A.2.1 Acute Toxicity Data on AMITRAZ Technical

Guideline No./ Study Type MRID No. Results Toxicity 

Category

870.1100 Acute oral toxicity 00041539 LD50: 531 mg/kg (M)

515 mg/kg (F)

III

870.1200 Acute dermal toxicity 00040862 LD50: > 200 mg/kg II

870.1300 Acute inhalation toxicity 00029963 LC50: 2.4 mg/L IV 

870.2400 Acute eye irritation 00040861 Non-irritating IV

870.2500 Acute dermal irritation 00040862 Non-irritating IV

870.2600 Skin sensitization 00029965 Not a sensitizer under conditions of 

study

N/A

Table A.2.2 Toxicity Profiles for Amitraz

Guideline 

Number

MRID No. Study Type Results

Sub-Chronic Toxicity

870.3100

MRID

00051784

90-Day Oral Toxicity -

Rat 

NOAEL = 3 mg/kg/day

LOAEL = 12 mg/kg/day based on irritability, 

excitability and reduced overall body weight gain.

No individual animal data for clinical signs and gross 

necropsy.

870.3150

MRID

00040345

90-Day Oral Toxicity -

Dog 

NOAEL = 0.25 mg/kg/day

LOAEL = 1.0 mg/kg/day based on CNS depression, 

decrease in pulse rate, increase in glucose in urine, 

hypothermia, neutrophilia of bone marrow, increased 

liver weights and increased extent of liver lesions.

Too few animals.

870.3200

MRID

00029972 21-Day Dermal Toxicity -

Rabbits

NOAEL = Cannot be determined

LOAEL = 50 mg/kg/day based on clinical signs 

(sedation) and a decrease in food consumption in 

males. Too few animals, concurrent infections, lack 

of information on the substance tested and limited 

histopathology.

870.3465

MRID

00029964

21-Day inhalation 

toxicity

rats

NOAEL = 1.8 mg/kg/day

LOAEL = 18 mg/kg/day (nominal) based on clinical 

signs of toxicity (irritation and neurological signs) and 

decreases in body weight and body weight gain. 

Limited individual animal data; analytical exposure 

concentrations not measured; purity of test material 

not reported and reporting incomplete in terms of the 

study protocol and environmental conditions. The 

particle size distribution was acceptable, thus the 
Amitraz Human Health Risk Assessment DP No.D407721

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Table A.2.2 Toxicity Profiles for Amitraz

Guideline 

Number

MRID No. Study Type Results

nominal concentrations were considered adequate.

Developmental Toxicity

870.3700

MRID

00029959

Developmental Toxicity -

Rat (gavage)

Maternal NOAEL = 3 mg/kg/day

LOAEL = 12 mg/kg/day based on decreases in body 

weight gain.

Developmental NOAEL = 12 mg/kg/day

LOAEL = > 12 mg/kg/day [HDT].

Very limited data, dosage period was from gestation

days 8-20.

870.3700

MRID 

44265902

Developmental ToxicityRat

Maternal NOAEL = 7.5 mg/kg/day

LOAEL = 15.0 mg/kg/day based on decreases in body 

weight and body weight gain.

Developmental NOAEL = 30 mg/kg/day

LOAEL = > 30 mg/kg/day [HDT].

870.3700

MRID

00029961

Developmental ToxicityRabbit (gavage)

Maternal NOAEL = 5 mg/kg/day

LOAEL = 25 mg/kg/day based on decrease in body 

weight gain and abortions

Developmental NOAEL = 5 mg/kg/day

LOAEL = 25 mg/kg/day based on decreased litter 

size, decreased implantations, increased postimplantation loss, abortions and decreased mean fetal 

body weight.

Too few litters, limited data, unclear method of 

dosing.

870.3700

MRID

44265901

Developmental Toxicity -

Rabbit 

Maternal NOAEL = not established

LOAEL = 3.0 mg/kg/day based on clinical signs.

Developmental NOAEL = 12 mg/kg/day

LOAEL = > 12 mg/kg/day (HDT).

Too few litters in two treated groups and pre-existing 

maternal respiratory infections.

Reproduction Toxicity
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Table A.2.2 Toxicity Profiles for Amitraz

Guideline 

Number

MRID No. Study Type Results

870.3800

MRID

00029960

One-generation 

Reproductive Toxicity -

Rat 

Parental/Systemic NOAEL = 3 mg/kg/day

LOAEL = 12 mg/kg/day based on decreased body 

weight gain.

Reproductive NOAEL = 12 mg/kg/day

LOAEL > 12 mg/kg/day (HDT).

Offspring NOAEL = 3 mg/kg/day

LOAEL = 12 mg/kg/day based on lower mean litter 

size at birth and on lactation day 4. 

870.3800

MRID

00029962

Three-generation 

Reproductive Toxicity -

Rat

Parental/Systemic NOAEL = 4.36/5.09 (M/F) 

mg/kg/day

LOAEL = 16.41/20.05 (M/F) mg/kg/day based on 

decreased body weight gain during the F0 premating 

period.

Reproductive NOAEL = 16.41/20.05 (M/F) 

mg/kg/day

LOAEL > 16.41/20.05 (M/F) mg/kg/day (HDT).

Offspring NOAEL = 1.29/1.58 (M/F) mg/kg/day

LOAEL = 4.36/5.09 (M/F) mg/kg/day based on 

decreased survival and mean litter size during 

lactation. Limited data provided, mating was not 1 

male to 1 female, no data on reproductive organs 

provided, litter data only provided for a few time 

points, and histopathology data not provided.

Chronic Toxicity

870.4100 MRID

00044586

Chronic Toxicity- Dog 

(2 years)

NOAEL = 0.25 mg/kg/day

LOAEL = 1.0 mg/kg/day based on CNS depression 

during first two days of dosing.

870.4300

MRID

00044585

Combined Chronic 

Toxicity/Carcinogenicity 

- Rat (2 years)

NOAEL = 2.5/0.97 (M/F) mg/kg/day

LOAEL = 10.18/3.13 (M/F) mg/kg/day based on 

clinical signs (M and F) and decreased body weight 

gain (M).

No evidence of carcinogenicity.

870.4300

MRID

00139552

Carcinogenicity - Mice

(2 years)

NOAEL = < 2.31/2.63 (M/F) mg/kg/day

LOAEL = 2.31/2.63 (M/F) mg/kg/day based on doserelated incidence of hyperplastic nodules, liver foci, 

stomach hyperkeratosis and spleen hematopoiesis.

Evidence of carcinogenicity: hepatocellular 

adenomas, carcinomas and combined; and lung 

adenomas, probably at dose levels above MTD.

Neurotoxicity

870.6200a NA Acute Neurotoxicity - Rat Waived by HASPOC

NA
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Table A.2.2 Toxicity Profiles for Amitraz

Guideline 

Number

MRID No. Study Type Results

870.6200b Sub-chronic 

Neurotoxicity - Rat

Waived by HASPOC

870.6300 NA Developmental 

Neurotoxicity - Rat A component of the EOGRT study

Genotoxicity

870.5100

MRID

00161009

Reverse Gene Mutation 

in Salmonella

typhimurium

Negative up to 1 mg/plate, with and without 

metabolic activation. 

870.5300

MRID

00161008

Forward Gene Mutation 

in mouse lymphoma cells Negative at 0.06-20 ug/ml with and without metabolic 

activation. HDT is highest non-cytotoxic dose.

870.5375

MRID

41795101

In Vitro Cytogenetics 

(Human Lymphocytes) Negative up to cytotoxic and/or insoluble 

concentrations.

870.5550

MRID

00161011

UDS Assay

(Human Embryonic Lung 

Fibroblast)

Negative up to cytotoxic concentrations, with and 

without metabolic activation.

Metabolism

870.7485

MRID

00160964 Metabolism - Rat

No sex differences in proportion of various 

metabolites recovered in 24-hour urine samples. 

Metabolic process saturated at the 100 mg/kg level. 

No unchanged parent material found in the urine. 

Major metabolites: N-(2,4-dimethylphenyl)-N-methyl 

formamidine, 4-formamido-3-methyl benzoic acid, 4-

acetamido-3-methyl benzoic acid and a polar fraction. 

The polar fraction was labile to acid hydrolysis, 

yielding conjugates of 4-amino-3-methylbenzoic acid, 

N-(2,4-dimethylphenyl)-N-methyl formamidine, 4-

formamido-3-methyl benzoic acid and 4-acetamido-3-

methyl benzoic acid. 

870.7485

MRID

46249601 Metabolism - Rat

Peak levels of amitraz reached in urine within 8 

hours: 78% of the dose in the urine and 9% in the 

feces by 98 hours. Peak levels of metabolite BTS 

27271 reached in urine within 24 hours: 89% of the 

dose in the urine and 4% in the feces by 96 hours. 

Highest residues of amitraz and BTS 27271 reported 

in the liver. Blood residue levels were 17.9 ppb for 

amitraz and not detectable for BTS 27271. Kidney 

residue levels were comparable with 18.0 and 24 ppb 

for amitraz and BTS 27271, respectively. 

Degradation products of amitraz and its metabolite, 

BTS 27271 were similar.

Dermal Penetration
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Table A.2.2 Toxicity Profiles for Amitraz

Guideline 

Number

MRID No. Study Type Results

870.7600

MRID

42133501 Dermal Penetration - Rats

The mean percent of dose absorbed: treatment site 

(2.98% at 24 hours, 1.41% at 120 hours); total 

absorbed (3.69% at 24 hours, 6.56% at 120 hours); 

total (6.67% at 24 hours, 7.79% at 120 hours). 

Non-Guideline Studies

NonGuideline 

Human Study

MRID

43283101 Acute Oral Toxicity

NOAEL = 0.125 mg/kg/day

LOAEL = 0.25 mg/kg/day based on dry mouth, 

drowsiness, decreased temperature, decreased blood 

pressure and decreased heart rate.

NonGuideline 

Human Study

MRID

00160964

46249601

Metabolism Study LOAEL = 0.25 mg/kg

NonGuideline 

Human Study

MRID 

44639401

Acute Dermal Toxicity NOAEL = 24 mg/kg (no LOAEL)

NonGuideline In 

Vitro Study

MRID

00161010

Cell Transformation Negative up to cytotoxic concentrations, with and 

without metabolic activation.

Summary of Toxicity Studies 

Single Oral Dose

A. Human - Double Blind Tolerance Study (MRID 43283101)

In a double-blind sequential dosing oral human tolerance study of Amitraz, (Lot No. not 

reported, purity 98.2%), 7 adult male volunteers were given encapsulated 0, 0.0625, or 0.125 

mg/kg doses in lactose over three 14-day periods. One volunteer was withdrawn from the study 

following development of a rash unrelated to treatment. The study subjects were 28.17 ± 3.54 

years in age and weighed 67.15 ± 3.15 kg. The study was approved by the Simbec Independent 

Ethics Committee and done according to the Helsinki and Hong Kong revisions (l989). Each 

dose was administered 30 minutes after breakfast with 150 ml water. Vital signs including: 

pulse, respiration rate, blood, pressure and temperature were taken at -1, -0.5, 1, 3, 6, 12, 24, and 

36 hours after dosing. ECGs were performed at -1, 1, 3, 6, 12, 24, and 36 hours after dosing and 

pupil responsiveness and psychomotor performance were evaluated pre-dose and at 2.5 and 8 

hours after dosing. Urine was collected at 0-36 hours and 36-60 hours after treatment and blood 

for clinical chemistry and hematology was collected prior to the start of the study and 36 hours 

after each phase of the study. 
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Table A1.4 Dosing Schedule for Amitraz Single Dose Human Study1

Treatment Phase 1 Phase 2 Phase 3

Placebo 2 2 2

0.0625 mg/kg 4 2 0

0.125 mg/kg 0 2 4

1 A total of six male volunteers completed the study. Each individual received all doses as noted above. The phases 

were separated by a two-week period.

Two subjects reported tiredness, pre-dose (0.125 mg/kg) for 9 and 8h respectively, but this was 

determined to be unrelated to treatment. Vital signs and ECG parameters were within normal 

limits. No clinically significant changes were seen in pupil response. Psychomotor performance 

results showed no significant differences between pre- and post-treatment. One subject had a 

slightly elevated total bilirubin at the post-study screen but it returned to normal limits within 

three weeks completion of the study. No treatment-related effects were found on hematology or 

urinalysis parameters. 

This was a well conducted study. The test material was adequately characterized, the study was 

subjected to an ethical review, the subjects were admitted to a clinical pharmacology unit in the 

evening before each dosing and remained there for 36 hours. Complete physical examinations 

were conducted before and after the study and multiple measurements relevant to the CNS 

depression effects seen in the animal studies were taken during the study. The Kruskal Wallis 

one-way analysis of variance was conducted for temporally related changes and the Wilcoxon 

Rank Sum was used to determine changes between phases. The subjects were followed up for at 

least 3 weeks following the study. This was a double blind study with the use of placebos and 

GLP guidelines were followed. The data from this study appears to be reliable and may be used 

for risk assessment purposes. No effects were observed; however, a NOAEL may be 

established. The level of confidence in the data set is high. Criticism of the study focused on the 

small number of subjects and that only males were tested. 

B. Animal Studies

Acute Mouse. (MRID 00029857). Groups of ten male and female CFLP mice were dosed by 

gavage with single oral doses of 10, 30, 100 or 300 mg/kg/day Amitraz respectively after 

measuring their temperature with a thermistor probe. Control groups of ten male and female 

mice were given the vehicle (0.25% cellosize) 0.1 ml/10 gm. Body temperature measurements 

were taken 1, 2, 3, 4, 7, and 24 hours after dosing and mean body temperature changes were 

calculated.

Doses of 10 and 30 mg/kg/day had weak hypothermic effects. Females appeared to be more 

sensitive than males at 30 mg/kg/day. Doses of 100 and 300 mg/kg/day had marked effect on 

females (p <.001). The maximum effect occurred 2 to 4 hours after dosing. At 24 hours postdosing the animals were completely recovered. Doses of 10, 30, 100 and 300 mg/kg/day 

produced hypothermia in female mice.

In a second study (MRID 00040326), groups of 5 male CS1 mice were orally dosed with 

Amitraz suspended in 10% acacia mucilage at levels ranging from 100 to 1600 mg/kg. Clinical 

signs of CNS depression were first observed 1 hour following dosing, starting at 400 mg/kg. 
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Mice given either 400 or 800 mg/kg recovered within 24 hours but those given 1600 mg/kg were 

ataxic for 48 hours and had ptosis for 72 hours. Tremors were also observed at 400 and 800 

mg/kg but not 1600 mg/kg. Deaths were only observed at 1600 mg/kg. No effects were 

observed at 200 mg/kg and below. 

Acute Cat. MRID 00001112. A single dose of 10 mg/kg/day Amitraz was given to each of 2 

cats, one male, one female. Two cats (un-dosed) were used a controls. All were observed for 24 

hours post-dosing. No deaths occurred. Indications of slight CNS depression were noted within 

2 to 6 hours, with complete recovery after 6 hours in both cats. No effects were observed on 

rectal temperature, pulse or respiratory rates. 

Acute Rat. MRID 00040326. Single oral doses of Amitraz suspended in 10% acacia mucilage 

were given to groups of 5 male Boots Wistar rats. Dose levels ranged from 50 to 1600 mg/kg. 

Beginning at a dose level of 400 mg/kg, clinical signs of CNS depression (inactivity, ataxia, 

prostration, splayed limbs, prostration, labored respiration, discharge around the eyes and partial 

closure of the eyelids) were observed; however, in some cases, continuous squeaking, 

excitability, tremors and hyperactivity occurred when the animals were handled. Recovery took 

up to 7 days. Post mortem findings in animals that died included bladder distension and enteritis. 

No effects were observed at 200 mg/kg and below.

Acute Guinea Pig. MRID 00040326. Groups of 3 female guinea pigs were given oral doses of 

Amitraz suspended in 10% acacia mucilage ranging from 25 to 800 mg/kg with a two-fold 

increment between doses. At 800 mg/kg, the animals exhibited hyperexcitability and two died 

over the first night. The survivor was depressed, had swollen eyelids and tremor and died the 

next day. At 400 mg/kg, hyperexcitability was also observed but there were no deaths. No 

effects were observed at 200 mg/kg and below.

Acute Rabbit. MRID 00040326. Two female NZW rabbits were given a single oral dose of 25 

mg/kg Amitraz suspended in 0.4% cellosize. Rectal temperature, heart rate, general appearance 

and behavior were recorded before and at various times after dosing. A second pair was used as 

a control for seven days, but then were dosed with 100 mg/kg Amitraz while the first pair 

became the control. At 100 mg/kg, depressed rectal temperature and heart rate were observed. 

One rabbit exhibited splayed hind legs and became prostrate with severe rales and nasal 

discharge. Recovery was not complete until day 3. The second rabbit exhibited similar 

symptoms although not as severe. No effects were observed at 25 mg/kg.

Acute Dog. MRID 00040326. Three pairs of dogs (1 per sex) were provided with single oral 

doses of Amitraz (4, 20 and 100 mg/kg) in gelatin capsules. Clinical examinations were 

conducted before and after dosing at regular intervals. At 100 mg/kg, both dogs exhibited 

clinical signs of CNS depression after 2 hours and ataxia after 4 hours. Rectal temperatures and 

pulse rates were subnormal. On day two, there were marked signs of CNS depression, ataxia, 

muscular weakness, uncontrolled vocal spasm and micturition. Some muscular spasm and 

aggression was observed in the male. There was poor venous blood pressure (difficulty in 

obtained blood samples). The male died but the female started to show signs of improvement by 

day 3 and had nearly recovered by day 4. At 20 mg/kg, both dogs exhibited signs of CNS 

depression and other similar signs that were observed at 100 mg/kg. After day 3, their appetites 
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began to return. At 4 mg/kg, no clinical signs were observed in either dog. Rectal temperature 

fell slightly. No other effects were observed.

Acute Baboon. MRID 00040333. Three pairs of baboons (1 per sex) were provided with single 

oral doses of amitraz (20, 100 and 250 mg/kg) in gelatin capsules. Clinical examinations were 

conducted before and after dosing at regular intervals. Clinical signs of CNS depression were 

observed at all dose levels in a dose-related manner. At 20 mg/kg, the clinical signs were brief 

and had subsided by the second day. Ataxia, muscular weakness and poor pulse were observed 

at both 100 and 250 mg/kg, although slightly less severe at 100 mg/kg. Decreases in rectal 

temperature were observed at both 100 and 250 mg/kg, although only once in one animal at 100 

mg/kg. The report stated that Athe clinical symptoms produced in baboons by acute dosage with 

[amitraz] were essentially similar to those encountered in dogs. However, baboons appeared less 

sensitive since the dosages required to elicit equivalent effects were roughly 2.5-5 times greater 

in baboons than in dogs.

Repeat Dose Studies

A. Human

There are no repeat dose studies in humans.

B. Animal

Clinical signs, indicative of neurotoxicity were observed in both dogs and rats. In rats and mice, 

excitable, nervous and aggressive behavior was observed but in dogs, signs of CNS depression 

and a dose-related decrease in body temperature were observed. The dog appears to be the most 

sensitive species; however, it should be noted that dosing was provided via gelatin capsules in 

the dog whereas in the rat it was through the diet. 

Developmental Rat. In a developmental toxicity study (MRID 00029959) amitraz (% a.i and, 

batch/lot# not provided) was administered to 11-13 Boots-Wistar rats/dose by gavage (assumed) 

in 0.4% cellulose at dose levels of 0, 1, 3, or 12 mg/kg bw/day from days 8 through 20 of 

gestation. No treatment related maternal effects were observed; however, in a 1-generation 

reproduction study where rats (same strain, laboratory and year) were administered the test 

material by gavage from gestation day I through lactation day 21, dams receiving 12 mg/kg/day 

gained 20% less weight than the control group during gestation. Utilizing data from that study 

(MRlD 00029960), the maternal LOAEL is 12 mg/kg bw/day, based on decrease in body weight

gain. The maternal NOAEL is 3 mg/kg bw/day. The developmental LOAEL is undetermined 

(>12 mg/kg/day). The developmental NOAEL is 12 mg/kg bw/day (HDT).

The developmental toxicity study in the rat is classified unacceptable guideline; and does not

satisfy the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD

414) in the rat. Very limited data were provided, the purity of the test substance was not provided 

and the dosage period was from gestation days 8-20 which excludes some important gestation days.

Developmental Rabbit. In a developmental toxicity study (MRID 00029961) amitraz (% a.i and, 
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batch/lot # not provided) was administered to 8-10 New Zealand White rabbits/dose by gavage 

(assumed) in 0.4% cellulose at dose levels of 0, 1, 5, or 25 m/lkg bw/day from days 6 through 18 

of gestation. No treatment-related maternal effects were observed at either 1 or 5 mg/kg/day. At 

25 mg/kg/day, the does gained less weight than the control group and 4 does aborted. The

maternal LOAEL is 25 mg/kg bw/day, based on decrease in body weight gain and abortions. The 

maternal NOAEL is 5 mg/kg bw/day. No treatment-related developmental effects were observed 

at either 1 or 5 mg/kg/day. At 25 mg/kg/day, decreased litter size, decreased implantations, 

increased post-implantation loss and decreased mean fetal body weight were observed. The 

developmental LOAEL is 25 mg/kg bw/day, based on decreased litter size, decreased 

implantations, increased post-implantation loss, abortions and decreased mean fetal body weight. 

The developmental NOAEL is 5 mg/kg bw/day. The developmental toxicity study in the rabbit is 

classified unacceptable guideline; and does not satisfy the guideline requirement for a 

developmental toxicity study (OPPTS 870.3700; OECD414) in the rat. Very limited data were 

provided, there is no indication that gavage dosing was used (it is assumed), there was a limited 

number of litters available for analysis (too few does), the purity of the test substance was not 

provided.

Reproduction Rat. In a 1-generation reproduction study (MRID 00029960) BTS 27419 (Amitraz 

(purity and batch number not provided) was administered to 13 Boots-Wistar rats/sex/dose (14 in 

the high dose) by gavage (assumed) at dose levels of 0, 1, 3 or 12 mg/kg bw/day in a volume of 1 

ml/100 g body weight from gestation days 1 through lactation day 21. At 12 mg/kg/day, the 

dams gained a mean of 20% (p< 0.001) less weight than the control group. No maternal effects 

were observed in any other treated group. No information was provided on males. The maternal 

systemic LOAEL is 12 mg/kg bw/day, based on decrease in body weight gain. The maternal

systemic NOAEL is 3 mg/kg bw/day. At 12 mg/kg/day, there was a slightly lower mean litter 

size at birth and on lactation day 4 (p <0.01). By lactation day 21, mean litter size was 

comparable to the control group. The offspring

LOAEL is 12 mglkg bw/day. The offspring NOAEL is 3 mg/kg bw/day. Note: The NOAEL

is lower than the original DER because the slightly lower mean litter sizes support the NOAEL,

LOAEL and effects observed in the multi-generation reproduction study (MRID 00029962). No 

reproductive effects were observed.

This study is acceptable, non-guideline and does not satisfy the guideline requirement for a

multi-generation reproduction study (OPPTS 870.3800; OECD 416) in the rat. It is only one

generation, only provides limited information and does not provide the purity of the test

compound.

Reproduction Rat. In a 3-generation reproduction study (MRID 00029962) BTS 27 419 (Amitraz , 

99.8% a.i., batch/lot# 2099DR) was administered to 10 male and 20 female Boots-Wistar rats/dose in 

the diet at dose levels of 0, 15, 50, or 200 ppm (equivalent to 0, 1.29, 4.36, or 16.41 mg/kg bw/day

for males and 1.58, 5.09 or 20.05 mg/kg bw for females). One litter was produced per generation.

At 16.41120.05 mg/kg/day, the FO parents gained less mean body weight than the control group

during the premating period (7% in males, 14% in females; statistically significant in females).

Body weights were not reported during either the gestation or lactation periods. Body weight

values were not measured at this dose level in succeeding generations because of a nearly

complete loss of the high dose group during the Fl generation. No other parental effects were

observed in any of the treated groups. The parental systemic LOAEL is 200 ppm (16.41 mg/kg 
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bw/day in males, 20.05 mg/kg bw/day in females), based on decreased body weight gain during the 

FO premating period. The parental systemic NOAEL is 50 ppm (4.36 mg/kg bw/day in males, 5.09 

mg/kg bw/day in females).

At 16.41/20.05 mg/kg/day, there was an almost complete loss of pups in the F1 generation during

the lactation period (5 remaining pups versus 115 remaining pups in the control group). In

addition, there was a reduction in mean litter size on day l (6.7 pups/litter versus 7.9 pups/litter

in the control group). At 4.36/5.09 mg/kg/day, in each generation, there was a higher number of

pup deaths than in the control group. By day 21, the mean litter size was smaller but was not

statistically significantly smaller until the final generation (p < 0.05).

The offspring LOAEL is 50 ppm (4.36/5.09 mg!kg bw/day), based on decreased survival

and mean litter size during lactation. The offspring NOAEL is 15 ppm (1.29/1.58 mg/kg

bw/day).

This study is unacceptable guideline and does not satisfy the guideline requirement for a 3-generation

reproductive study (OPPTS 870.3800); OECD 416 in the rat. Very limited data were provided, 

mating was not 1 male to 1 female (only 10 males used/dose group/generation), no data on 

reproductive organs were provided, litter data were only provided for a few time points, and

histopathology data were not provided.

Subchronic Dog. In a 90-day oral toxicity study (MRID 00040345) Amitraz (% a.i. and batch/lot 

# not provided) was administered to 2 beagle dogs/sex/dose in capsules at dose levels of 0, 0.25, 

1.0, or 4.0 mg/kg bw/day for 90 days. Clinical signs were monitored; body weight and 

temperature, hematology, clinical chemistry, urinalysis and organ weight parameters were 

measured; and gross and microscopic examinations were conducted.

At 1.0 mg/kg/day and above, transient central nervous system depression, decrease in pulse rate, 

increase in glucose in the urine, neutrophilia of the bone marrow, hypothermia, increases in 

absolute and relative liver weights and an increase in the extent of liver lesions were noted. At 

4.0 mg/kg/day, ataxia, emesis and catarrhal conjunctivitis were also observed. The LOAEL is 

1.0 mg/kg/day, based on CNS depression, decrease in pulse rate, increase in glucose in the urine, 

hypothermia, neutrophilia of the bone marrow and increased liver weights and extent of liver 

lesions. The NOAEL is 0.25 mg/kg/day.

Chronic Dog. In a chronic toxicity study (MRID 00044586) Amitraz (97.8-99.8 % a.i. and 

batch/lot #’s 2093DH and 2099DH) was administered to 4 beagle dogs/sex/dose in gelatin 

capsules at dose levels of 0, 0.1, 0.25, or 1.0 mg/kg bw/day) for 2 years. Clinical signs were 

monitored; body weight and temperature, hematology, clinical chemistry, urinalysis and organ 

weight parameters were measured; and gross and microscopic examinations were conducted.

At 1.0 mg/kg/day, all 8 dogs had CNS depression on the first two days of dosing; however, the 

data on clinical signs were not submitted. There was a dose-related decrease in body 

temperature after dosing that lasted approximately 6 hours; however, body temperatures 

essentially remained in the normal range. The LOAEL is 1.0 mg/kg/day, based on CNS 

depression during the first two days of dosing. The NOAEL is 0.25 mg/kg/day.

Carcinogenicity Rat. In a combined chronic / carcinogenicity study (MRID 00044585) BTS 27 

419 (97.8-99.8 % a.i, batch/lot #’s 2093DH, 2099DH) was administered to 40 Ash-Wistar SPF 
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rats/sex/dose in the diet at dose levels of 0, 0.77/0.97, 2.5/3.13 or 10.18/12.59 mg/kg bw/day, 

males/females, respectively) for 2 years. There was no treatment-related effect on survival. The 

number of rats reported as excitable, nervous or aggressive was increased in the 3.13 mg/kg/day 

(females) and 10.18 mg/kg/day and 12.59 mg/kg/day groups (males and females); the females 

were more frequently affected. Body weight gain was decreased in the 10.18 and 12.59 

mg/kg/day group at weeks 0-12 for both males and females, weeks 12-24 for females, and weeks 

24-36 and 0-104 for males. Food consumption was reduced in both the males and females in the 

10.18 and 12.59 mg/kg/day group at the beginning of treatment but then was comparable to the 

control group. There were no treatment-related effects on clinical pathology parameters or 

necropsy findings. The incidence of tumors was comparable between the treated and control 

groups. The LOAEL is 10.18 mg/kg/day (males, based on clinical signs and decreased body 

weight gain) and 3.13 mg/kg/day (females, based on clinical signs). The NOAEL is 2.5 

mg/kg/day (males) and 0.97 mg/kg/day (females). At the doses tested, there were no treatmentrelated increases in tumor incidence when compared to controls. Dosing was considered 

adequate based on clinical signs and decreased body weight gain.

Carcinogenicity Mouse. In a carcinogenicity study (MRID 00139552) Amitraz (88.2-100.8% 

a.i., batch #’s 34732Y, 52221, 56105) was administered to 100 control and 75 treated B6C3F1 

mice/sex/dose in the diet at dose levels of 0, 25, 100 or 400 ppm (equivalent to 0, 2.31/2.63, 

9.61/10.77, or 44.65/50.13 mg/kg bw/day) for 104 weeks. At 2.31/2.63 mg/kg/day and above, a 

dose-related increase in the incidence of hyperplastic nodules, basophilic and telangiectatic foci 

was observed in the liver of females, accompanied by an increased incidence of stomach 

hyperkeratosis and spleen hematopoiesis in males.

At 9.61/10.77 mg/kg/day and above, hyperactivity and aggressive behavior were observed in 

males during the first 12 weeks of the study. Cutaneous lesions, as evidence of fighting were 

also observed. By week 52, mean body weight gain was reduced by 29% in males and 32% in 

females (p<0.01). A 6% decrease in food consumption was observed in males during weeks 1-

12 at both the mid- and high dose levels (p<0.05) and during weeks 13-25 at the mid-dose level 

(p<0.01). An increase in food consumption was recorded at the high-dose level in males during 

weeks 26-104 (9-13%). In females, 5-11% decreases in food consumption were observed during 

weeks 1-19 (p<0.05), followed by increases in food consumption during weeks 31-58 and 93-

104. The bone marrow myeloid/erythroid ratio was significantly reduced (p<0.001) in females 

(22% and 26% at the mid- and high-dose levels, respectively).

At 44.65/50.13 mg/kg/day, piloerection and hunched posture were observed in males during 

weeks 2-4. Food utilization was lower for both sexes during the first 24 weeks of the study 

(approximately 35%-42% lower). Mean body weight gain was reduced by week 52 in males and 

females (29% and 32%, respectively, p<0.01). The bone marrow myeloid/erythroid ratio was 

significantly reduced (p<0.001) in males (24%).

Males had a significant positive trend in mortality with incremental doses of amitraz.

The LOAEL is 2.31/2.63 based on telangiectatic foci in the liver of females, accompanied by an 

increased incidence of stomach hyperkeratosis and spleen hematopoiesis in males. The NOAEL 

is less than 2.31/2.63 mg/kg/day. In females there were significant dose-related positive trends in 
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hepatocellular adenomas, carcinomas and in combined adenomas/carcinomas. Females also had 

a significant increase in the pair-wise comparison of controls and the highest dose group in 

hepatocellular adenomas, carcinomas and in combined adenomas and/or carcinomas. Males had 

a significant dose-related positive trend in lung adenomas. In addition, males had a significant 

increase in the pair-wise comparison of controls and the highest dose group in lung adenomas. 

Reduction in body weight gain and a significant positive trend in mortality in male mice suggest 

that the highest dose tested was excessive. In females, however, the highest dose was high but 

not excessive since it was not life-threatening.

Dermal Studies

A. Human - Double Blind Tolerance Study (MRID 44639401)

In a double-blind sequential dosing study with a randomized crossover and placebo group, eight 

healthy male volunteers 32.0 ± 6.8 years and 76.6 ± 9.3 kg (2 subjects per dose) were given a 

total dose of either 0, 8, 16 or 24 mg/kg Amitraz (Lot No. B570R, Batch No. 459732, purity = 

100%) applied dermally as four equal doses of 0, 2, 4 or 6 mg/kg in an aqueous 1:1 slurry every 

2.5 hours over 10 hours according to a random schedule. Starch, 1:1 in water, was used as a 

placebo. The purpose of the study was to find a no effect level for acute pharmacological effects 

following administration by repeated dermal doses of amitraz. The volunteers were evaluated 

for vital signs, ECG, and psychomotor measurements (pupil critical flicker threshold and 

reaction time in a choice scenario), hematology, clinical chemistry and urinalysis before and 

after dosing. The parameters were selected on the basis of the test material’s pharmacological 

activity (sedation, hypotension and effects on body temperature in humans following oral 

dosing).

Vital signs and ECG parameters were normal for all subjects. No clinically significant changes 

were seen in pupil response. Psychomotor performance results such as choice reaction time and 

critical flicker fusion demonstrated no significant differences. None of the out-of-range ECG 

events were considered clinically significant by the investigating physician. Random out of 

normal range values for clinical chemistry were without biological significance. No treatmentrelated effects were found in hematological or urinalysis results. No effects were observed; 

however, a NOAEL may be established. 

In April 2006, the Human Study Review board (HSRB) reviewed the dermal study conducted in 

human subjects and determined the study to be ethically sound, and concluded that the dermal 

study should not be used for deriving the POD since no toxicity was seen in the dermal study (the 

HSRB questioned whether the methods used were the most sensitive) and there was a concern for 

dermal loading (i.e., the Panel believed that there was overloading of the chemical which may 

have resulted in less chemical absorbed) (HSRB, 2006).

B. Animal

Dermal Rabbit. In a 21-day dermal toxicity study (MRID 00029972), BTS 27 419 (Amitraz in 

acetone: % a.i. and batch/lot # not provided) was applied to the shaved skin of 4 New Zealand 

White rabbits/sex/dose at dose levels of 0, 50 or 200 mg/kg bw/day, 6 hours/day for 5 days/week 

during a 21-day period.
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At 200 mg/kg, sedation was observed in both sexes, starting after the second or third dose. In 

males, a slight to moderate erythematous reaction that persisted throughout the study was 

observed only in males and was accompanied by desquamation of the skin and subcutaneous 

hemorrhage in one male. This male died on the last day of treatment. The 3 surviving males 

steadily lost weight throughout the study. Three out of 4 of both sexes had decreased food 

consumption, beginning in either the first or second week and continuing until either death or 

study termination. Three females died on days 15, 17 and 20. Decreased testicular weights were 

observed. This decrease corresponded with tubular degeneration observed in the microscopic 

examinations.

At 50 mg/kg/day, the males were sedated, starting after the 3rd or 4th dose. Slight to moderate 

erythematous reactions were observed in the treated area of the skin. One male had some 

desquamation and subcutaneous hemorrhage during the final week of treatment. One male 

stopped eating shortly after the treatment commenced and was found dead on day 6. One of the 

3 surviving males lost weight while the other two showed little change in weight, even with 

decreased food consumption in the second and third weeks of dosing. 

Too few animals were tested per dose group, there were concurrent infections and a lack of 

information on the substance tested and limited histopathological investigations were conducted. 

There were indications of infection (bacterial and/or parasitic) in a few animals in all groups. It 

is not known how this may have affected the outcome of the study.

The LOAEL is 50 mg/kg/day, based on clinical signs and a decrease in food consumption in 

males. The NOAEL cannot be determined.

The dermal irritation LOAEL is 50 mg/kg/day based on slight to moderate erythematous 

reactions in the treated area of the skin in 3 out of 4 males accompanied by some desquamation 

and subcutaneous hemorrhage in 1 of the 3 males. The dermal irritation NOAEL cannot be 

determined.

Dermal Absorption Rat. In a dermal absorption study (MRID 42133501) groups of male 

Sprague-Dawley rats were exposed to a single dermal dose of 1 .0 mg/kg 14-C amitraz for 24 or 

120 hours. Urine and feces were collected up to 120 hours post dosing. Elimination of 14-C 

amitraz derived radioactivity as a percentage of the administered dose was minor via urine and 

feces, with the majority of the dose recovered at the dosing site. Metabolism appeared similar 

from urine analyzed at 24 hours from dermally and orally exposed rats, but data were 

inconclusive. Dose-dependency of absorption was not addressed. Levels of Amitraz derived

radioactivity in the carcass appeared low at 120 hours. The mean percent of dose absorbed at the 

treatment site was 2.98% at 24 hours and 1.41% at 120 hours; total absorbed was 3.69% at 24 

hours and 6.56% at 120 hours; total was 6.67% at 24 hours and 7.79% at 120 hours.

Inhalation Studies.

A. Human Studies
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None. 

B. Animal Studies

Inhalation Rat. In a subchronic inhalation toxicity study (MRID 00029964) [amitraz, (% a.i., 

batch/lot # not provided)] was administered to 6 CFHB rats/sex/concentration by dynamic whole 

body exposure at nominal concentrations of 0, 1.8, 18, 180 mg/kg/day (dust) for 6 hours per day 

for 14 days over a period of 3 weeks. Analytical concentrations were not measured.

No treatment-related effects were observed at 1.8 mg/kg/day. A decrease in body weight gain 

was observed in females (66% of the control group). No statistical data or standard deviations 

were provided. An examination of the individual animal data does not indicate any significant 

outliers; however, the mean body weight was 94% of the control group and no other parameters 

indicated any effects at this concentration level.

At 18 mg/kg/day and above, the animals showed signs of mild dyspnea with occasional 

sneezing; intermittent blinking and licking of lips; hyposensitivity to noise; hypersensitivity to 

touch and aggressive behavior. Decreases in body weight gain were observed in both sexes (54 

and 49% of control for males and females, respectively). 

At 180 mg/kg/day, dyspnea and eye irritation were more severe. Labored movements, increased 

nasal secretion and polyuria with brown discoloration of the urine were noted during exposures. 

In several rats, slight coma and body tremors were observed following exposure. Decreases in 

body weight and body weight gain were observed in both sexes when compared to the control 

group. The rats lost weight at this dose level. There was a significant reduction of packed cell 

volume (hematocrit), hemoglobin and erythrocyte count and an increase in the number of 

neutrophils in both sexes. A low mean corpuscular hemoglobin content and decrease in the 

number of lymphocytes was found in the blood of male rats. There was also a significant 

reduction in total protein levels in both sexes. In males, a low albumin concentration and a 

decreased albumin/globulin ratio were observed. In females, a low urea level and a fall in the 

alpha and beta fractions of globulins were noted. 

No treatment-related macroscopic or microscopic lesions were observed at any concentration 

level. The LOAEL is 18 mg/kg/day, based on clinical signs of toxicity (irritation and 

neurological signs) and decreases in body weight and body weight gain. The NOAEL is 1.8 

mg/kg/day.

Metabolism Studies

A. Human (MRID 00160964, 46249601).

In a metabolism study (MRIDs 00160964 and 46249601) radiolabeled Amitraz (1,5-di(14C-2,4-

dimethylphenyl)-3-methyl-1,3,5-triazapenta-1,4-diene) purity >95%, Batch #s REM-XLVI-85 

and VA) was orally administered to rats, mice, baboons and humans. Rats and mice were given 

1, 10, or 100 mg/kg [14C]-Amitraz at 1, 10 or 100 mg/kg body weight in corn oil by gavage. In 

addition, another group of mice were pretreated with 15 mg/kg/day and 60 mg/kg/day amitraz in 

the diet for six weeks before being given a single gavage dose of 10 mg/kg of [14C]-amitraz. 
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Baboons were given 10 mg/kg [14C]-amitraz by gavage while human volunteers received a single 

0.25 mg/kg dose of [14C]-amitraz by capsule. 

Table A1.5 Dosing Groups for Oral Amitraz Metabolism Studies

Species Dose of Labeled Amitraz (mg/kg) Number/Gender

Rat* 1, 10, 100 2-3 M and 1-3 F

Mouse (naïve only)* 1, 10, 100 0-4 M, 1-4 F

Baboon 10 1 M, 1 F

Human 0.25 2M

* Number of animals varied by dose group and experiment (two different experiments were run in mice 

and rats). In addition, the separate mouse experiment that was pre-treated is not reported here.

Humans appear to be the most sensitive species to the neurological effects of amitraz as shown 

by dry mouth, drowsiness, disorientation, decreased temperature, bradycardia, and slightly pale 

appearance after treatment with 0.25 mg/kg persisting up to 12 hours after dosing. These effects 

were not found in rats, mice, or baboons, but, subjective effects would be difficult to evaluate in 

animal species. Following treatment, the majority of the radiolabel was excreted in the urine, 

with 54.9-73.7%, excreted within 24 hours of treatment. In further studies on human urine 

samples, ~43.5% of the dose was excreted within 12 hours, with 62.9% excreted within 24 hours. 

No major differences in the amount of radiolabel excreted between sexes or species were noted. 

The overall metabolic profile was similar across sex and species. Polar metabolites, likely 

representing conjugates (type not reported), were the predominant metabolites identified, 

contributing 52-64% of the total with BTS 39 098/BTS 31 158 contributing 15-27%. 

The average proportion of metabolites identified in the urine following a single 1, 10, or 100 

mg/kg oral dose of radiolabeled amitraz was similar in rats and mice at doses of 1 mg/kg and 10 

mg/kg. When the dose was increased to 100 mg/kg, the percentage of polar metabolites 

decreased for both rats and mice and the metabolite BTS 27 271 increased. This suggests 

metabolic saturation and shifting in the metabolic pathway. In rats and mice pretreated with 100 

ppm amitraz in the diet for three weeks followed by three weeks of 400 ppm amitraz in the diet, 

no effect on the relative concentrations of the metabolites were found. 

This study is a metabolism study. The test chemical was radiolabeled, so the purity is > 95%. 

Only 2 males were tested. Potential confounding factors (i.e. medications, alcohol or nicotine 

use) were not discussed in the study. No control group was in the design and no statistical 

analysis was conducted. Since similar effects were observed in both human subjects, it is 

possible to set a LOAEL using this study for the purposes of risk assessment. 

B. Animal

See A. above.
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Appendix B. Physical/Chemical Properties

Table B.1.1 Physicochemical Properties of Amitraz.

Parameter Value Reference

Melting point/range 86-87 C Amitraz RED, 03/95

pH of 1% aqueous suspension N/A (low solubility; decomposes in water) Amitraz RED, 03/95

Density or specific gravity 1.128 g/mL at 20 C Amitraz RED, 03/95

Water solubility <1 ppm at 20-25 C Amitraz RED, 03/95

Solvent solubility At 20-25 C

xylene 66.6 g/100 mL

acetone 50.0 g/100 mL

methanol 2.38 g/100 mL

Amitraz RED, 03/95

Vapor pressure 3.4 x 10-4

Pa at 25 C Amitraz RED, 03/95

Octanol/water partition coefficient (Kow) 3.0 x 105

at 25 C (pH 5.8) CBRS No. 3975, 7/21/88, H. Fonouni.
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Appendix C. Review of Human Research

The amitraz residential risk assessment relies in part on data from studies in which adult human 

subjects were intentionally exposed to a pesticide or other chemical. These data, which are 

subject to ethics review pursuant to 40 CFR 26, have received that review, and are compliant 

with applicable ethics requirements. For certain studies that review may have included review 

by the EPA Human Studies Review Board (HSRB). Descriptions of data sources as well as 

guidance on their use can be found at http://www.epa.gov/pesticides/science/handler-exposuredata.html and http://www.epa.gov/pesticides/science/post-app-exposure-data.html.

An acute oral human study and a human metabolism study were used in assessing toxicological 

points of departure for the amitraz risk assessment (MRIDs 43283101, 00160964). These studies 

were reviewed by EPA (J. Carley, 3/06) and the HSRB (4/06) and determined to be ethically 

acceptable.
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Appendix D. Summary of Amitraz US and International Tolerances and Maximum 

Residue Limits (MRLs)

Table D.1.1 Summary of Amitraz US and International Tolerances and Maximum Residue 

Limits (MRLs)

US Canada Mexico2 Codex

Residue Definition:

40 CFR 180.287

Amitraz (N'-[2,4-

dimethylphenyl]-N-[[(2,4-

dimethylphenyl)imino]methyl]]-

N-methylmethanimidamide) 

and its metabolites containing 

the 2,4-dimethylaniline moiety 

calculated as the parent

N,N-bis(2,4-

xylyliminomethyl)methylamine, 

including the metabolite N-(2,4-

dimethylphenyl)-N'-methyl 

formamidine

amitraz Sum of amitraz and 

N-(2,4-

dimethylphenyl)-

N’-

methylformamidine 

calculated as N-

(2,4-

dimethylphenyl)-

N’-

methylformamidine.

Commodity Tolerance (ppm) /Maximum Residue Limit (mg/kg)

Commodity US Canada Mexico2 Codex

Cattle, fat 0.1

Cattle, meat 0.02 0.053

Cattle, meat 

byproducts 0.2 0.2 (kidney; liver) 0.23

Cotton, undelinted 

seed1 1.0 1 0.5

Hog, fat 0.1

Hog, kidney 0.1 0.2 0.2 (edible offal)3

Hog, liver 0.1 0.2 0.2 (edible offal)3

Hog, meat 0.05 0.053

Hog, meat 

byproducts 0.3

Milk 0.03 0.01 (*)3

Milk, fat 0.2

Pear 0.5 (pome fruit)

Cherries 0.5

Cucumber 0.5

Oranges 0.5

Peach 0.5

Sheep meat 0.13

Tomato 0.5

Apples 0.5

There are no US registrations on cottonseed as of May 3, 2006.

Available information for Mexico is dated (2004). Mexico adopts US tolerances or Codex MRLs for 

export purposes.

The MRL accommodates external animal treatment.