Amitraz Human Health Risk Assessment DP No.D407721

t l NIT E D STAT ES ENVIR ON MRNTAL PROT ECTI ON AG EN CY
WASHINGTON, I>.C. 204()(}

MEMORANDUM

Date: Janua ry 8

, 2013 O FFI CE OF
CHEMICAL SA FETY AND
POLLUTION PREVEN TION

SUBJECT: Amit r az. Aggregate Hum an Hea l th Risk Assessment for Sect i on 3 New Use i n Beehives

.

PC Code : I 0620 I

Decisi on No.: NA

Petition No.: 9F7673 DP Barc ode: 0407721

R egistration No.: 87243- R Regulatory Action: Section 3

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

TX RN o.: NA CAS No.: 33089-61-1

40 CFR: 1 80.287 MRIDN o.: NA

FROM:

THROUGH:

TO:

and Danett e Drew

, Chemist/Ri s k Assessot_ 

### 'u4_**~** Wade Bri tton

, MPH, Indu stria l Hygienist 

## /P' (l'fJ

_ _

_ -
William Ir win, PhD, DABT, Toxicologist /, 

### r**~** 

# OrJ:

_ Q...

### ~ Risk Assessme n t Branch V**W~-7** 

### / 1 

### J/

r>-. Health Effects Division (7509P) ·'**~** v 1 

## L

Office of Pesticide Program s

Mic h ael Metzger, Branch

# Chief**~~~ ~**  () 

# k[

-

Risk Assessment Branch V / VII / · Hea lth Effects Division (7509P)

Office of Pesticide Program s

Autumn Metzge r
, CRM

Insecticid e Rodenticide Branch

Stacey Groce, CRM

Risk In tegration, Minor Use & Emer gency Response Branch Reg i stration Division (7505P)

Office of Pesticide Program s

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

beehives.

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

Page 2 of 48 

### 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 Databas e

................................ ................ 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

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

Page 3 of 48 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)

................................ ................................ ................................ .............. 48Amitraz Human Health Risk Assessment DP No.D407721

Page 4 of 48 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 as sessment 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 (D 406097) . Th e exposure assumption s 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. A n E xtended 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

. T he 10X FQPA Safety Factor is retained in the form of a data base Uncertainty Factor (UF DB ) to account for the

lack of the EO G RT 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 sys tem (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 human s 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 0.0 0125 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 FQP A fac tor for the data gap (EOGRT study) and 1X for

the inter

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

- s pecies factor is

reduced to 1X. A m argin of e xposure (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 ap proach ( 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 doseAmitraz Human Health Risk Assessment DP No.D407721

Page 5 of 48 (cPAD) to protect for cancer concerns wh en 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 a nalysis 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 thro ugh the current and proposed uses. The acute dietary (food o nly)

exposures estimates are below HED’s level of concern.

There are no residential (non

- occupational) exposures expected from the beehive use so a non

occupational risk assessment is not requ ired 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 documen t 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 exposure s 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

.

Occupa tional (d ermal 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 recommend s tolerance s 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 D 406097 , 10/18/12 ( ID#:10SD04 Updated Section 18 Emergen cy Exemption for the Use of Amitraz in Beehives in South Dakota ).

The Agency has determined that an Extended One

- Generation Reproduction Toxicity (EOGRT)Amitraz Human Health Risk Assessment DP No.D407721

Page 6 of 48 study is required to evaluate the reproductive, neuro

- and immuno

- toxic potential of amitraz.

The E OGRT 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 exposu re on the developing nervous system.
Cohort 3: assesses the potential impact of chemical exposure on the developing immune system.

T he 10X FQPA Safety Factor is retained in this assessment in the form of a data base Uncertainty Factor (UF DB ) to account for the lack of the EO G RT study

.

The Agency has also determined that a “Triple p ack” 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 viv o 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 i s 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 t he 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 recommend s tolerance s for residues of total amitraz of 0.2 ppm in honey and 9 ppm in honeycomb for the proposed S ection 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)

, includin g 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 convertibleAmitraz Human Health Risk Assessment DP No.D407721

Page 7 of 48 to 2,4

- dimethylaniline, expressed as the stoichiom etric 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 Econ omical Cooperation and Development (OECD) calculation procedure using the field trial data points from the 1 5 day pre

harvest interval ( PHI ) field tria l 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 ) f or 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 In troduction

3.1 Chemical Ide ntity

Table 3.1 Amitraz Nomenclature

Chemical structure

Common name AmitrazAmitraz Human Health Risk Assessment DP No.D407721

Page 8 of 48 Table 3.1 Amitraz Nomenclature

Molecular Formula C 19 H 23 N 3 Molecular Weight 293.42

IUPAC name N

- methylbis(2,4

- xylyliminomethyl)amine

CAS name N'

- (2,4

- dimethylphenyl)

- N

- [[(2,4

- dimethylphenyl)imino]methyl]

- N

methylmethanimidamide

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 Patte rn

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 v arroa 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. S trips 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.Amitraz Human Health Risk Assessment DP No.D407721

Page 9 of 48 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 post

application dermal or inhalation exposure. Dermal exposure resulting from use of these

pre packaged 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 ha s 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 (no n

- 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 (ha ndling) 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 post

application exposure to treated dogs, a dermal assessment was performed for adul ts 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 Populatio ns 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 subgrou ps 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 assessmen ts 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 andAmitraz Human Health Risk Assessment DP No.D407721

Page 10 of 48 for toddlers, youths, and adults entering or playing on treated a reas 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 lif estyle 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, DP 406097 ).

4.1 Summary of Toxicological Effects

Multiple studies in various mammal ian 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

p aralysis 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 in halation 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. Multipl e 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 sy stem (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

excitabi lity 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 huma n 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 stud ies, 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

- gen eration 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 wasAmitraz Human Health Risk Assessment DP No.D407721

Page 11 of 48 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 Potent ial” 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 carcinogen ic 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 amitra z 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 foun d 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 tha t 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 dose s 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 w ere 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 (pu lse, 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, a nd psychomotor performance wereAmitraz Human Health Risk Assessment DP No.D407721

Page 12 of 48 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 me tabolism 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

- formamid o

- 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

benzo ic 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 huma n 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)

T he proposed and exi sting use patterns for amitraz may result in dietary and residential exp osure 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 Sa fety Factor ( for the protection of infants and children ) be retained in the form of a data base uncertainty factor (UF DB ), due to multiple toxicology data deficiencies for amitraz (i.e. reproduction, immunotoxicity, and DNT studies). An Extended One

- Gener ation 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, EP A 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 i n 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; r ete ntion 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 sensitiv ity in the young of no greater than 4X.

4.2.1 Completeness of the Toxicology DatabaseAmitraz Human Health Risk Assessment DP No.D407721

Page 13 of 48 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 t he EOGRTS will contain a DNT component and other studies have demo nstrated 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 d ata base uncertainty factor (UF DB ) 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). Th e 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 b ecause 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 dos e 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 con tain specific neurotoxicity studies. The dietary and residential assessments are based on reliable exposure data and will not under

estimate exposure.

The Agency has determined that an Extended One

- Generation Reproduction Toxicity (EOGRT) study is requir ed to evaluate the reproductive, neuro

- and immuno

- toxic potential of amitraz. The EOGRT study will provide comprehensive evaluation of three cohorts of F 1 animals:

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

an F 2 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.Amitraz Human Health Risk Assessment DP No.D407721

Page 14 of 48 T he Agency has retained the 10X FQPA Safety Factor in the form a data base uncertainty factor (UF DB ) 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 ex posure 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 co llars 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, represe nts 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 h ave 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 a nd 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 i ntra

- 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

. A n 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 adequat e 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 sk in (Bartek et al., 1972). OPP has reviewed a number of studies comparing dermal absorption in rats ( in vivo and in vitro methods)Amitraz Human Health Risk Assessment DP No.D407721

Page 15 of 48 and in vitro studies with human skin. These studies suggest that rat skin is typically at least 2.5 times more permeable th an 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 subje cts 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 = 100 1

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 cross over study in human subjects.

LOAEL = 0.25 mg/kg based on dry mouth, drowsiness, decreased temperature, decreased blood pressure and decreased heart rate.Amitraz Human Health Risk Assessment DP No.D407721

Page 16 of 48 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 d uration. b= UF of 100 (10x for intra

- species variation, and 10x FQPA factor for data gap for EOGRT study and 1x for inter

species extrapolation). Since the PODs used are from studies conducted in human subjects, the inter

- species extrapolation factor is r educed 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, lemon s, citrus, cotton, and pears, and

animal metabolism studies with cattle an d 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. T he 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 assess ment.

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 L ifescience 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 withAmitraz Human Health Risk Assessment DP No.D407721

Page 17 of 48 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 t wo 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) /a ddition 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 f o r 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 Stri ps Proposed Label Data 2

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

S torage 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 r esidues of amitraz are stable in cottonseed for 13.5 months, citrus for 18 months and in animal muscle, fat, and milk forAmitraz Human Health Risk Assessment DP No.D407721

Page 18 of 48 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 potentia l 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 b ased 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 a mitraz 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 hone y the residues at study day 57

, i.e. 1 5 days after treatment (DAT), from MRID

48341001 w ere 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 a ny 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 99.9 th percentile of exposu re. The general population is estimated to be exposed at 0.000199 mgs/kg/day, or 16 % of the aPAD.Amitraz Human Health Risk Assessment DP No.D407721

Page 19 of 48 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 (de rmal 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 suggest s 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.

An assessment of the amitraz

- impregnated pet collar residentia l 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) Re sidenti a l 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 assessme nt 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 appear s early after dosing and reverse s rapidly; in humans theAmitraz Human Health Risk Assessment DP No.D407721

Page 20 of 48 effects were reversed within 12 hours (acute bolus dose). (See detailed discuss ion in Section

3.0 of this document .)

3) Fur Residue Transfer Data: In the 2006 assessment a carbaryl pet col lar 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 cons istent 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 carbar yl 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 su bmitted residue transfer study for a different chemical to refine residential post

application exposure/risk estimates. This exposure study, which was done for a registered propoxur pet collar (MRID 48589901), us es 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

- en d default fraction application rate (F AR ) transferred recommended (2%) in the updated 2012 Residential SOPs when chemical

- specific data are unavailable. The F AR from the propoxur study is 0.1%. The default F AR relies on exposure data from multiple, liqui d 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 indi vidual than that of pet collars which are designed for slow release over time; as a result, the use of the

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

It is unclear, however, exactly what effect differing ch emicals 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 lub ricants 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 com pounds. 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 bin d 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.Amitraz Human Health Risk Assessment DP No.D407721

Page 21 of 48 6.1 Residential Handler Exposure

- Pet Collar

Residential hand ler 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 submis sion 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 spot

on applications (MRID 44433303) as a surrogate to esti mate 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). T his 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 produ ct 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 a re not of concern MOEs>100) for all exposure durations.Amitraz Human Health Risk Assessment DP No.D407721

Page 22 of 48 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 ris ks (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 t heir 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

- speci fic 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 conduct ed 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 co llar exposure assessment and offers a considerable refinement from the high

- end default F AR recommendation; the default F AR 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

Scenario 1 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

MOE 5 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 480Amitraz Human Health Risk Assessment DP No.D407721

Page 23 of 48 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

F aihands DE SA H HR F

m ET Replen

ishment

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/cm 2 ) Fraction

of hand

mouthed Exposure

Time (hours/ day) # Replen

ishment 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 390Amitraz Human Health Risk Assessment DP No.D407721

Page 24 of 48 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 390Amitraz Human Health Risk Assessment DP No.D407721

Page 25 of 48 6.3 Risk Characterization of Resi dential 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

fract ion 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 assess ment 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 int o 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 a pproach 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 t o 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 correspond s 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 (F AR ) is used in conjunction with the application rate to estimate TR. Based on recommendation of the Treated Pet SOP, a 2% screeni ng level F AR 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., aero sol 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 p erformed 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 d etermined. Of the 8 pet residue transfer studies used to establish

F AR , 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 un ique. 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. F or example, the labeled amount of a spot

- on product can be used directly in the assessment of exposure/riskAmitraz 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 re lease 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 majo rity 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 its elf 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 ing redient 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 wil l 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 upo n 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 do g and caring for pe ts 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 ti me 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 assu med 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 t o treated pets, transfer coefficients (TCs) can be defined as animal surface area contact per unit time (cm 2 /hr). The transfer coefficient used to assess exposure to a dog treated with the amitraz collar was derived from a study representing application a nd 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, th e 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

Page 27 of 48 6.4 Residential Bystander Post

- applicatio n 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 (ad d) 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 P AD), 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 c umulative, 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 assessmen t 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, a n aggregate MOE of 100 or greater does not exceed HED’s level o f

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 resul t in higher estimated exposure levels and

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

assessment, r esidential 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 95 th percentile of exposure. While aggregation using an average background exposure would more appropriately reflect expected exposures, in the absence of a

chronic diet ary assessment, use of acute exposures at the 95 th 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.Amitraz Human Health Risk Assessment DP No.D407721

Page 28 of 48 7.1 Aggregate Risk (All Durations)

For adu lts, 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 (1 to < 2 yrs ), c ombining 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 t o Medium Dog Collar plus Dietary)

Population All Durations

NOAEL mg/kg/day LOC 1 Max

Allowable Exposure 2
mg/kg/day 95 th

Percentile

Food Exposure mg/kg/day Residential

Exposure mg/kg/day 3 Total

Exposure mg/kg/day 4 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 = 95 th Percentile Food Exposure + Residential Exposure. (Soderberg, D 383216)

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 t oxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has not

assumed that amitraz h as a common mechanism of toxicity with other substances. For information regarding EPA’s efforts to determine which chemic als 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 post

appl ication dermal or inhalation exposure. The Apivar beehive s trips 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 late x gloves by applicators which will further reduce the potential for exposure . After six weeks, at the time of disposal, residues have significantlyAmitraz 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 neg ligible potential for inhalation exposure, at any time, to the applicator or during post

application activities. Because of the very low potential for dermal or inhalation exposure, quantitative occupational risk assessments are not required for this prop osed 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 Sout h Dakota, D 406097.

W. Britton, 10/11/12, Amitraz: O ccupational 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, D 383216

.

W. Irwin et al., 3/10/10, Amitraz: Human Health Assessment Sc oping Document in Support of Registration Review, D367925.

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

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

D 310227

.

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 58 35 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 , "Kinetic s and equilibrium of sol ute diffusion into human hair", Ann Biomed Eng, July 2012 (Epub ahead of print)Amitraz Human Health Risk Assessment DP No.D407721

Page 30 of 48 A ppendix 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

yes d

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 assessm ent. 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) studyAmitraz Human Health Risk Assessment DP No.D407721

Page 31 of 48 A.2 Toxicity Profile s

Table A.2.1 Acute T oxicity Data on AMITRAZ Technical

Guideline No./ Study Type MRID No. Results Toxicity Category

870.1100 Acute oral toxicity 00041539 LD 50 : 531 mg/kg (M) 515 mg/kg (F) III

870.1200 Acute dermal toxicity 00040862 LD 50 : > 200 mg/kg II

870.1300 Acute inhalation toxicity 00029963 LC 50 : 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 mater ial not reported and reporting incomplete in terms of the study protocol and environmental conditions. The particle size distribution was acceptable, thus theAmitraz Human Health Risk Assessment DP No.D407721

Page 32 of 48 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 Toxicity

Rat 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 Toxicity

Rabbit (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 post

implantation loss, abortions and decreased mean fetal body weight. Too few litters, limited data, unclear method of dosing.

870.3700 MRID

4 4265901 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 ToxicityAmitraz Human Health Risk Assessment DP No.D407721

Page 33 of 48 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 dose

related incidence of hyperplastic nodules, liver foci, stomach hyperkeratosis and spleen hematopoiesis. Evidence of carcinogenicity: hepatocellular adenomas, carcinomas and combined; and lung
adenom as, probably at dose levels above MTD.

Neurotoxicity

870.6200a NA Acute Neurotoxicity

- Rat Waived by HASPOC

NAAmitraz Human Health Risk Assessment DP No.D407721

Page 34 of 48 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

- meth yl 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 ac id.

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. High est 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. Degradatio n products of amitraz and its metabolite,
BTS 27271 were similar.

Dermal PenetrationAmitraz Human Health Risk Assessment DP No.D407721

Page 35 of 48 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

Non

Guideline 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.

Non

Guideline Human Study MRID

00160964

46249601 Metabolism Study LOAEL = 0.25 mg/kg

Non

Guideline Human Study MRID

44639401 Acute Dermal Toxicity NOAEL = 24 mg/kg (no LOAEL)

Non

Guideline 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 withdr awn 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 Inde pendent Ethics Committee and done according to the Helsink i 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 an d blood for clinical chemistry and hematology was collected prior to the start of the study and 36 hours after each phase of the study.Amitraz Human Health Risk Assessment DP No.D407721

Page 36 of 48 Table A1.4 Dosing Schedule for Amitraz Single Dose Human Study 1

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 respon se. 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 stud y. 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 p harmacology 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 t aken 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 s tudy. 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

gavag e 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 mea surements 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 post

dosing 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 40 0 mg/kg.Amitraz Human Health Risk Assessment DP No.D407721

Page 37 of 48 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,

pros tration, 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 hand led. 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 nigh t. 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 sev en 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

dose s 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 b lood 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 appetitesAmitraz Human Health Risk Assessment DP No.D407721

Page 38 of 48 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

tem perature were observed at both 100 and 250 mg/kg, although only once in one animal at 100 mg/kg. The report stated that A the 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 sign s, 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 appe ars 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; howeve r, 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 dur ing 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 NO AEL 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,Amitraz Human Health Risk Assessment DP No.D407721

Page 39 of 48 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 obse rved 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 tr eatment

- 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

classi fied 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 me an 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. Th e 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 effec ts 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 s ucceeding 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/kgAmitraz Human Health Risk Assessment DP No.D407721

Page 40 of 48 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 i n 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 m g/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 requ irement 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 we re 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 dos e 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 conduc ted.

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 e xtent 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 o f 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 c hronic / carcinogenicity study (MRID 00044585) BTS 27 419 (97.8

- 99.8 % a.i, batch/lot #’s 2093DH, 2099DH) was administered to 40 Ash

- Wistar SPFAmitraz Human Health Risk Assessment DP No.D407721

Page 41 of 48 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, r espectively) 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 tumo rs 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 treatment

related 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 carcinogen icity 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 44.6 5/50.13 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 sto mach 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 lev el (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 dur ing 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 m ales 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 inAmitraz Human Health Risk Assessment DP No.D407721

Page 42 of 48 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 tr end 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 pharmacologi cal 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 pharmacologi cal activity (sedation, hypotension and effects on body temperature in humans foll owing 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 sign ificant 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 treatment

related effects w ere 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 bat ch/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.Amitraz Human Health Risk Assessment DP No.D407721

Page 43 of 48 At 200 mg/kg, sedation was observed in both sexes, starting afte r 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. Th ree 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 3 rd or 4 th dos e. 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 co ncurrent 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 ma y 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 s tudy (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. Do se

- 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 StudiesAmitraz Human Health Risk Assessment DP No.D407721

Page 44 of 48 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 individua l 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 dys pnea 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 wer e 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), he moglobin 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 macros copic 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.

Metab olism Studies

A. Human (MRID 00160964, 46249601). In a metabolism study (MRIDs 00160964 and 46249601) radio labeled Amitraz (1,5

- di( 14 C

- 2,4

- dimethylphenyl)

- 3

- methyl

- 1,3,5

- triazapenta

- 1,4

- diene) purity >95%, Batch #s REM

- XLVI

- 85 and VA) was orally administ ered to rats, mice, baboons and humans. Rats and mice were given 1, 10, or 100 mg/kg [ 14 C]

- 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

t he diet for six weeks before being given a single gavage dose of 10 mg/kg of [ 14 C]

- amitraz.Amitraz Human Health Risk Assessment DP No.D407721

Page 45 of 48 Baboons were given 10 mg/kg [ 14 C]

- amitraz by gavage while human volunteers received a single 0.25 mg/kg dose of [ 14 C]

- amitraz by capsule.

Table A1.5 Dosing Gro ups 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 ho urs 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 w ithin 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 radio labeled 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 wee ks 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

p ossible to set a LOAEL using this study for the purposes of risk assessment.

B. Animal

See A. above.Amitraz Human Health Risk Assessment DP No.D407721

Page 46 of 48 Appen dix 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 (K

ow ) 3.0 x 10 5 at 25 C (pH 5.8) CBRS No. 3975, 7/21/88, H. Fonouni.Amitraz Human Health Risk Assessment DP No.D407721

Page 47 of 48 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 compli ant
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

- exposure

data.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

w ere reviewed by EPA (J. Carley, 3/06) and the HSRB (4/06) and determined to be ethically acceptable.Amitraz Human Health Risk Assessment DP No.D407721

Page 48 of 48 Appendix D

. Summary of Amitraz US and International Tolerances and Maximum Residue Limits (MRLs)

Table D.1.1 Summary of Amitraz US and International To lerances and Maximum Residue Limits (MRLs)

US Canada Mexico 2 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 Mexico 2 Codex
Cattle, fat 0.1
Cattle, meat 0.02 0.05 3

Cattle, meat byproducts 0.2 0.2 (kidney; liver) 0.2 3

Cotton, undelinted

seed 1 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 0.05 3

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 0.1 3

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.