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June 1, 2010, regarding the insecticide amitraz. Specifically, the correspondence from PCRM 

questions some of the requirements for animal testing for amitraz (Registration Review docket 

for Amitraz; Docket ID EPA-HQ-OPP-2009-1015), as listed below. 

The Agency appreciates the comments of PCRM and has reviewed the suggestions with careful 

consideration (PCRM comments listed below followed by the Agency’s response). 

Comment 1: 

The Summary Document indicates that the Agency may request a series of animal 

tests. However, we believe data may already exist either with the technical registrants or other 

sources that could fulfill some of the Agency’s information needs. We provide 

some references and encourage the Agency to further explore other areas so as to 

minimize costs as well as duplicative testing that will cause suffering and death to 

hundreds of animals. 

• (GLN 870.3100) Sub-chronic (90-day) Oral Toxicity Study in Rats 

Response 1: 

Agency: Thank you for your comment. The Agency is committed to reduce, refine and 

ultimately replace animals for toxicity testing where practicable. Toward that goal, the 

Agency is asking that the toxicity parameters that would normally be included in a 

separate sub-chronic oral study be included and covered by an extended one-generation 

toxicity study protocol. This will save animals and still provide the necessary insight 

sought from both study types. The Agency has looked at your references and others for 

areas where additional efficiencies might be rendered; however for the toxicity effects of 

concern, the Agency believes that the animal tests currently requested are necessary to 

meet our obligations to ensure the safety of pesticides under FIFRA, FFDCA and FQPA. 

Comment 2: 

We recommend that the technical registrants find the information from the previous study 

including the necessary individual animal data, in lieu of conducting another study especially 

since the purpose of this study will be to remove the uncertainty factor that is providing 

additional protection. Considering that humans are the most sensitive species tested (according to 

the Human Health scoping document) this should only provide more of a reason to retain 

uncertainty factor. 

We also encourage the company to combine this study with the sub-chronic 

neurotoxicity study (GLN 870.6200) as the Agency recommends. 

In advance of performing these dermal repeated-dose studies, we urge the Agency and 

the registrants to consider whether extrapolating from oral toxicity value is possible, 

given the current knowledge of the chemical’s dermal absorption factor and local 

effects. The Agency should consider requesting in vitro dermal penetration testing (see 

OECD Test Guideline 428 for example) to confirm this estimate of the extent of dermal 
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penetration likely for Amitraz, instead of in vivo dermal penetration. Results can also be 

confirmed using bridging data from other appropriate organotins. 

Finally, we are wondering why the requirements for additional dermal data exist if according to 

the Summary Document, “Based on the 2.4 g ai pet collar use, all 

residential exposure scenarios, including toddler dermal and incidental oral (hand-to-mouth) 

exposures (MOE of 97), were not of concern to the Agency.” 

Conducting the dermal toxicity studies listed would have serious repercussions for the 

welfare of animals, and flies in the face of OPP’s move toward more thoughtful data 

requirements. 

• (GLN 870.3200) 21/28-Day Dermal Toxicity 

• (GLN 870.3250) Sub-chronic (90-day) Dermal Toxicity 

Response 2: 

Agency: As noted in our response to the first comment the Agency is committed to 

exploring ways to reduce, refine and ultimately replace the use of animals for toxicity 

testing. The Agency has launched efforts to do this by our strategic direction for new 

pesticide testing and assessment approaches (www.epa.gov/pesticides/science/testingassessment.html). And while the Agency is moving in that direction, at the current time the 

Agency must follow 40 CFR part 158 that requires these animal studies to fully evaluate 

the hazards of chemicals to humans, especially the young, depending on the human 

exposure scenarios. Currently, Agency policy is not to accept in vitro dermal absorption 

data without in vivo supporting data, based on the NAFTA Dermal Absorption Group 

Position Paper. Studies tailored to human exposure scenarios are necessary because routeto-route extrapolation from oral exposure may not provide data which are protective to 

humans. In some instances, toxicity from dermal absorption of a chemical is more toxic 

than from oral absorption, due to liver detoxification of oral chemical exposure; a 

detoxification system which is bypassed via dermal exposure. Regarding an attempt to 

build on previous studies, the studies must be for related compounds. Amitraz is not 

related to organotins and therefore, such studies cannot be bridged. The Agency would 

also note that several amitraz products are marketed to be applied dermally to animals, 

thus the animal testing can also serve to protect many more animals (livestock and pets) 

than those animals utilized in the safety assessments. 

Comment 3: 

Using the following link, under the “Animal Toxicity Studies” tab we found that Hayes, 

W.J. et al i

 summarized many studies for Amitraz including a 21-day dermal study in 

rabbits, short-term inhalation in rats, 90-day repeat dose in dogs, and 90-day repeat 

dose in rats. We implore the Agency to further invest these references. 

http://toxnet.nlm.nih.gov/cgi-bin/sis/search/r?dbs+hsdb:@term+@na+AMITRAZ

• (GLN 870.3465) Sub-chronic (90-day/28-day) Inhalation Toxicity 

 

Response 3: 
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Agency: Thank you for your suggestion. The Agency looked at these studies and found 

them useful but not sufficient for our need to evaluate all important human exposure 

scenarios. The studies described by Hayes were not sufficient to cover the human exposure 

scenarios relevant to amitraz use. For example current amitraz exposure scenarios require 

sub-chronic inhalation studies due to the use of spray wands for product application to 

animals, which can generate mists capable of being inhaled for intermediate-term 

durations of exposure. Studies by the relevant route of exposure and for the relevant 

exposure duration are important, e.g., inhalation exposure bypasses liver detoxification 

processes and is potentially more toxic than oral exposures. Furthermore, new studies to 

be conducted after the 1991 Hayes et al book reflect the most current advances in scientific 

testing methods, thus potentially providing more protective endpoints to humans. 

Comment 4: 

Hayes et al ii summarizes a pre-natal developmental toxicity in rats and rabbits. Study 

authors found that “rats who received 12 mg/day of Amitraz from day one of pregnancy 

until the young were weaned at 21 days old had a reduced number of young born and 

alive at day four.” They concluded that, “although there have been reproductive effects 

observed in laboratory animals at some dose levels, likely human exposures are very 

much less than those which produced effects. These effects are unlikely in humans 

under normal circumstances.” A more in-depth summary can be found here: 

http://pmep.cce.cornell.edu/profiles/extoxnet/24d-captan/amitraz-ext.html. 

Additionally, the following three studies could be used to fulfill the data requirements. 

The effects of prenatal Amitraz exposure on the physical and behavioral parameters of 

rats were studied.iii Then a follow up study was conducted to determine the effects of 

postnatal Amitraz exposure on physical and behavioral parameters in rats, whose 

lactating dams received the pesticide (10 mg/kg) orally.iv The final study investigated the 

potential adverse effects of Amitraz on the initiation and maintenance of pregnancy in 

Sprague-Dawley rats as well as its effects on embryo-fetal development after maternal 

exposure during the entire pregnancy period.v

 

• (GLN 870.6200a) Neurotoxicity Battery (acute studies) 

• (GLN 870.6200b) Neurotoxicity Battery (sub-chronic studies) 

• GLN 870.3700) Pre-Natal Developmental Toxicities – Rabbit 

Response 4: 

Agency: Again, thank you for your thoughtful suggestions. They are consistent with the 

practice of the Agency to take exposure scenarios into account in our risk assessments and 

in designing toxicity studies to inform our risk assessments. Because amitraz exhibits 

clinical signs of neurotoxicity and sedation, the Agency concludes that the current studies 

are insufficient and that, for instance, additional detailed neurotoxicity information beyond 

the behavioral study is necessary to fully evaluate the hazards of amitraz to humans and 

ensure safety. These neurotoxicity studies will be included in the comprehensive extended 

one-generation protocol which makes more efficient use of animals. 
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Comment 5: 

As previously stated we encourage the registrants to combine the (870.3100) 90-Day 

Oral Toxicity study in rats and the sub-chronic neurotoxicity study listed, if conducted. 

A GHS classification document classifies Amitraz into a category 2 CNS toxicant.vi

“Specific target organs/systemic toxicity following single exposure” and “specific target 

organs/systemic toxicity following repeated exposure” based on data in animals and 

humans. The agency should examine these studies to ensure that they will not meet the 

agencies requirements for this endpoint. 

Additionally, a series of studies investigating the possible neurotoxicity of Amitraz was 

conducted on Long-Evans rats following acute and short-term repeated exposures to 

the pesticide.vii Part four of the series assessed the toxicity syndrome using a functional 

observational battery in rats.viii

We acknowledge that the two-generation reproduction and health fertility effects study is 

not listed on the Summary Document but is listed in the Human Health Scoping 

Document and wanted to clarify what the agency is requesting regarding this matter. We are 

happy to see that the registrants will include protocols for a modified one-generation 

reproduction study. Once the study is confirmed it may be used to satisfy data requirements for 

the DNT study and immunotoxicity studies. 

Additionally, we found a 3-generation reproductive test conducted in both rats and 

rabbits saying that Amitraz is not a reproductive toxicant.ix Again, the Agency should 

examine these studies to ensure that they will not meet its requirements for this 

endpoint. If not, then we support use of modified one-generation reproduction study in hope it 

will be used successfully used in re-registration process. 

We hope that the reproduction study mentioned above satisfies the immunotoxicity data 

needs for Amitraz. If it does not, then we recommend investigating a study we found 

which analyzed the effects of Amitraz in four-week old outbred male Wistar rats for 

specific immune functions.X

In conclusion, we have provided the Agency with quite a bit of information regarding 

existing studies done on animals for the data gaps listed for Amitraz. In our search we 

found a plethora of information on this pesticide and encourage the Agency to further 

investigate our findings (and possibly find even more) within the framework of 

minimizing duplicative animal tests. 

• (GLN 870.6300) Developmental Neurotoxicity Study 

• (GLN 870.3800) Two Generation Reproduction and Fertility effects 

• (GLN 870.7800) Immunotoxicity 

Response 5: 

Agency: The Agency appreciates PCRM suggestions for data sources to potentially 

evaluate the safety of amitraz to humans, livestock and pets. The many data sources listed 
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by PCRM will be carefully evaluated for scientific adequacy, quality and relevance during 

the next risk assessment for amitraz. The Agency is working to implement the extended 

one-generation reproduction study to conserve animal utilization for toxicology 

assessments. 

In conclusion, the Agency is striving to minimize animal testing with amitraz. In that 

respect, the Agency has been communicating with the registrants to develop a very efficient 

“extended one-generation reproduction protocol” which will greatly minimize replication of 

testing and animal usage. This protocol will combine multiple tests into one combined 

comprehensive study utilizing the same animals, to greatly conserve animals. The toxicology 

studies that will be combined include reproduction, immunotoxicity, neurotoxicity, 

developmental, clinical chemistry, hematology, urinalysis, oral toxicity, etc. It is the mission of 

the US EPA to protect the public from pollution and ensure chemical safety. Unfortunately, the 

toxicology data base for amitraz has multiple deficiencies, thus several animal studies are 

required to fulfill federal regulations 40 CFR §158.