i 

## Response to the External Peer Review of

“Technical Review of Acetonitrile (Methyl Cyanide)”

January 24 , 201 3

United States Environmental Protection Agency Office of Environmental Information Office of Information Access and Analysis

TABLE OF CONTENTS

I. BACKGROUND

................................ ................................ ................................ ................... 1

II. CHARGE TO THE PEER REVIEWERS

................................ ................................ ............. 1

III. RES P ONSE TO HUMAN HEALTH PEER REVIEW COMMENTS

................................ 2 A. General Impression

................................ ................................ ................................ ................ 2 B. Response to Charge Questions

................................ ................................ ............................... 6 C. Specific Observations

................................ ................................ ................................ .......... 3 5

IV. RESPONSE TO CHEM

- FATE & ECOTOXICOLOGY PEER REVEW COMMENTS

.. 4 0 A. General Impressions

................................ ................................ ................................ ............. 4 0 B. Response to Charge Questions

................................ ................................ ............................. 4 7 C. Specific Observations

................................ ................................ ................................ .......... 59

APPENDICE: External Peer Review of EPA’s Draft Technical Review of Ace tonitrile (Methyl Cyanide)..........................................................................................

.......................... 771 I. BACKGROUND AND DOCUMENT FORMAT

In response to a petition to delist acetonitrile for the Toxics R elea s e Inventory (TRI) , the EPA’s Office of Information Analysis and Access (OIAA) conducted a full hazard assessment of acetonitrile. Assessment findings were captured in the EPA draft report entitled Technical Review of Acetonitrile (Methyl Cyanide). Eight senior scientists were selected to peer review t he draft report. Within this document (Response to Peer Review) , the reviewer’s names have been masked. Five of the scientist s (Commenter One, Commenter Two, Commenter Three, Commenter Four and Commenter Fiv e), had expertise in human health hazard assessment s (human health toxicologists)

. Commenter Six, Commenter Seven

, and Commenter Eight were ecotoxicologists, selected for their expertise in environmental fate and ecological hazard
assessment s

.

II. PEER REVIEW CHARGE

The draft Technical Review of Acetonitrile (Methyl Cyanide) consisted of four chapters :

Chapter One

--- Integrated Technical Summary Chapter Two

--- Chemistry and Environmental Fate Chapter Three

--- Human Health Hazard Assessment Chap ter Four

--- Ecological Hazard Assessment

The eight reviewers were asked to thoroughly review and comment on the content and scope of the draft report, respond to a series of charge questions, and assess whether the report reflects the
current state

- of

- the

- science.

T he five human health toxicologist were asked to review Chapter Three, (Human Health Hazard Assessment), and any relevant sections of Chapter One (Integrated Technical Summary). The specific charge questions they were to address were :

1 . Has the available information supporting the hazard assessment of acetonitrile been objectively described? Are there any additional studies that should be included?

2. The U.S. EPA has determined that mortality is the critical endpoint for acetonitrile as described in the IRIS “Toxicological Review of Acetonitrile” (1999). Mortality has been observed in a 13

- week subchronic study as well as in developmental and reproductive toxicity studies. Mortality was not observed in a 2

- year chronic s tudy conducted at doses below those that caused mortality in the 13

- week subchronic study. Based on the totality of available evidence, particularly the 13

- week and 2

- year studies, is it reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure? Please explain.

The three ecotoxicologists were asked to review Chapter Two (Chemistry and Environmental Fate) and Chapter Fou r (Ecological Hazard Assessment ) and any relevant sections of Chapter One (Integrated T echnical Summary). The specific charge questions they were to address were:2 Chapter Two, Chemistry and Environmental Fate

1. Has an objective (accurate, clear, complete, and unbiased) and technically accurate description of acetonitrile been provided in terms of chemical and physical properties?

2. Does the report objectively address the fate of the chemical in the environment including both transport between environmental compartments (atmosphere, aquatic and terrestrial) and transformation (e.g., de gradation), including both abiotic and biotic processes where relevant?

Chapter Four, Ecological Hazard Assessment

1. Has the available information on the ecological toxicity of acetonitrile been objectively described ? Are there any additional studies t hat should be included?

2. Is the conclusion presented in section 4.1, “Acetonitrile does not present a concern for adverse effects on the environment” supported by the data/information presented?

Sections III and IV of this report capture the peer revie wer’s comments and the EPA response to these comments. Section III focuses on Chapter Three (Human Health Hazard Assessment) peer review comments and is divided up into three subsections: A. General Impressions, B. Response to the Charge Questions and C. Specific Observations. Section IV focuses on both Chapter Two (Chemistry and Environmental Fate) and Chapter Four (Ecological Hazard Assessment) peer review comments and is also divided up into three subsections: A. General Impressions, B. Response to the Charge Questions and C. Specific Observations.

Note that for subsection B. ( Response to Charge Questions ), the comments are individually identified and contain a “Ch” specifying the chapter the question is related two and a “Q” indicating which peer r eview charge question is being addressed. For example the “Ch2” in
comment IV

- B

- Ch2

- Q1

- 2 found in section IV B below indicates the comment is relevant to Chapter Two (Chemistry and Environmental Fate) of the draft Technical Review of Acetonitrile (Methyl Cyanide) . The “Q1” identifies the comment is addressing question 1 of the specific peer review charge questions for Chapter Two.

III. RESPONSE TO HUMAN HEALTH CHARGE

The reviewers of the Human Health information provided a portion of comments that were editorial in nature. These comments were considered by the U.S. EPA and addressed in the document where appropriate, but were not included in this response section.

A. General Impressions

Comment III

- A

- 1: Commenter One Chapter Three describes in some details the studies available on ACN

- induced human and animal acute, sub

- acute, and chronic toxicities. However, the data available are relatively old information that is derived from accidental human ingestion of ACN and relatively few idealized

a nimal studies in mice rats and rabbits. Also, the interpretations of the results are based on somewhat obsolete mechanistic information.3 EPA Response III

- A

- 1: As noted by this and other reviewers, the Technical Review of Acetonitrile (Methyl Cyanide) has incorporated all available studies of acetonitrile. The EPA agrees that many of the studies are older but several more recent studies are also included. The mechanistic information used in the technical review has been revised.

Comment III

- A

- 2: Comm enter One The overall conclusion that “ACN

- induced acute, sub

- acute and chronic toxicities and subsequent mortality is due to the amount of un

- detoxified cyanide moieties alone” is not valid because of the current understanding of the molecular mechanisms of actions of cyanide ions and other reactive metabolites of ACN.

EPA Response III

- A

- 2: Based on the comments of the reviewers, the EPA has revised its overall conclusion that “ACN

induced acute, sub

- acute and chronic toxicities and subsequent mortality i s due to the amount of

un

- detoxified cyanide moieties alone .” The current state of science does not allow for the pharmacokinetic modeling necessary to accurately predict human body burdens of acetonitrile or tissue specific release of cyanide moieties. Therefore, the Technical Review of Acetonitrile (Methyl Cyanide) has been modified, removing this language from the document.

Comment III

- A

- 3: Commenter Two The intent of this technical report is not clear. It is stated that it is related to the review of a petition to remove acetonitrile from the list of chemicals subject to reporting requirements.

EPA Response III

- A

- 3: The intent of the document has been stated on page ii of the Technical Review of Acetonitrile (Methyl Cyanide) . This statement has b een modified to provide clarity.

Purpose of this Hazard Assessment for Acetonitrile

Before a chemical can be proposed for addition to or deletion from section 313 of the Emergency Planning and Community Right

- To

- Know Act (EPCRA) of 1986, EPA performs a hazard assessment on the chemical. The statutory chemical listing/delisting criteria of EPCRA section 313(d)(2) are generally based on human health and environmental hazards. The analysis focuses on identifying and characterizing a chemical’s potential f or toxicity to humans and the environment. This technical report provides an overall assessment and review of the toxicity of acetonitrile, which includes a review and consideration of both human health and environmental hazards. It includes an assessme nt of the severity of the effect(s) and the dose or concentration levels at which the effect(s) were observed. This report contains several chapters including chemistry and environmental fate, human health hazards, and ecological toxicity. An “Executive Summary” is included to provide the user with a brief review of the key findings presented in the following chapters of the report

Additionally, the Executive Summary, which has now replaced the Integrated Technical Summary

, details the purpose and focus of the Technical Review of Acetonitrile (Methyl Cyanide).4 In the 2002 petition, BP claimed that acetonitrile does not satisfy the criteria for adverse acute or chronic health effects or adverse environmental effects. The critical issue of the 2002 peti tion is the assertion that the mortality reported in the NTP (1996) 13

- week study was an acute effect. EPA’s technical assessment of the mortality reported in the NTP study along with a review of other human health and environmental hazards and the enviro nmental fate of acetonitrile are provided in this report.

Comment III

- A

- 4: Commenter Two A major concern is how consistent this document is with the rationales that underlie other regulatory activities of the Agency. This document does make reference to IRIS reviews of acetonitrile and the analysis appears consistent with what is provided in the IRIS document. The present document makes the conclusion that the effects of acetonitrile are related to its conversion to cyanide. The conclusions of this docu ment are not consistent with the Office of Ground Water and Drinking Water's (OGWDW) Consumer Factsheet on Cyanide, where effects of cyanide on thyroid function and neurological effects which are related to its conversion to thiocyanate. Unfortunately, OG WDW has no transparent link to the criteria document that supported the cyanide regulation. It is my recollection that the concern over thyroid effects of cyanide largely relied on human data related to exposure to cyanogenic compounds (i.e. similar
to ac etonitrile) in the diet that lead to inhibited thyroid hormone synthesis. This literature needs to be revisited and an explicit determination made as to its applicability to the problem the present document is supposed to address. I recognize that many o f the papers that address this issue are old and may well be confounded with other socioeconomic factors. Nevertheless, they need to be explicitly considered in this document because of the huge emphasis that is placed upon the interpretation that the tox icity of acetonitrile is attributable to its conversion to cyanide.

EPA Response III

- A

- 4: Efforts have been made to review the draft Technical Review of Acetonitrile (Methyl Cyanide) for concurrence with other EPA offices and documents. No apparent inconsistencies exist between this and other Agency documents. Specifically regarding the Office of Ground Water and Drinking Water's (OGWDW) Consumer Factsheet on Cyanide, the statement that weight loss, thyroid effects

, and nerve damage

, are long

- term health effects related to cyanide exposure from drinking water does not contradict the conclusions made based on human and animal research of inhaled acetonitrile.

Comment III

- A

- 5: Comment er Three I would recommend on first occurrence of description of the cyanide molecular defect (p. 2, line 5), use of terminology “ chemical reduction of oxygen” to distinguish from “reduction” meaning loss of quantity.

EPA Response III

- A

- 5: The terminology “ reduction of oxygen ” in the Integrated Technical Summary , (now the Executive Summary) , is no longer a part of the text in question.

Comment III

- A

- 6: Commenter Four This reader was left, however, with the distinct impression that certain data pertaining to acetonitrile pharmacokinetics and mortality were not simply being reported in an objective, unbiased manner, but rather, were being marshaled to support the Agency’ s principal assertion regarding acetonitrile that “it is reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure”, without appropriate and balanced consideration of the alternatives.5 While this lack of balance is dis cussed in greater detail below, I believe this Agency position is altogether unjustified by the available information. In my opinion, the data presented and summarized in this draft chapter point convincingly toward mortality arising solely from intense ( high concentration) acetonitrile exposures of quite short durations (from hours to at most a few weeks). Thus, if excessive mortality was to be seen in a chronic study, and it has not, it would be reasonably anticipated to occur during and as a consequenc e of the first few weeks of acetonitrile exposure. This view is consistent with what is known of the kinetics of acetonitrile (and cyanide) uptake, distribution, metabolism, and elimination in laboratory animals and humans.

Comment III

- A

- 7: Commenter Fiv e The health assessment chapter is repetitive and does not provide a thorough, integrated hazard characterization with a weight

- of

- evidence analysis. It also is not sufficiently rigorous, complete and transparent to meet current scientific standards and r egulatory guidance.

EPA Response III

- A

- 6 and III

- A

- 7: In light of this and other reviewer’s comments, the technical review has been amended to provide a more balanced discussion of these areas. In response to the reviewer’s comments regarding mortality a s the result of chronic exposure, the Agency has more clearly defined the specific data leading to this conclusion in the Technical Review of Acetonitrile (Methyl Cyanide) . Specific attention has been paid to the mortality occurring in the NTP (1996) 13

- w eek mouse study.

Comment III

- A

- 8: Commenter Five Table 3

- 1 is misleading. First, no statistical significance is reported. A single death or even two deaths in a dose group would not be considered an effects level even if higher rates of mortality

occu r at higher doses because the incidence is too low to differ significantly from control rates and could have occurred by chance. Second, in developmental studies, all fetotoxic or reproductively toxic effects occurred at maternally toxic doses and thus ac etonitrile is not a fetal/embryo toxicant. Third, some studies showing no treatment effect reported in the text are not summarized in this table (e.g., Hazelton 1983). I discuss these issues under the Charge Questions.

EPA Response III

- A

- 8: The EPA agre es with the concerns regarding the statistical significance of a single death. Therefore Table 3

- 1 has been revised to reflect the higher NOAEL and LOAEL values for the NTP (1996) 90

- day inhalation studies. For further discussion of the relevance of mort ality in this study, please see Comment III

- B

- CH3

- Q1

- 20(b). The observations of developmental toxicity were also observed at doses at which maternal toxicity or mortality were observed. However, it is inadequate to assume that the developmental effects r esult only from maternal toxicity, and the results may indicate that both lifestages, the adult and developing offspring, are sensitive to the dose level (U.S. EPA, 1991). Table 3

- 1 has been extended to include the studies included in the text and have bee n reorganized to follow the relevant sections based on duration of exposure.

Comment III

- A

- 9: Commenter Five The conclusions are not supported by the data.

EPA Response III

- A

- 9: This comment does not provide enough detail to allow for a thorough response. More information regarding the conclusions of the Technical Review of Acetonitrile (Methyl Cyanide)6 can be found in Section 3.11.

Comment III

- A

- 10: Commenter Five Human relevance is inadequately characterized.

EPA Response III

- A

- 10: Human relevance has been specifically considered in response to this and other reviewer’s comments. Details on human relevance can be found in Section 3.10 Human Relevance of the Animal Data , of the Technical Review of Acetonitrile (Methyl Cyanide).

Section 3.10. “ In the absence of human data associating chronic exposure of humans with increased risk of death, the toxicity observed in animals following repeated exposures to acetonitrile is a ssumed to be relevant to humans. Case studies reporting mortality in humans after acute accidental poisoning describe effects consistent with those observed after exposure to inorganic cyanide and other saturated aliphatic nitriles exposures. Animal stud y findings suggest that the toxic effects of acetonitrile may be correlated with the metabolic release of cyanide and/or formaldehyde, a process that would also occur in humans. ”

B. Response to Charge Question s

Chapter Three, Charge Question 1. Has th e available information supporting the hazard assessment of acetonitrile been objectively described? Are there any additional studies that should be included?

Comment III

- B

- Ch3

- Q1

- 1: Commenter One To my knowledge, there are no new studies on ACN adverse activities. There are, however, several recent studies that describe the molecular mechanisms of toxicities of the cyanide moieties and how cyanide actually interferes with mitochondrial respiratory chain mechanisms. Also, there are modern studies that des cribe the molecular mechanisms of other ACN metabolites such as formaldehyde (see below).

EPA Response III

- B

- Ch3

- Q1

- 1: The concerns regarding molecular mechanisms of toxicity through cyanide interference with mitochondrial respiration has been addressed i n the Technical Review of Acetonitrile (Methyl Cyanide). The reviewer’s suggestion to expand the discussion of molecular mechanisms of formaldehyde has not been incorporated into the technical review. This discussion was not added given the uncertainty o f the acetonitrile toxicokinetics and the relevance of applying the information addressing the molecular mechanisms of formaldehyde to acetonitrile toxicity.

Comment III

- B

- Ch3

- Q1

- 2: Commenter Two While the data are sparse, this review does not make adequa te use of the available data to form the conclusions of the report. One example is the handling of the genotoxicity data. A second was the failure to utilize the limited metabolism data to support their hypothesis that the lethal effects (and other effec ts) of acetonitrile were attributable to the generation of cyanide as a metabolite. A third example, addressed further in the response to the next question, is that potential effects of acetonitrile on thyroid function were not adequately discussed and eva luated in this review.

EPA Response III

- B

- Ch3

- Q1

- 2:7 The discussion of genotoxicity data (Section 3.6) has been amended to incorporate suggestions from several reviewers’ comments. The revised version provides a more complete discussion of the available d ata.

The reviewers’ comments on the relationship between cyanide bioaccumulation and lethality were considered; however, it was determined that the information on metabolism and mode of action were not sufficient to identify a mode of action for acetonit rile toxicity. Section 3.2.3 focusing on the metabolism of acetonitrile, has been added to the Technical Review of Acetonitrile (Methyl Cyanide)

.
The discussion of thyrotoxic effects ha s been expanded in Section 3.9 Thyroid Toxicity.

Comment III

- B

- Ch3

- Q1

- 3: Commenter Two The data appear to clearly establish that acetonitrile produced chromosome aberrations and aneuploidy in several test systems. The document treats these as “only marginal effects”, which is a prejudicial editorial comment ma de without discussing why the data were marginal. The sentence talks about “potential” of acetonitrile being demonstrated. If the results were statistically significant, one should conclude that it produced the effect. It is entirely possible that these data can be dismissed by a more thorough discussion of the data, including questions of consistency among tests measuring the same endpoint, that is, comparing the doses used in positive vs. negative effects in tests measuring the same endpoint. That dis cussion should come to a judgment based on the data as a whole. As this section now stands, it comes to no conclusions and largely leaves the issue open.

EPA Response III

- B

- Ch3

- Q1

- 3: The EPA has modified the discussion of genotoxic effects of acetonitril e in concurrence with the reviewer’s recommendations. The genotoxic data is largely negative, with some notable exceptions highlighted in the draft Technical Review of Acetonitrile (Methyl Cyanide). The overall data indicate that acetonitrile is not a po int mutagen, but does interfere with chromosomal segregation. The positive effects evidenced in acetonitrile studies are generally seen at in vitro dose levels that are equivalent to or above those inducing mortality in mouse and rats whole organism studi es. Therefore, acetonitrile genotoxicity is not considered to be among the most sensitive endpoints.

“3.6 GENOTOXICITY
Acetonitrile results in gene mutation assays have been predominantly negative, with some notable exceptions. In mutation assays w ith a variety of Salmonella typhimurium strains, both with and without metabolic activation, acetonitrile has failed to produce a measurable effect (U.S. EPA, 1999). Similarly, acetonitrile did not produce mutations in Saccharomyces cerevisiae D7 (U.S. EP A, 1999). Both sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells and gene conversions in S. cerevisiae were increased in the absence of S9 metabolic activation, but not in the presence of S9 (U.S. EPA, 1999). In contrast, chromosomal aberrations in CHO cells were increased in the presence of S9, but not in the absence of S9 (U.S. EPA, 1999). Further, effects in chromosomal aberration assays have demonstrated interference with chromosome segregation both in vivo and in vitro using the Drosophila melanogaster model system (U.S. EPA, 1999). Increases in micronucleated erythrocytes have been reported in mice treated with acetonitrile via inhalation (NTP, 1996) and intraperitoneal injection (Schlegelmilch et al.,8 1988); however, the incid ence of micronucleated polychromatic erythrocytes in bone marrow and peripheral blood was not affected in groups of male and female mice (5/sex) given single intraperitoneal injections of 100 or 125 mg/kg acetonitrile in corn oil (Jones et al., 2001). Pos itive micronucleus assays can indicate either clastogenic activity or interference with chromosome segregation. Sehgal et al. (1990) obtained in vitro evidence that acetonitrile does inhibit microtubule assembly in taxol

- purified Drosophila or mouse micro tubules. Acetonitrile induced aneuploidy in treated mature oocytes of D. melanogaster females exposed either as larvae or as adults (Osgood et al., 1991a,b). Further, acetonitrile induced mitotic aneuploidy in S. cerevisiae exposed to 5% acetonitrile (Zi mmermann et al., 1985). There are no data on the capacity of acetonitrile to produce heritable genetic mutations in humans or animals. The overall genotoxicity data are inconsistent, and indicate that acetonitrile is not a point mutagen, but does interfe re with chromosomal segregation (U.S. EPA, 1999). Genotoxicity following exposure to acetonitrile is not considered to be among the most sensitive endpoints.”

Comment III

- B

- CH3

- Q1

- 4: Commenter Two The metabolism and pharmacokinetic data on acetonitrile and its metabolites are limited, but there are sufficient details available to allow more defensible conclusions to be made related whether mortality is the key event. Some of elements of this discus sion are provided on page 29 of the document, but the discussion of the data is interspersed with a series of speculative comments that detract from the proper use of these data (e.g. “overwhelming enzymatic detoxifying mechanisms” and “reconversion of thi ocyanate to cyanide”). These statements should not be made without specifically discussing how those two things might occur or have been shown to occur. I would strongly suggest the inclusion of a diagram that lays out the metabolism of acetonitrile to c yanide and thiocyanate. In that diagram, all the important routes of elimination of acetonitrile and these two metabolites should be estimated. That will mean some exploration of the cyanide and thiocyanate literature to obtain the important quantitative data (hopefully in humans as well as rats and/or mice).

EPA Response III

- B

- CH3

- Q1

- 4: As noted in EPA Response III

- B

- CH3

- Q1

- 12, there is a dearth of quantitative data. Therefore, the draft Technical Review of Acetonitrile (Methyl Cyanide) has been revised and speculative comments have been removed to more fully defend the EPA’s conclusions regarding mortality. These extensive changes were made throughout the Executive Sumnmary (formally the Integrated Technical Summary ), and the Human Healt h Hazard chapters.

As suggested, we have incorporated a diagram of the acetonitrile metabolic pathway to provide a more complete description of the potential modes

- of

- action. Specific quantitative data regarding the rates of conversion are not included. Estimation of the elimination routes for acetonitrile and cyanide is beyond the scope of the technical review and not sufficiently supported by the scientific literature.

Comment III

- B

- CH3

- Q1

- 5: Commenter Two As pointed out, the half

- life of acetonitril e in humans poisoned with acetonitrile was 32 hours while that of cyanide is about 15 hours. Therefore, the slow conversion of acetonitrile will not allow substantive amounts of cyanide to accumulate at low doses. Thus, it would appear that the “detoxifi cation mechanism” alluded to is rhodanese conversion of cyanide to thiocyanate. The authors need to explore the kinetics of this latter conversion to determine what systemic concentrations of cyanide will begin to saturate this enzyme. Until that occurs, there will not be a sharp accumulation of cyanide. The idea that the reconversion of thiocyanate back to cyanide9 can contribute to this build

- up also needs to be supported. As written, the implication is that the elimination of thiocyanate is slower tha n that of cyanide, but limited quantitative data are provided to support this contention (i.e. simply stated that thiocyanate is not entirely eliminated between doses). This needs to be confirmed, not simply be a suggestion by an author (line 1, page 30). To get accumulation cyanide, it needs to be established from the literature that this is the main route of cyanide elimination and that the relative rate of cyanide generation is more rapid than its elimination by rhodanese. One way to slow this convers ion is to build up levels of thiocyanate. However, that is simply a question approaching equilibrium concentrations of cyanide and thiocyanate in the systemic circulation. It does not mean that there is a net conversion of thiocyanate conversion back to cyanide, but that they are at equilibrium (or more properly in a steady state that is controlled by the rate of thiocyanate elimination).

EPA Response III

- B

- CH3

- Q1

- 5: The scientific literature does not provide enough data to further explore the kinetics o f the metabolism of acetonitrile. In response to these comments, EPA has revised the discussion of the mode of action related to the chronic toxicity of acetonitrile. The proposed overwhelming of the rhodanese detoxification pathway has been removed from the technical review. A more thorough diagram of the acetonitrile metabolic pathway has been included. These changes are incorporated into and the Human Health Hazard chapter of the draft Technical Review of Acetonitrile (Methyl Cyanide).

Comment III

- B

- CH3

- Q1

- 6: Commenter Two The question of whether acetonitrile would be likely to produce inhibition of iodide uptake and thus decrease thyroid hormone synthesis will depend upon the systemic concentrations at which this steady

- state establishes itself with continued treatment.

EPA Response III

- B

- CH3

- Q1

- 6: An expanded discussion of possible thyrotoxicity following exposure to acetonitrile, including this comment, has been incorporated into the Technical Review of Acetonitrile (Methyl Cyanide).

3.9. Thyr oid Toxicity

“ Thiocyanate does not demonstrate high acute toxicity, but has been shown to be a potent inhibitor of iodide transport into the thyroid (Klaassen, 2008). Long

- term ingestion of elevated levels of cyanide can result in sustained serum levels of thiocyanate, leading to inhibition of iodide uptake by the sodium

- iodide symporter in the thyroid gland, reduced production of thyroid hormones T4 and T3, and subsequent thyrotoxicity (Klaassen, 2008). Whether acetonitrile exposure and the subsequent m etabolism to thiocyanate is likely to produce this inhibition of iodide uptake, and thus decreasing thyroid hormone synthesis, will depend on the systemic concentrations at which the steady

- state establishes itself with continued treatment. The nature of iodine uptake into the thyroid gland and thyroid hormone production may contribute to the lack of overt signs of thyrotoxicity, such as alterations in systemic thyroid stimulating hormone, thyroxine, and triiodothyronine levels, as demonstrated in the NTP (1996) studies. Since acetonitrile exposure was administered 6 hours/day, 5 days/week, temporary activation of the sodium

- iodide transporter may have occurred because of thiocyanate clearance during periods of non

- exposure. In addition, the thyroid gland stores thyroglobulin as colloid within the gland (Klaassen, 2008), and this reservoir has the potential to serve as a reserve for the production of thyroxine, thus maintaining circulating levels. Neuronal pathology that would suggest hypothyroidism was not observed in the NTP10 (1996) studies. This suggests adequate thyroid hormone (T 3 and T 4 ) during development. It should be noted that the animals used in the NTP (1996) 13

- week and 2

- year mouse and rat studies were approximately 6 weeks old when they began exposure. Current literature suggests rat neuronal development is sensitive to thyroi d hormone action from shortly after birth through post natal day 20

- 30 (Oppenheimer and Schwartz, 1997). The exposure periods in the NTP (1996) studies, therefore, appear to occur outside the proposed critical window for neuronal thryroid hormone sensitiv ity. In addition to direct effects on the thyroid, a decrease in thyroid hormones following exposure to cyanide, mediated through the cyanide metabolite thiocyanate, may result in reproductive toxicity effects (U.S. EPA, 2010). As stated in the Toxicologi cal Review of Hydrogen Cyanide and Cyanide Salts (U.S. EPA, 2010), “research exists to suggest that reproductive tissues in developing and adult animals are responsive to alterations in thyroid hormone levels.” ”

Comment III

- B

- CH3

- Q1

- 7: Commenter Three How ever, I believe addition information on clinical symptoms from the reviewed studies should be presented and explicitly used to provide some evidence for equivalence of mode of action of lethality from acute, short term and subchronic exposure to acetonitri le. The rationale for calling for this additional material is detailed below in paragraph 4 of Section II.2. Summary of clinical symptoms from the Argus 1984 study, however, should be qualified by disclosure of the Study Director’s (Dr. A.M Hoberman) ob servation, expressed Apr 1, 2002, to Mr. D.E. Strother, that gastrointestinal irritation and decreased food consumption were coincident adverse effects.

EPA Response III

- B

- CH3

- Q1

- 7: The draft Technical Review of Acetonitrile (Methyl Cyanide) has been modi fied to reflect the reviewer’s comments relating to similarities in human clinical presentation and animal study observations, as well as discussion of potentially confounding factors in the Argus Research Laboratories study.

Section 3.10.

“

Acetonitrile has been shown to cause death in rabbits, mice, rats, and humans (U.S. EPA, 1999). The molecular mechanisms underlying the cause of death for the various species are thought to be the same across species, and the clinical symptoms of exposure in cases of accidental or occupational human exposure are similar to those observed in studies using laboratory animals (WHO, 1993; U.S. EPA 1999). These symptoms of acetonitrile poisoning include neurotoxic effects (i.e. ataxia, tremors and convulsions) commonly as sociated with cyanide poisoning. This evidence strongly supports equivalent modes of action for acetonitrile lethality upon acute exposure for test animals and humans, and by extension suggests relevance of acetonitrile lethality due to chronic exposures in humans, as seen in animals from the NTP (1996) subchronic mouse inhalation study. ”

Section 3.5. “ It should be noted that an observation made by the Study Director, Dr. A.M. Hoberman, expressed April 1, 2002, to Mr. D.E. Strother, stated that gastroin testinal irritation and decreased food consumption were coincident adverse effects. ”

Comment III

- B

- CH3

- Q1

- 8: Commenter Three Brief mention should be given to alternative or complementary mechanisms, such as an expansion of the comment on thyroid toxicity from thiocyanate (p. 31, lines 12

– 15). If the authors believe these mechanisms are not relevant, their reasoning should be given.11 EPA Response III

- B

- CH3

- Q1

- 8: The technical review has been revised to present a more balanced discussion of the human heal th hazard associated with acetonitrile, specifically in the area of thyrotoxicity. See Sections 3.8 and 3.9 of the draft Technical Review of Acetonitrile (Methyl Cyanide)

.

Comment III

- B

- CH3

- Q1

- 9: Commenter Four Most of the information presented in the dr aft chapter has been objectively described. However, there are specific areas where there seems to be a lack of balance. In Section 3.2, Kinetics and Metabolism, for example, the lower measured affinity for cyanide (higher Km) of rhodanese from human na sal tissues and lower measured maximal rate (smaller Vmax) of cyanide metabolism, relative to that in rat nasal tissues, were highlighted as key results from the study by Lewis et al. (1991) (see p 29 lines 1

- 8). A comment follows immediately that humans are thus “less able [than rats] to detoxify cyanide to thiocyanate,” with the implication that humans would thus be at greater risk of cyanide

- induced toxicity.

However, the draft chapter neglects to point out that Lewis et al. were unable to saturate t he human rhodanese enzyme despite the use of potassium cyanide concentrations of 500 mM and greater. In fact, Lewis et al. reported that the rhodanese enzyme exhibited activation, not saturation, in one of their samples, consistent with previous work by J arabak and Westley (1974) (cited in Lewis et al., 1991). This suggests that a simple Michaelis

- Menten model of cyanide metabolism fails to adequately describe the actual dose

- response relationship obtained by Lewis et al., particularly at high concentrati ons (see their Figure 2)! Such enzyme induction in human nasal tissues and, possibly, liver would also, presumably, make humans less sensitive to cyanide exposure than rats at least at sufficiently high concentrations where the rat metabolic response is

s aturated but the human response is not.

Lewis et al. (1991) also provide highly relevant discussion regarding the high “maximum detoxi fi cation” capacity for cyanide metabolism observed in the rat nasal cavity: “This capacity is sufficient to completely me tabolize a maximum of 2800 ppm inhaled hydrogen cyanide ...” This high detoxi fi cation capacity may explain in part the reduced sensitivity of rats, compared to mice, to cyanide

- induced toxicity that was observed in animals exposed to high acetonitrile

conce ntrations in the NTP subchronic inhalation study (NTP 1996).

EPA Response III

- B

- CH3

- Q1

- 9: The EPA appreciates this further discussion of the Lewis et al. (1991) data. A more complete and balanced discussion of the human and rat studies has been included.

3.2.3., Metabolism

“

Acetonitrile conversion to cyanide appears to be about 10

- fold greater in rat nasal ethmoturbinate microsomes than in liver microsomes (Dahl and Waruszewski , 1989), due to high P450 content in the rat nasal cavity (U.S. EPA, 1999). The nasal cavity of the rat, in particular the olfactory region, also has a high concentration of rhodanese (Dahl and Waruszewski, 1989). Rhodanese has also been found in the res piratory epithelium of human nasal tissue; however, Lewis et al. (1991) showed that the rhodanese in human nasal tissue had a lower affinity (higher K

m ) for cyanide and a lower rate (lower V max ) for cyanide metabolism than rhodanese from rat nasal tissue. K(m) is the concentration of substrate which permits the enzyme to achieve half V(max)(the maximum rate of the reaction. Taken together these data suggest that cyanide may be detoxified to thiocyanate more rapidly in rat nasal tissue than in animal speci es with lower12 amounts or bioactivity of the enzyme, such as humans (U.S. EPA, 1999). Lewis et al. (1991) further reported an induction of rhodanese activity and an inability to saturate rhodanese enzymatic activity at high potassium cyanide concentrations using human nasal tissue in vitro. This was in contrast with a previous study that showed no substrate induction of rhodanese activity and saturation of rhodanese activity at high substrate levels using rat nasal tissue in vitro. Due to the dynamic natur e of rhodanese activity it is not possible to directly compare the human and rat capacity or rate of cyanide conversion to thiocyanate via rhodanese. Further confounding the issue, Lewis et al. (1991) reported that rhodanese activity was statistically l ower in smokers than in nonsmokers, and individual K m and V max values suggest that rhodanese in smokers may have a decreased affinity for cyanide. It is postulated by the authors that “increased exposure to cyanide or other pollutants in the tobacco smoke appears to have suppressed enzyme activity.” If chronic exposure to cyanide does result in decreased rhodanese activity, it may provide a mechanism by which cyanide would accumulate after chronic exposure to acetonitrile. ”

Comment III

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- 10: C ommenter Four Section 3.2 also fails to mention that CYP2E1, the P450 enzyme responsible for the metabolism of acetonitrile to cyanide, is an enzyme that is inducible by a variety of substrates, including ethanol, c.f., Lu and Cederbaum, Free Radic Riol Me d 2008, 44(5)723

- 38. This property could lead to significantly higher rates of cyanide production from inhaled acetonitrile, as well as shortening of the half

- life of acetonitrile in the body.

EPA Response III

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- 10: This information has been integ rated into Section 3.2.3., Metabolism, of the Technical Review of Acetonitrile (Methyl Cyanide)

.

Section 3.2.3. “The cytochrome P450 2E mixed

- function oxidases are responsible for the biotransformation of many xenobiotics and are highly inducible at the gene level by many substrates, including alcohol (Lu and Cederbaum, 2008).”

Comment III

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- 11: Commenter Four Section 3.2 of the draft ends with two highly speculative paragraphs. They assert that the slow metabolism of acetonitrile to cyanide, spe cies differences in the tissue distribution of rhodanese, and the slow elimination rate of the thiocyanate, at least in some species, when taken together, strongly suggest that acetonitrile from one day’s dose is not totally eliminated before the next day’ s dose in repeat dose studies (in what species?, at what doses?, after how many doses?), leading to an accumulation of both acetonitrile and cyanide “to the point of overwhelming detoxifying mechanisms.” However, I am aware of no direct evidence that this cascade of events occurs during chronic exposure to acetonitrile, thus producing excess mortality as a chronic endpoint, as is stated in Section 3.3 of the draft document.

EPA Response III

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- 11: The statements made in the two referenced paragraphs have been removed from the Technical Review of Acetonitrile (Methyl Cyanide)

.

Comment III

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- 12: Commenter Four I was also surprised by the primarily qualitative nature of the discussion of acetonitrile13 pharmacokinetics. This is a topic that cries out for quantitative treatment with a PBPK model, yet a brief search of the literature using PubMed failed to identify any recent work in this area. Perhaps this is a significant data gap that needs to be filled with additional research.

EPA Response III

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- 12: The Agency recognizes that further elucidation of acetonitrile pharmacokinetics, complete with a PBPK model, would provide quantitative data for use in this hazard assessment. Unfortunately the information to inform a quantitative analysi s of the pharmacokinetics is not available in the
peer

- reviewed literature.

Comment III

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- 13: Commenter Five There is a lack of objectivity in the health hazard assessment. The hypothesis or postulated mode of lethality is presented in the first paragraph of the summary. Data are subsequently presented in a manner which supports this position, without providing a thoughtful discussion and integration of all available data and consideration of alternatives.

EPA Response III

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- 13: As detaile d in response to previous reviewer’s comments, the technical review has been revised and reorganized to present a more balanced discussion of the human health hazard associated with acetonitrile. A more thorough discussion of the available animal mortalit y data caused by acute or chronic exposure (NTP 1996) has replaced the speculative discussion postulating the mode of lethality. Extensive modifications of the Technical Review of Acetonitrile (Methyl Cyanide) have been made throughout Executive Summary ( formally the Integrated Technical Summary ), and the Human Health Hazard Assessment chapter

.

Comment III

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- 14: Commenter Five Although new data are not available, existing data are not framed in accordance with the currently

- accepted standards of hazard characterization. In particular, insufficient information is presented on mode of toxic action and the human relevance of animal studies, and a weight

- of

evidence narrative is incomplete. There is also a reasonable lack of objectivity, as indicat ed by the almost

- complete focus on mortality with little discussion of other health outcomes (e.g., liver, lung, forestomach, blood), incomplete presentation of toxicokinetics and mode of action data, and lack of rigor in assessing and interpreting finding s from individual studies in accordance with current standards and regulatory guidance.

EPA Response III

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- 14: The mode of toxic action has been further reviewed by the EPA. See Section 3.8., Potential Modes of Action, for a discussion of the mode of action information available for acetonitrile.

The human relevance of animal studies has been expanded and included in Section 3.10., Human Relevance of Animal Data, of the Technical Review of Acetonitrile (Methyl Cyanide).

The description and analy sis of the effects observed in animals following acute, subchronic, and chronic exposure to acetonitrile have been expanded upon within the Technical Review of Acetonitrile (Methyl Cyanide) . The Conclusion of the Technical Review of Acetonitrile (Methyl C

yanide) has also been modified to improve transparency. Mortality was a main focus of the assessment because EPA had previously reviewed acetonitrile in response to a petition to delist and the only chronic effect identified as sufficient to support listi ng was mortality. The petitioner focused their second petition on this effect, thus EPA’s focus was on this effect.14 Comment III

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- 15: Commenter Five There is a developmental toxicity study discussed on p. 18 of the NTP (1996) report on the toxi cology and carcinogenesis of acetonitrile, in which cyanide and acetonitrile were measured in Sprague

- Dawley rats exposed to acetonitrile at concentrations of 100, 400 or 1200 ppm for 6 hours/day on gestation days 6

- 19. The only reported effects were “a f ew” maternal deaths at 1200 ppm and one at 400 ppm. Analysis indicated “significant exposure

- related concentrations of acetonitrile in the blood of all exposed groups and the presence of cyanide in the blood of the 1200 ppm group (cyanide was detected at a very low level in one 400 ppm rat at each time point but “could not be quantified”

– presumably because it was too low). And: “The concentration of cyanide in the blood of 1200 ppm rats declined from approximately 2 mg/mL on GD 8 to approximately 0.8 mg /mL on GD 18, while acetonitrile concentration remained essentially constant during the same period...due to induction of rhodanese.” These data do not support the hypothesis that cyanide bioaccumulates and are consistent with other data that also do not in dicate bioaccumulation. This study should be added, as should others referenced in WHO/EHC (1993), which address blood levels of acetonitrile, cyanide, and/or thiocyanate.

EPA Response III

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- 15: EPA has revised the discussion of mode of action rel ated to the chronic toxicity of acetonitrile and the proposed overwhelming of the rhodanese detoxification pathway, leading to cyanide bioaccumulation, has been removed from the technical review. These changes are incorporated into the Human Health Hazard Assessment chapter of the draft Technical Review of Acetonitrile (Methyl Cyanide). In addition, the references from WHO/EHC (1991) highlighted by the commenter were reviewed and incorporated into draft Technical Review of Acetonitrile (Methyl Cyanide) where appropriate.

Comment III

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- 16: Commenter Five References which might be useful for a more integrated assessment of acetonitrile toxicity include the following documents.

WHO. 2004. World Health Organization. Concise International Chemical Assessment Document (CICAD): 61. Hydrogen Cyanide and Cyanides: Human Health Aspects. Geneva, Switzerland.

Way J.L. 1984. Cyanide intoxication and its mechanism of antagonism. Ann Rev Pharmacol and Toxicol. 24:451

- 481.

Sylvester, M. and C. Sander. 1990. Immunohistochemical localization of rhodanese. Histochem. J. 22(4):197

- 200.

Melnick, R.L., Roycroft, J.H., Chou, B.J., Regan HA and Miller. RA 1990. Inhalation toxicology of isoprene in F344 rats and B6C3F2 mice. Environ. Health Perspe ct. 86:93

- 98.

U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. [ See section on writing a weight

of

- evidence hazard narrative, using a modified set of Bradford Hill (1965) criteria]

Boobis AR, Doe JE, Heinrich

- Hirsch B, Meek ME, Munn S, Ruchira wat M, Schlatter J, Seed J, Vickers C. 2008. IPCS framework for analyzing the relevance of a noncancer mode of action for humans. Crit Rev Toxicol. 38 (2):87

- 96.15 Hill AB. The en vironment and disease: association or causation? Proc Royal Soc Med 1965; 58:295

- 300.

NAS 2005. Health Effects of Perchlorate Ingestion. National Research Council. Washington, D.C. [ Thyrotoxicity induced by ions which competitively inhibit iodide uptak e in the thyroid gland

– as thiocyanate does

– is thoroughly discussed in this report .]

EPA Response III

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- 16: As noted in this and other reviewer’s comments “new data are not available” specific to acetonitrile. The studies listed by this reviewe r, which do not provide acetonitrile

- specific information, have been reviewed by the EPA and have been considered in the development of the Technical Review of Acetonitrile (Methyl Cyanide)

.

Comment III

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- 17: Commenter Five Toxicokinetic and mo de of toxic action data are incompletely summarized.

EPA Response III

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- 17: See specific responses to subsections ( a) through ( f) of III

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- 17.

Comment III

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- 17(a): Acetonitrile is more slowly metabolized to cyanide than cyanide s alts or some other nitriles (WHO/EHC 1993; Hartung 1982). Therefore, during acetonitrile exposure, the body/tissue burden of acetonitrile is also increased. Both human case reports of poisoned individuals and animal studies show that acetonitrile is distr ibuted fairly uniformly throughout the body (EHC 1993).

EPA Response III

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- 17(a): The distribution of acetonitrile described above in humans and animals has been revised in section 3.2.2., Distribution, of the Technical Review of Acetonitrile (Methyl Cyanide).

Section 3.2.2.

“

Acetonitrile is rapidly, widely, and fairly uniformly distributed throughout the body in both humans and laboratory animals (U.S. EPA, 1999; WHO, 1993). A human autopsy of an individual who died 2 days following inhala tion of acetonitrile vapors found cyanide in the brain, spleen, lungs, and kidneys, but not liver (Amdur, 1959; WHO, 1993). Animal studies have shown hydrocyanic acid (HCN, i.e., hydrogen cyanide), a metabolite of acetonitrile, has been found in the brain , heart, kidney and spleen of rats after inhalation of acetonitrile vapors (Haguenoer et al., 1975). Whole

- body autoradiography in male mice injected intravenously with acetonitrile radiolabeled with 14 C in the methyl group indicated that radioactivity wa s widely distributed throughout the body (e.g., liver, thymus, reproductive organs, nasal secretions, oral cavity, esophagus, and stomach contents) (U.S. EPA, 1999). Additionally, oral exposure of animals resulted in metabolites found primarily in the spl een, stomach, and skin (U.S. EPA, 1999)

. ”

Comment III

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- 17(b): Acetonitrile is also excreted unchanged in urine and in expired air. Therefore, the clinical and subclinical effects are likely to be a result of the combined effects of the parent m olecule and its cyanide metabolite (Hartung 1982; EHC 1993). Further, animal studies do not indicate that16 either acetonitrile or cyanide has the potential for bioaccumulation (EHC 1993; ATSDR 1999).

EPA Response III

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- 17(b): The EPA has included t he information regarding the direct excretion of acetonitrile in urine and expired air.

“In addition, acetonitrile may also be excreted unchanged in the urine or in expired air, although at extremely low levels.”

In response to this and other reviewers’ comments, the EPA has deleted the text in the document regarding the accumulation of acetonitrile, cyanide, and thiocyanate as part of the potential mode of action of acetonitrile toxicity.

Comment III

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- 17(c): Released cyanide is metabolized to thiocyanate by rhodanese, a sulfur transferase enzyme, which is readily excreted in the urine. Rhodanese is an inducible enzyme and levels of rhodanese increase in response to increasing blood and tissue cyanide concentrations. Sites with the greatest abundance of rhodanese are located to maximize conversion of cyanide to thiocyanate following both oral and inhalation exposure (Sylvester and Sander, 1990 ). Detoxification of cyanide by rhodanese exhibits zero

- ord er kinetics relative to cyanide; the concentration of sulfur

containing donor molecules is the rate

- limiting factor.

EPA Response III

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- 17(c): The reviewers comments have been incorporated into section 3.2.3., Metabolism, of the Technical Review o f Acetonitrile (Methyl Cyanide).

Section 3.2.3. “Cyanide is further oxidized and conjugated to thiocyanate, a less toxic compound that is excreted in urine, but one that has been shown to interfere with thyroid function (Hartung, 1982). Conversion is me diated by rhodanese , a sulfurtransferase found in liver and human nasal respiratory mucosa (Hartung, 1982, Lewis et al., 1991). Rhodanese is an inducible enzyme and levels of rhodanese increase in response to increasing blood and tissue cyanide concentrat ions. Sites with the greatest abundance of rhodanese, such as hepatocytes in close proximity to the blood supply to the liver, bronchial epithelial cells, and proximal tubule cells of the kidney, are located to maximize conversion of cyanide to thiocyanat e following both oral and inhalation exposure ( Sylvester and Sander, 1990 ).”

Comment III

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- 17(d): The role of metabolism is crucial in determining the acute toxicity of ingested cyanide. Both the total amount administered and the rate of absorption are important (U.S. EPA 1992) because acute toxicity results from rapid ingestion of high doses of cyanide at a rate that exceeds the body’s capacity for metabolism (detoxification). In contrast, s low absorption of the same or greater total oral load of cyanide may allow for complete metabolism. Unmetabolized cyanide can be both acutely and chronically toxic, although cyanide is not known to accumulate in the body.

The principal features of the acute toxicity profile for cyanide are its severe toxicity, including lethality, by all routes of administration, with a steep rate

- dependent dose

– effect curve. The toxic effects of cyanide ion in humans and animals are gener ally similar and are believed to result from inactivation of cytochrome oxidase and inhibition of cellular respiration during the17 terminal reaction of the electron transport chain. This prevents the formation of adenosine triphosphate (ATP) via oxidative phosphorylation. Although acute oral doses of cyanide cause cardiovascular, respiratory, and neurophysiological changes, the brain appears to be the organ most sensitive to acute cyanide toxicity (WHO 2004). Death from cyanide poisoning is believed
to re sult from central nervous system depression, subsequent to inhibition of brain cytochrome oxidase activity (WHO 2004).

Thiocyanate is not acutely toxic; approximately 60

- 80% of an ingested dose of inorganic cyanide is converted to thiocyanate, the majorit y of which is rapidly excreted in the urine. However, elevated long

- term ingested cyanide can result in sustained serum levels of thiocyanate leading to inhibition of iodide uptake at the level of the sodium

- iodide transporter in the thyroid gland, subseq uent thyrotoxicity and resulting neurotoxicity (iodide deficiency

- associated neuropathy and mental retardation).

U.S. EPA’s hazard assessment suggests that: “With repeated exposure to acetonitrile, it is possible that continued generation of cyanide from the existing body burden of acetonitrile, coupled with cyanide generated from new exposures, results in cyanide levels that exceed the capacity of enzymes to metabolize cyanide to thiocyanate.”

However, if this is the case, then thiocyanate levels in bot h urine and blood would be expected to be significantly elevated and abnormalities in the thyroid gland (e.g., goiter) and thyroid hormone homeostasis (i.e., increased TSH and/or T4) would be expected to occur. Chronic exposure to cyanide, resulting in e xcessive production of thiocyanate, can interfere with iodine uptake by the thyroid gland, leading to enlargement of the thyroid (e.g., goiter) and abnormal levels of thyroid hormones (increased TSH and/or T4). The most prominent health hazard of chronic exposure to elevated levels of cyanide is thyrotoxicity resulting in neurotoxicity. There is no evidence of either thyroidal or neurotoxic effects that could be attributed to prolonged or repeated cyanide exposure in either the subchronic or chronic NTP a cetonitrile studies.

The potential effects of thiocyanate, which are associated with chronic cyanide toxicity, are not discussed in this assessment. The only thyroid

- related finding was noted in the subchronic rat study by NTP (1996) and was reported a s a statistically significant increase in T3 in the absence of any changes in T4 or TSH. This is puzzling. T3 is the bioactive thyroid hormone and is converted from T4 in situ by specific iodinase enzymes (NAS 2005). A change in iodide uptake in the thy roid gland because of the competitive inhibition of thiocyanate usually results in an increase in TSH and/or a change in T4. A change only in T3 might indicate an increased energy or metabolic tissue demand, as could occur with cyanide

- induced oxygen depr ivation, which is met by increased conversion of stored T4 to T3.

EPA Response III

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- 17(d): This reviewer’s comments have been considered and incorporated throughout Chapter 3 of the Technical Review of Acetonitrile (Methyl Cyanide)

. The potenti al effects of extended thiocyanate exposure have been discussed in EPA’s response to comment III

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- 6 and in Section 3.9., Thyroid Toxicity, of the Technical Review of Acetonitrile (Methyl Cyanide)

. The Technical Review of Acetonitrile (Methyl Cyanid e) has been revised to remove accumulation of cyanide as a potential mode of action. An understanding of the exact mode of action for a toxicological effect is not required in order to identify an effect as being of concer n in a hazard identification analysis. In the case of acetonitrile, the mode of action of noncancer toxicity is unknown, although limited information is available to support different hypotheses.18 Comment III

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- 17(e): The results from both the su bchronic and chronic studies do not support the following assumptions :

“With repeated exposure to acetonitrile, it is possible that continued generation of cyanide from the existing body burden of acetonitrile, coupled with cyanide generated from new ex posures, results in cyanide levels that exceed the capacity of enzymes to metabolize cyanide to thiocyanate” and

“As some deaths from acetonitrile did not occur immediately, but instead occurred after longer exposure, the results suggest that certain key biologic processes may be gradually overwhelmed or gradually degraded in function such that the body can no longer successfully counter the toxic effects of cyanide generated from acetonitrile” (p.35).

Based on the findings, the above statements are not b iologically plausible scenarios. If there had been cumulative toxicity resulting from repeated

- dosing, then the results should indicate a temporal relationship between effect and exposure

- duration. If Concentration x Time (C x T) effects are invoked as a possible explanation for the mortality findings, an increase in mortality with increasing exposure duration should have also been observed. This did not occur. The animal deaths in the subchronic study were concentrated in weeks 1

- 3; a gradual degradati on in function or an overwhelming of key biologic processes would predict that mortality would increase in subsequent exposure weeks, especially in the higher dose groups. Similarly, mortality would be expected in the 2

- year chronic study at lower concent rations.

It is understandable that U.S. EPA is concerned about possible mortality associated with a cyanide

- containing compound because mortality is a very frank, irreversible effect and cyanide has a steep acute concentration

- response curve. Lethality is well known as an acutely toxic response. However, it is the dose rate that determines the acute lethality of cyanide. The weight

- of

- evidence shows that a lower dose rate for a longer period of time is detoxified and it is only when the detoxificatio n system is acutely overwhelmed that lethality occurs. The data on mortality in the subchronic studies, in conjunction with the significant amount of knowledge on the mode of toxic action, suggest that the observed findings were due to acute toxicity. I n this context, the MTD was exceeded in the higher doses. Repeated dosing (6 hours/day, 5 days per week

– note that it was not continuous) is better construed as a series of acute toxicity doses with regard to lethality as an endpoint.

The pattern of responses under very high atmospheric concentrations of acetonitrile for a 6

- hour period is strongly supportive of an acutely toxic response. The clustering of deaths at the beginning of the study, in conjunction with an absence of mortality in the chroni c study and in the Hazelton (1983) subchronic studies, indicates that there is no CxT response. What is known about the acute and chronic modes of toxic action of cyanide provide further evidence in support of assigning mortality to acute, rather than chr onic, toxicity.

With regard to U.S. EPA’s statement that mortality was not observed in the chronic studies because exposure concentrations were significantly lower than those in the subchronic studies, I note that the acetonitrile concentrations in the chronic study are still orders of magnitude greater than relevant environmental concentrations. This is also a tautological argument.19 EPA Response III

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- 17(e): The two assumptions made by EPA and highlighted by the reviewer as “not biologically plausible” have been deleted.

In response to the reviewer’s assessment of the mortality data, the mortality data from these studies has been expanded within the draft Te chnical Review of Acetonitrile (Methyl Cyanide)

, including a discussion of dose selection, pertinent to the reviewers comments on the CxT response. In addition, the EPA does not agree that, “The data on mortality in the subchronic studies, in conjunction with the significant amount of knowledge on the mode of toxic action, suggest that the observed findings were due to acute toxicity”. Th e evidence from the NTP (1996) studies support the conclusion that long

- term repeated acetonitrile exposure can cause mortality in mice, with chronic toxicity likely occurring at a lower dose than mortality occurring following subchronic exposure. In addi tion, the mode of action for acetonitrile toxicity following long

- term repeated exposure is unknown.

Further, the Technical Review of Acetonitrile (Methyl Cyanide) is a hazard review. It is not a risk assessment. Therefore, the reviewers observation tha t the acetonitrile concentrations in the NTP (1996) chronic study are orders of magnitude greater than relevant environmental concentrations, have no bearing on this document. As noted in the general comments for this chapter, this document has been devel oped solely and specifically to support listing/delisting decisions for EPCRA section 313.

Comment III

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- 17(f): The pattern of dosing would also predict mortality as an acutely toxic response, not as a chronic

one.

The dosing regime was 6 hour s/day, 5 days/week for 13 weeks. Thus, animals were not exposed for 18 hours a day. There was also a non

- exposure period of approximately 60 hours over the weekend (Friday evening to Monday morning). Given what is known about cyanide, its toxicity and m odes of toxic action and detoxification, cyanide levels would have been significantly reduced during non

- dosing periods. Mortality is thus an acute response. Chronic effects, if they occurred, would be manifested in organ toxicity, such as liver toxicity , and would be associated with repeated oxygen deprivation in tissues.

EPA Response III

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- 17(f): It should first be noted that chronic studies often implement a repeated

- dose structure incorporating a non

- exposed period in the interim. The stud y design implemented by NTP (1996) is not an acute exposure, but rather a subchronic and chronic exposure duration. It is the EPA’s contention that the mortality observed in the NTP (1996) mouse inhalation study provides evidence that, above a threshold l evel, long

- term repeated acetonitrile exposure leads to mortality, and this mortality due to chronic exposure occurs at doses below those that cause mortality due to the acute exposures. In addition, mortality due to acute exposure to acetonitrile

occurs at doses greater than the doses at which mortality was observed in the subchronic study

.
In assessing mortality following acetonitrile exposure, the patterns in the timing of death across exposures demonstrates the chronic nature of the effect. In the NT P (1996) 13

- week mouse inhalation study, mortality was observed in the 800 and 1600 ppm treatment groups. The first incidence of mortality in the 800 ppm treatment group was not observed until day 20, and single deaths continued on days 21, 45, 69 and 89 of the 13

- week study. This pattern of mortality is20 dissimilar to that observed in the 1,600 ppm treatment group of the NTP (1996) 13

- week mouse inhalation study, where initial deaths were observed in the first week and all mice died by day 21. Based on t he observed pattern of death in the 800 ppm treatment group of the NTP (1996) 13

- week inhalation study, beginning at the end of the third week and extending through the termination of the study, it can be reasonably anticipated that additional acetonitrile

- induced mortality would have continued beyond the termination of the study and the sacrifice of surviving animals. Because the mortalities extended from the third week of the study to study termination, the data indicates that the mortality observed in t he 800 ppm treatment groups is not due to a single acute exposure to sufficiently high acetonitrile concentrations, but rather is best explained as being the result of long

- term repeated exposures.

Comment III

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- 18: Commenter Five Human relevan ce is not adequately addressed. Human relevance is noted in only one sentence and framed as a default statement (i.e., in the absence of data to the contrary, animal studies are assumed to be relevant to humans). The state

- of

- the

- science has progressed s ignificantly since the times when this type of sentence would be considered sufficient for inter

- species extrapolation (see Boobis et al. 2008 cited above). Human relevance topic should be expanded to indicate that there are sufficient data to indicate wh ether there are sufficient data to conclude that the mode of toxic action of acetonitrile and cyanide are qualitatively similar among species, including humans. How well are the quantitative differences among species (including absorption and metabolism) described? Further, as implied on p. 36, lines 7

- 10, if effects observed in animals are only likely to occur at very high acetonitrile atmospheric exposures in humans (which are orders of magnitude above environmentally relevant concentrations), is there human relevance?

EPA Response III

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- 18: Human relevance has been revised and is addressed in Section 3.10. Both mode of action among species and relative potencies among species are discussed. The Technical Review of Acetonitrile (Methyl Cyanide ) is a hazard review and the issue of environmentally relevant concentrations of acetonitrile is not included.

3.10. Human Relevance of Animal Data

“

Acetonitrile has been shown to cause death in rabbits, mice, rats, and humans (U.S. EPA, 1999). The molecular mechanisms underlying the cause of death for the various species are thought to be the same across species, and the clinical symptoms of exposure in cases of accidental or occupational human exposure are similar to those observed in studies using laboratory animals (WHO, 1993; U.S. EPA 1999). These symptoms of acetonitrile poisoning include neurotoxic effects (i.e. ataxia, tremors and convulsions) commonly associated with cyanide poisoning. This evidence strongly supports equivalent modes of action for acetonitrile lethality upon acute exposure for test animals and humans, and by extension suggests relevance of acetonitrile lethality due to chronic exposures in humans, as seen in animals from the NTP (1996) subchronic mouse inhalation study

. Death was observed in rabbits at dose (30 mg/kg

- day) much lower than that which caused mortality in rats, mice, and hamsters. Species differences in sensitivity are due to variability in toxicokinetic and toxicodynamic parameters. The toxicokinet ic parameters include binding affinity (K m ) and metabolic rate (V max ) for cyanide metabolism. These toxicokinetic parameters dictate the ability of species to detoxify acetonitrile and its metabolites, particularly cyanide moieties. Variability between s pecies is further compounded by the tissue specific inducible nature of the enzyme responsible for converting acetonitrile to hydrogen cyanide21 and/or formaldehyde (cytochrome P450) and the enzyme responsible for converting hydrogen cyanide to thiocyanate ( rhodanese). A study comparing the rhodanese activity of rats and humans observed that the respiratory epithelium of human nasal tissue had a lower affinity (K m ) for cyanide and a lower rate (V max ) for cyanide metabolism than rhodanese from rat nasal tissu e (Lewis et al., 1991). This suggests that humans may be more sensitive to cyanide than rats because cyanide may be detoxified to thiocyanate more rapidly in rat nasal tissue than in animal species with lower amounts or bioactivity of the enzyme (U.S. EPA , 1999) . No estimation of effects on sensitive human populations is currently available (e.g. fetal effects, genetic polymorphisms, individual variation). In the absence of human data associating chronic exposure of humans with increased risk of death, th e toxicity observed in animals following repeated exposures to acetonitrile is assumed to be relevant to humans. Case studies reporting mortality in humans after acute accidental poisoning describe effects consistent with those observed after exposure to inorganic cyanide and other saturated aliphatic nitriles exposures. Animal study findings suggest that the toxic effects of acetonitrile may be correlated with the metabolic release of cyanide and/or formaldehyde, a process that would also occur in humans

. ”

Comment III

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- 19: Commenter Five The weight

- of

- evidence narrative does not consider all data and is incomplete. This hazard assessment does not meet current quality standards for integration and synthesis of all available data. U.S. EPA (2005) provides an updated framework for a weight

- of

- evidence narrative evaluation, based on the Bradford Hill criteria for causality in epidemiology (Hill 1965).

Briefly, in a weight

- of

- evidence narrative, all data is considered simultaneously and evaluated fo r (1) strength of association; (2) consistency of association; (3) specificity of association; (4) temporality of association; (5) dose

- response or “biological gradient”; (6) biological plausibility; (7) coherence; and (8) other factors.

EPA Response III

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- 19:

Th e acetonitrile technical report provides an overall assessment and review of the toxicity of acetonitrile as it applies to listing/delisting decisions of EPCRA section 313(d)(2), which includes a review and consideration of both human heal th and environmental hazards. This technical review is also specifically focused on evaluating the scientific data relevant to the 2002 request for the delisting of acetonitrile under EPCRA section 313. It assesses the scientific soundness of the readily available data/studies including an assessment of the severity of the effect(s) and the dose or concentration levels at which the effect(s) were observed.

Comprehensive reviews of acetonitrile and cyanide toxicity have been published by the EPA IRIS pro gram, ATSDR, and the World Health Organization. This technical review is neither meant to revise nor replace these review documents. The weight of evidence narrative described by the commenter, and cited by the reviewer as U.S. EPA (2005), is intended for use in the analysis of a chemical’s human carcinogenic potential. As this document presents a hazard identification conclusion based on noncancer effects, the weight

- of

- evidence analysis, as described by this reviewer, is beyond the scope of the Technical Review of Acetonitrile (Methyl Cyanide).

Comment III

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- 20: Commenter Five There is a lack of rigor in assessing and interpreting individual study findings.22 EPA Response to III

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- 20: See specific subsections (a) through (d).

Comme nt III

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- 20(a): The health hazard assessment focuses on mortality and does not give sufficient consideration to other potentially adverse health effects, or lack thereof (see below).

EPA Response III

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- 20(a): Mortality was a main focus of th e assessment because EPA had previously reviewed acetonitrile in response to a petition to delist and the only chronic effect identified as sufficient to support listing was mortality. The petitioner focused their second petition on this effect, thus EPA’ s focus was on this effect. A more complete discussion of the effects observed following acute, short

- term, subchronic, and chronic exposure to acetonitrile, as well as a presentation of the developmental toxicity studies, has been added to the Technical Review of Acetonitrile (Methyl Cyanide).

Comment III

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- 20(b): No statistical significance evaluation has been conducted on any of the reported studies. As a result, the LOAEL/NOAELs of various studies are overestimates.

The statistical signifi cance of cancer findings is presented and discussed. A major omission is lack of consideration of statistical significance of noncancer findings. Current guidance mandates an evaluation of the statistical significance of all findings.

Dose groups in which one or two animals die are not effect

- levels because as compared to concurrent (or historical) controls, the increase in mortality would not significantly differ from chance. This increase would not be either statistically or toxicolog ically significant. Although mortality is a frank effect, the death of one or two animals in a dose group is usually considered a random event which can also occur in control animals, even if there is a significant increase in mortality incidence at highe r doses. Therefore, a dose level in which 1 or 2 deaths occur does not constitute an effects level.

In the NTP (1996) 13

- week rat studies, the mortality incidence for males was 0/10 in each of the 0, 100, 200 and 400 ppm groups, 1/10 in the 800 group (one death occurring in week 1), and 6/10 in the 1600 group (four deaths occurring during week 1, one death occurring during week 2, one death occurring during week 4). For females, the mortality incidence was 0/10 in each of the 0, 100, 200, 400 and 800 ppm groups, and 3/10 in the 1600 ppm group (one death occurring during week 1, two deaths occurring during week 2). In the 13

- week mouse studies, the mortality incidence in males was 0/10 in each of the 0, 100, 200 and 400 ppm groups, 1/10 in the 800 ppm group (death occurring sometime during study weeks 6

- 13), and 10/10 in the 1600 ppm group (two deaths occurring during week 1, eight deaths occurring during week 3). In females, the mortality incidence was 0/10 for the 0, 100 and 200 ppm groups, 1/10 in the 400 ppm group (occurring during week 2), 4/10 in the 800 ppm group (one occurring during week 2, two occurring during week 3, and one occurring during weeks 6

- 13), and 10/10 in the 1600 ppm group (four occurring during week 1, six occurring during week 3).

Inspection of these data for statistical significance suggests that (1) the data would be statistically positive in a trend test; and (2) pairwise comparisons would indicate a significant effects level of23 1600 ppm for rats and 800 ppm for mice, not 8 00 ppm for rats and 400 ppm for mice.

EPA Response III

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- 20(b): The EPA agrees that statistical significance of deaths within the various treatment groups should be addressed and recognizes the fact that a single death within a test group may not b e statistically significant. The NOAEL and LOAEL identifications for the NTP (1996) subchronic study were updated in the draft Technical Review of Acetonitrile (Methyl Cyanide) to reflect this comment. In addition, the description of noncancer observatio ns, including statistical significance has been extended.

Comment III

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- 20(c): A fuller discussion of adverse findings, including mortality, is needed for completeness, comprehensiveness and transparency.

NTP (1996) reports that that (1) the maj ority of deaths occurred during the first two weeks of exposure; and (2) the spectrum of lesions reported in animals dying early was similar to those encountered in early

- death animals in other NTP studies (in addition to being lesions consistent with prev ious descriptions of acetonitrile toxicity). Thus, it is reasonable to assume that these early deaths were associated with overdosing. Overdosing with a range of compounds typically results in clinical signs of neurotoxicity which may be followed by deat h.

NTP (1996) also notes that (1) the character of the forestomach lesions (i.e., hyperplasia with and without inflammation, focal ulceration) observed in mice surviving to the end of the study has also been seen in mice in other NTP inhalation studies (Melnick et al., 1990), and (2) this suggests that the effect may not be specifically related to acetonitrile exposure but rather to “some other factor associated with the conditions of exposure” (p.55). The most likely explanation for this histopathology is incidental ingestion which would be increased by grooming

- and

- swallowing behavior during whole body exposure.

It is useful to note that acetonitrile is water

- soluble and is also irritating to the skin and the respiratory tract. Acetonitrile is also rapidly and efficiently absorbed through skin and the gastrointestinal tract. Therefore, given the multi

- route exposure pathways (inhalation + dermal + incidental ingestion), the internal acetonitrile concentrations are likely to have been much higher tha n target inhalation concentrations in animal studies.

EPA Response III

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- 20(c): The Technical Review of Acetonitrile (Methyl Cyanide) has been revised to incorporate a more complete discussion of adverse effects observed in the NTP (199 6) studies. A full discussion of the mortality from the 13

- week and 2

- year studies is included in the text and Figure 3

- 2. Forestomach effects are also discussed.

Although whole body exposure does allow multiple routes of exposure, exposure via inhala tion is the primary route. Further, this study design utilized a target ambient air concentration, and not a “ target inhalation concentration”. In addition, there is no evidence supporting the reviewers comment that “the internal acetonitrile concentrations are likely to have been much higher than target inhalation concentrations.”

Comment III

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- 20(d): Other adverse effects:24 Subchroni c (NTP 1996) rodent studies : In rats, clinical signs of neurotoxicity during week 1 were hypoactivity and ruffled fur at 800 and 1600 ppm; ataxia and abnormal posture and clonic convulsions in animals who died during the first week. Changes in the following organ weight were observed: (1) significantly decreased absolute and relative thymus weights in males and females at 800 and 1600 ppm; (2) significantly increased absolute te stis weight at 1600 ppm; and (3) significant increases in the absolute or relative weights of the heart, liver and kidney at 800 and 1600 ppm; an increase in absolute heart weight also occurred at lower doses (≥ 200 ppm). Other effects included: (1) a

no nresponsive, normocytic and normochromic anemia in both sexes at 1600 ppm and in females at 800 ppm; and 20 decreased serum triiodothyronine (T3) concentrations in 1600 ppm females in the absence of alterations in serum throxine (T4) and thyroid

- stimulatin g hormone (TSH). No histopathology unrelated to early death was observed in any animals.

In mice, clinical signs of neurotoxicity occurred only during week 1 and consisted of hypoactivity and a hunched, rigid posture at 800 and 1600 ppm. As all animals in the 1600 ppm group died early, 800 ppm was the highest subchronic dose tested for systemic effects in mice. Relative to concurrent controls, the following statistically significant organ weight changes were observed: (1) increased absolute liver weights in males at ≥ 200 ppm and in females at 800 ppm; (2) increased relative liver weights in all exposed male groups and in females at ≥ 400 ppm. Histopathology demonstrated the presence of microscopic lesions in the liver (i.e., hepatocellular cyptoplasmic vacuolation in both sexes at 400 and 800 ppm) and in the anterior forestomach (i.e., focal and multifocal hyperplasia/lesions/ulceration at exposure concentrations ≥ 400 ppm in males and ≥ 200 ppm in females).

Based on these effects, the critical effect s for subchronic (systemic) toxicity in mice are hepatic and stomach. Forestomach histopathology was observed in all but the lowest dose group in females. Using this end point, the LOAEL and NOAEL would be 200 and 100 ppm, respectively. However, humans d o not have a forestomach and thus the human relevance of these lesions is questionable. A more relevant endpoint is hepatic effects. Acetonitrile and its active metabolite cyanide have been shown to induce hepatic effects in other studies (concordance of information across studies). An increase in liver weight was observed at exposure concentrations which were less than or equal to the exposures at which liver histopathology was observed. This pattern of hepatic events is consistent with a cumulative
to xic effect resulting from repeated

- dosing, and indicative of a concentration x time (C x T) effect. In contrast, there is no indication of a cumulative mortality effect.

Chronic studies : No mortality was observed in the chronic study in either rats exp osed to vaporized acetonitrile at concentrations of 0, 100, 200 or 400 ppm (equivalent to 0, 168, 335 or 670 mg/m3) or in mice exposed to 0, 50, 100 or 200 ppm (equivalent to 0, 84, 168 or 335 mg/m3). Hepatoxicity was observed in male, but not female, r ats. There was also a marginally significant increase in hepatic tumors (combined adenomas and carcinomas) in male, but not female, rats. In mice, a dose

- related increase in the incidence of hyperplasia of the forestomach epithelium was observed in both males and females (NTP 1996).

Developmental studies: Developmental effects were only observed at maternally toxic doses. This fact is not highlighted in the hazard assessment, as it is in WHO’s Environmental Health Criteria document (WHO25 1993). Accord ing to current hazard and risk assessment guidelines, developmental effects observed at maternally toxic doses are assumed to be indirect (due to maternal toxicity). Therefore, acetonitrile would not be considered a developmental or reproductive toxicant according to current hazard and risk assessment guidelines. If mortality is statistically elevated at the highest dose tested, then it would be considered treatment

- related. However, developmental toxicity at that dose level would be attributed to matern al toxicity. Many compounds induce some degree of mortality at the highest dose tested.

EPA Response III

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- 20(d): The description and analysis of the effects observed in animals following acute, subchronic, and chronic exposure to acetonitrile, as well as the developmental toxicity studies, have been expanded upon within the Technical Review of Acetonitrile (Methyl Cyanide) . The EPA disagrees with the commenter and has retained mortality as the critical effect from the NTP (1996) subchronic study. EPA recognizes that there were additional subchronic and chronic effects observed; however, mortality was a main focus of the assessment because EPA had previously reviewed acetonitrile in response to a petition to delist and the only chronic effect iden tified as sufficient to support listing was mortality.

With regard to the developmental toxicity studies, EPA recognizes that developmental effects were observed at maternally toxic levels and this has been highlighted in the Technical Review. However, it is inadequate to assume that the developmental effects result only from maternal toxicity, and the results may indicate that both lifestages, the adult and developing offspring, are sensitive to the dose level (U.S. EPA, 1991).

B. Chapter Three, Char ge Question 2.

The U.S. EPA has determined that mortality is the critical endpoint for acetonitrile as described in the IRIS “Toxicological Review of Acetonitrile” (1999). Mortality has been observed in a 13

- week subchronic study as well as in developmen tal and reproductive toxicity studies. Mortality was not observed in a 2

- year chronic study conducted at doses below those that caused mortality in the 13

- week subchronic study. Based on the totality of available evidence, particularly the 13

- week and 2

- y ear studies, is it reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure? Please explain.

Comment III

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- 1: Commenter One The answer to this question is: Yes. Based on the available human and animal data, I would anticipate that ACN could cause mortality as a result of chronic exposure, given the right species and the right period of exposure. My interpretation of the data that are available and presented in this document is outlined below.

The data indica te that ACN causes death in rabbits, mice and rats. It causes death in rabbits at much lower doses than that which was administered in the two year studies to mice and rats. Due to the similarities in metabolic enzymes between rabbits and humans and due to the fact that humans are less able to than rats to detoxify cyanide, I would expect that health effects and mortality in humans, following human exposure to ACN, would be closer to that observed in rabbits than to that observed in mice and rats. Therefore , I would26 anticipate that human would be more sensitive to ACN actions than mice and rats. Thus, the data derived from the 2 year chronic exposure of rats and mice to low concentration of ACN should not be used to un

- list ACN from the current EPA regulatio ns.

Furthermore, most, if not all, of the conclusions are based on the assumption that the lack of apparent lethality from ACN in rats and mice in the two year studies using low concentrations is due to the ability of rats and mice to detoxify ACN metabol ites, particularly cyanide moieties.

The lack of such an apparent endpoint (mortality) of adverse effect does not mean that there were not any adverse effects that had occurred on the molecular levels within the experimental animal species utilized in the se studies.

To explain this statement, I would like first to describe ACN metabolism. ACN is known to be oxidized by Cytochrome P450 2E enzyme system or by other oxidizing enzymes to yield a reactive intermediate, methyl cyanohydrine. The latter intermediate is in equilibrium with equal molar concentrations of cyanide ions and formaldehyde.

CH 3 CN

------------ CH 2 OHCN

--------- CH 2 O + HCN

Cyanide ions had been implicated in most aliphatic nitriles’ toxicity including that of ACN. The other metabolite, formaldehyde (CH 2 O) has not been discussed in the interpretation of ACN toxicity and lethality. Formaldehyde on the other hand has its own set of toxic actions. Aliphatc aldehydes, in general, are known to block the activities and funct ions of several enzymes via the formation of Schiff’s base with the amino groups in the enzymatic reactive sites. Also, CH 2 O is known to deplete anti

- oxidants in the cellular environment leading to cellular oxidative stress and oxidative damage. Both cyanide and aldehyde are known to alter mitochondrial chain reactions leading to mitochondrial membrane damage and the release of Cytochrome C, which activates apoptosomal enzymes, leading to the break down of nuclear DNA and the induction of cellular apoptosis. Because cellular apoptosis is a slow process, it is expected that mortality caused by cellular apoptosis to be a slow pr ocess during the life span of the cell. During these processes, tissues and organ degeneration may constitute a signal for tissue or organ injury that leads to mortality.

Acute exposure to ACN will yield large number of formaldehyde molecules and cyanide ions. Both will cause severe oxidative stress and cause massive blockage of respiratory chain reactions involved in oxygen metabolism leading to the sudden accumulation of reactive oxygen species, cellular, tissue, and organ damage, and subsequent death. Even in acute processes, delayed death usually occurs as a result of the sudden accumulation of cell, tissue and organ damage that could be caused by sudden increase in oxidative damage and interference with oxygen metabolism.

Sub

- acutely, the processe s of damage accumulation may take a while longer and when enough damage accumulates, organ malfunction may occur leading to death as indicated in the 13

- week ACN sub

- acute studies.

If exposure occurs at lower doses (such as that in the chronic studies) , eventually enough molecular damage will accumulate at a longer time period (may be more than the two year period used in the studies conducted in the least sensitive species; rat and mice but a shorter period in the more sensitive species such as the rab bits and humans).27 Therefore, I don’t believe that the lack of mortality indicated in the available chronic studies in rats and mice (2 year) justify the un

- listing of ACN from the criterion described because:

a. In the chronic studies described, highly insensitive species (rats and mice) were used. Thus, the doses that would produce mortality following chronic exposure would be high.

b. The duration of two years used for the studies described may not be enough to observe mortality in the species used.

c. Due to the known toxicity of ACN metabolites, CN and CH 2 O, molecular damage may have occurred in these species that may lead to altered physiological functions and other chronic disorders without the induction of death.

d. Due to the known teratogenic activitie s and developmental effects of ACN, and because of the sensitivity of developing fetuses to in

- utero exposure to very low levels of environmental chemicals, it is anticipated that molecular damage may occur in the fetus at low levels of exposure. Such mole cular damage during fetal development may lead to developmental abnormalities and impaired offspring that are susceptible to various diseases.

EPA Response III

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- 1: The reviewer’s description of acetonitrile metabolism is greatly appreciated. This information has been incorporated into the Technical Review of Acetonitrile (Methyl Cyanide). Figure 3

- 1 has been added to clarify the acetonitrile metabolic process. In addition, text was added to Section 3.8., Potential Modes of Action, describing inh ibition of additional biochemical processes, other than cytochrome oxidase, by cyanide.

3.8. Potential Modes of Action

“ The available data indicate that the observed mortality following acute exposure to acetonitrile is mediated through its metabolism t o cyanide (Hartung, 1982; WHO, 1993; U.S. EPA 1999). Cyanide inhibits a number of enzyme systems, the main target of which is cytochrome c oxidase, the terminal oxidase in the mitochondrial respiratory chain (Ballantyne, 1987). Cyanide has long been know n to react with trivalent iron of cytochrome c oxidase in mitochondria and block the reduction of oxygen needed for cellular respiration, thus leading to cytotoxic anoxia (Albaum et al., 1946). The acute toxicity of acetonitrile is believed to be mediated , in part, through this mechanism. If the conversion of acetonitrile to cyanide leads to systemic cyanide concentrations that bind the cytochrome c oxidase for a critical period, it may lead to death. Although acute oral doses of cyanide cause cardiovasc ular, respiratory, and neurophysiological changes, the brain appears to be the organ that is most sensitive to acute cyanide toxicity (WHO, 2004). Death from acute exposure to cyanide is believed to result from central nervous system depression, subsequen t to inhibition of brain cytochrome c oxidase activity (Way, 1984). The role of metabolism may be crucial in determining the acute toxicity of acetonitrile via cyanide production. Mortality may result from the absorption and metabolism of high doses of acetonitrile into cyanide at a rate that exceeds the body’s capacity for detoxification. In contrast, slow absorption of the same or greater total load may allow for complete metabolism and not cause mortality due to acute exposure. Acetonitrile is metab olized to inorganic cyanide but the conversion occurs slowly compared to other nitriles (Hartung, 1982), which may explain the delay in onset of acute symptoms (U.S. EPA, 1999). The mode of action for acetonitrile toxicity following chronic exposure is un known,28 although the metabolism of acetonitrile to its reactive metabolites is likely required for toxicity. For chronic acetonitrile exposure to cause mortality through the inhibition of cytochrome c oxidase by cyanide, an accumulation of systemic cyanide reaching a critical level may be necessary to bind enough cytochrome c oxidase to cause inhibition of cellular respiration. Currently, evidence in humans or animals is not available demonstrating, either qualitatively or quantitatively, that systemic cya nide accumulates over time due to chronic acetonitrile exposure. In addition, there is currently not enough data in the scientific literature to quantitatively construct a physiologically

- based pharmacokinetic (PBPK) model of acetonitrile toxicokinetics i n humans. It is also plausible that other biological systems and enzymes, besides cytochrome oxidase, may be inhibited by cyanide (Way, 1984), leading to the accumulation of cellular damage over time and the delayed acetonitrile

- induced mortality obse rved in the NTP (1996) mouse study. Lethality from adverse effects of repeat chemical exposures can also result from cumulative, irreversible damage to a critical molecular/cellular target such that the summation of damage eventually exceeds a threshold f or non

- viability of the organism (Klaassen, 2008). However, data demonstrating an accumulation of cellular damage with subsequent cell death following exposure to acetonitrile or cyanide is not available. ”

Comment III

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- 2: Commenter Two The descri ptive data do suggest that a few adverse effects occur at inhalation doses of acetonitrile below those that induce mortality. However, the document does not adequately explore the occurrence of effects that were anticipated in the subchronic and chronic e xperiments conducted by the National Toxicology Program. As indicated in the general comments above, the metabolic conversion of acetonitrile, first to cyanide and then to thiocyanate, was expected to produce effects on thyroid function. Thyroid hormone measurements were made in the 13

- week study and there was some suggestion of the expected effects on thyroid hormones in male and female rats at the 1600 ppm dose with a trend in the dose

- response data in lower doses. Moreover, there was evidence of incre ased C

- cell hyperplasia in the thyroid of female rats at all doses (statistical analysis not explicitly presented). As these were expected toxicities of acetonitrile they should have been discussed in the document and a formal assessment made of whether t hese data were relevant to humans (there are reasons for not considering rat data in where effects on thyroid function. Rats are much more sensitive to such effects than humans). In considering these effects on the thyroid, the Agency should consider whe ther the available reproductive and developmental toxicity studies were conducted in a fashion that would allow effects of modified thyroid function on development to be evaluated. My review of these papers indicates that no treatments beyond the 20 th ges tational day were performed. The critical period for thyroid

- dependent development events within the brain of the rat occurs in the postnatal period. Therefore, I suspect the Agency would ordinarily conclude that this is a critical data need and would mo dify their assessment to account for the deficiencies in the database.

EPA Response III

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- 2: The description and analysis of the effects observed in animals following acute, subchronic, and chronic exposure to acetonitrile, as well as the developmental toxicity studies, have been expanded within the Technical Review of Acetonitrile (Methyl Cyanide ) . In addition, the discussion of acetonitrile induced thyrotoxicity has been expanded within the technical review. As stated previously, the Technical Review of Acetonitrile (Methyl Cyanide) is a hazard review and not a risk assessment; therefore, it do es not have a vehicle for modifying the assessment to account for implied data needs.29 Comment III

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- 3: Commenter Two The only reference to the possibility of thyroid effects is a reference to Hartung et al. (1982) in referring to the effects of t hiocyanate. Thiocyanate is known to be a potent inhibitor of iodide transport in humans as well as experimental animals. Therefore, it is critical that the document consider whether there has been an adequate experimental evaluation of risks that would a rise from the generation of this important metabolite that arises in the metabolism of acetonitrile.

The evaluation of toxicities that could be potentially attributed to anti

- thyroid effects of the metabolite, thiocyanate, should be discussed and taken to a transparent conclusion in the “Conclusions” section, that is found at the beginning of the document.

EPA Response III

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- 3: The discussion of potential thyroid effects has been expanded in Section 3.9. Thyroid Toxicity, of the Technical Review of Acetonitrile (Methyl Cyanide). See Response to Comment III

- B

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- 17.( d). The evaluation of toxicity attributed to anti

- thyroid effects was not described in the conclusion section, which instead focused on the observed mortality, because effects indicating thyrotoxicity were not observed in the studies of acetonitrile expos ure.

Comment III

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- 4: Commenter Three This reviewer believes that the available science supports the conclusion that acetonitrile can be reasonably anticipated to cause mortality as a result of chronic exposure. This opinion is based upon interpre tation that mortalities in rodents observed after repeat exposures during the subchronic 1996 NTP study occurred as a consequence of cumulative adverse effect of the chemical, rather than as an isolated, immediate response to one of the latter doses. Henc e, it is reasonable to project that the same qualitative adverse effects would continue to accrue with additional dose applications upon extension of the exposure period. This would manifest as additional, later deaths in a chronic protocol at the lower e ffective dose level of the subchronic study. Lack of direct determination of this anticipated result is a consequence of failure to include the subchronic LOAEL (400 ppm for mice; 800 ppm for rats) in the chronic study; absence of mortality with a 2

- fold lower dose does not preclude mortality occurring at the subchronic LOAEL.

EPA Response III

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- 4: The EPA agrees with these comments and has included this reasoning in the Technical Review of Acetonitrile (Methyl Cyanide).

Comment III

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- 5: Commenter Three Definitive evidence that mode of action for acetonitrile lethality upon acute exposure is equivalent for test animals and humans suggests relevance to irreversible chronic health effects to humans; however, confidence in this opinion is les sened by a lack of direct evidence that the same mode of action mediates lethality from repeat dose, short term or subchronic exposure to acetonitrile in rodents as from high dose acute exposure to acetonitrile.

EPA Response III

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- 5: An understandi ng of the mode of action for a toxicological effect, which may be supportive of the critical effect identified in certain scenarios, is not required to identify a critical effect for use in a hazard identification analysis. In the case of acetonitrile, th e mode of action of noncancer toxicity is unknown; although limited information is available to support different hypotheses.30 Comment III

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- 6: Commenter Three Another important issue for CERCLA listing is relevance of rodent mortality from chro nic acetonitrile exposure to humans. Extrapolation of rodent toxicities to humans is best supported by demonstrating equivalent mechanisms. It is clear from observations taken from emergency medicine that acute acetonitrile poisoning in humans occurs via the same mechanism as acute animal lethality, i.e., through cytochrome P450

- dependent production of cyanide and consequential inhibition of mitochondrial cytochrome c oxidase, especially that of central nervous system (CNS) respiratory control centers. T he Technical Review suggests (p 5, line 2), that repeat exposure to acetonitrile will result in “continued generation of cyanide from the existing body burden of acetonitrile, coupled with the cyanide generated from new exposures” such that detoxication ca pacity for cyanide will be exceeded. The implication is that toxic effects from repeat dose mediated through the same mode of action, i.e., through cyanide, as acute effects. If so, relevance to humans is highly feasible. However, there is no direct
exp erimental evidence presented to support equivalent mode of action for lethality from both exposure protocols. Information on blood and tissue cyanide levels after repeated acetonitrile doses associated with lethality would strengthen the conclusion that bi ologically equivalent doses of reactive metabolite occurs with both acetonitrile exposure protocols. Comparable inhibition of target tissue (brain) cytochrome oxidase of rodents from high

- dose acute and lower, repeat

- dose acetonitrile exposures would indi cate an equivalent molecular mechanism. In contrast, the 1996 NTP report (p. 18) describes a decrease in blood cyanide concentration with time in a 1994 NTP inhalation developmental study (cited in the Technical Review as Mast et al., 1994) from GD8 to GD 18 and suggests induction of rhodanese may be responsible. If so, thiocyanate, which has known thyroid effects, would be expected to increase. A reduction in serum T 3 was reported in high dose rats of the 13

- wk study. Also, one lesion characteristic of low

- dose, chronic cyanide exposure, demyelination, was not reported in the 13

- wk histopathology of the 1996 NTP report (p. 32) although brain was included in the tissues examined. Collectively, these data gaps and additional observations cause this review er to question whether lethality from low repeat

- dose exposures occurs through the same mode of action as high

- dose acute acetonitrile poisoning and to be less confident about extrapolation of mortality from chronic acetonitrile exposure to humans.

EPA Re sponse III

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- 6: The extrapolation of animal toxicities to humans, as well as a discussion of relative sensitivities, is included in section 3.10., Human Relevance of Animal Data. In Section 3.8., Potential Modes of Action, the mode of action for both acute exposure and c hronic exposure is addressed. However, the proposed mode of action of cyanide accumulation through repeated acetonitrile exposures has been removed from the document.

Comment III

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- 7: Commenter Three The technical reviewers support similar mode of action of acetonitrile lethality in test animals vs. humans by comparing clinical symptoms (ataxia, convulsions and abnormal posture; p, 2, line 11). This same sentence, however, is supportive of similar modes of action of lethality from acute and rep eat dose exposures, but the information is not apparent because it is unspecified that the comparison is between repeat dose animals (NTP 1996; p. 32, line 2) and acute poisonings of humans. Although these symptoms are nonspecific, a similar approach coul d be taken to provide some support for similarity of mode of action of lethality from acute vs. repeat dose acetonitrile exposure in animals. For example, Wilhite (1981) describes clinical symptoms of acute acetonitrile toxicity that include neurotoxic ef fects of tremors and convulsions. Such clinical symptoms of acute toxicity could be added to section 3.4.1. Similarly, clinical symptoms of the31 affected dams in the repeat dose, short term rabbit study (Argus 1984, p IV

- 2) include ataxia, decreased motor activity and loss of righting reflex could be mentioned as qualified with Study Director Hoberman’s observation, expressed Apr 1, 2002, to Mr. D.E. Strother, that gastrointestinal irritation and decreased food consumption were coincident adverse effects. Although not as strong as a demonstration of equivalent biological dose at the target, explicit comparison of similar clinical symptoms indicating neurotoxicity observed in the acute, short term and subchronic studies should be presented to provide some s upport for same mode of action (i.e., cyanide inhibition of CNS cytochrome oxidase) of lethality in the three protocols.

EPA Response III

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- 7: The Technical Review of Acetonitrile (Methyl Cyanide) details the human relevance (Section 3.10.) of effe cts observed in animal studies of different durations. EPA agrees with the reviewer’s comments regarding a mutual mode of action across species for acute exposures and possible extension to repeat dose exposures. Clinical symptoms of exposure are refere nced to the USEPA (1999) and WHO (1993) reports. However, acetonitrile

- specific information is not available to determine the mode of action of acetonitrile toxicity following chronic exposure, and mode of action was not a consideration in the identifica tion of the critical effect.

Comment III

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- 8: Commenter Three The technical review has very convincingly presented evidence indicating that the effect of any one interim daily dose of a repeat dose protocol is likely augmented by residual effects f rom remaining fractions of uncleared acetonitrile from the preceding one or two days’ administrations. Summarized information also makes a strong argument that a nearly continuous production of cyanide ensues. However, it has not been demonstrated that t he accumulated tissue dose of cyanide can approach levels of disposition associated with high dose acute exposure causing lethality. Alternatively, lethality from adverse effects of repeat dose can also result from cumulative, irreversible damage to a cri tical molecular/cellular target such that the summation of damage eventually exceeds a threshold for non

- viability of the organism. Lack of lethality in the 2

- yr bioassay at 50% of the effective subchronic dose suggests that a threshold for the accrual of these lesions exists. Continued cyanide production at lower levels after termination of daily acetonitrile exposure may contribute to the irreversibility of lesions by inhibiting tissue repair. Cellular anoxia from cyanide inhibition of cytochrome oxida se could have lasting effects subsequent to toxicant clearance analogous to the oxidative damage that occurs upon reoxygenation of a previously hypoxic tissue. These collective effects may or may not be equivalent mechanistically to that contributing to l ethality upon high dose acute exposure. However, if accrual of functional molecular/cellular defects is relevant, it is unclear why some evidence of their presence was not detected histopathologically. Perhaps detection would require sample collection fro m nonmorbid animals, since target tissue (brain) is prone to artifacts upon death and delayed immersion fixation. Histopathology of the 13

- wk studies of 1996 NTP study was restricted to animals that died on trial; hence, information from nonmorbid animals was not available.

EPA Response III

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- 8: The assertion that the toxicokinetic data necessary to adequately support the conclusion that accumulated tissue doses of cyanide can approach levels of disposition associated with high dose acute exposure causing lethality has been removed from the Technical Review of Acetonitrile (Methyl Cyanide) . A discussion of accumulated irreversible cellular damage due to the inhibition of other biochemical processes by acetonitrile metabolites has been added as one of the potential modes of action.32 Comment III

- B

- CH3

- Q2

- 9: Commenter Three Lethality from adverse effects of repeat dose can also result from cumulative, irreversible damage to a critical molecular/cellul ar target such that the summation of damage eventually exceeds a threshold for non

- viability of the organism. Lack of lethality in the 2

- yr bioassay at 50% of the effective subchronic dose suggests that a threshold for the accrual of these lesions exists. Continued cyanide production at lower levels after termination of daily acetonitrile exposure may contribute to the irreversibility of lesions by inhibiting tissue repair. Cellular anoxia from cyanide inhibition of cytochrome oxidase could have lasting effects subsequent to toxicant clearance analogous to the oxidative damage that occurs upon reoxygenation of a previously hypoxic tissue. These collective effects may or may not be equivalent mechanistically to that contributing to lethality upon high dos e acute exposure. However, if accrual of functional molecular/cellular defects is relevant, it is unclear why some evidence of their presence was not detected histopathologically. Perhaps detection would require sample collection from nonmorbid animals, s ince target tissue (brain) is prone to artifacts upon death and delayed immersion fixation. Histopathology of the 13

- wk studies of 1996 NTP study was restricted to animals that died on trial; hence, information from nonmorbid animals was not available.

E PA Response III

- B

- CH3

- Q2

- 9: Section 3.8., Potential Modes of Action, of the Technical Review of Acetonitrile (Methyl Cyanide) has been modified to reflect this comment

.

Comment III

- B

- CH3

- Q2

- 10: Commenter Four I agree completely with the conclusion that mo rtality is the critical effect that must be addressed in any risk assessment of acetonitrile.

EPA Response III

- B

- CH3

- Q2

- 10: Although this is a technical review of acetonitrile and not a risk assessment, the EPA agrees that mortality is the critical effect associated with acetonitrile exposure and is therefore the primary focus of the Technical Review of Acetonitrile (Methyl Cyanide).

Comment III

- B

- CH3

- Q2

- 11: Commenter Four However, this (mortality in the NTP (1996) studies) is an effect that arises as a c onsequence of cyanide

- induced toxicity, which is itself an acute or subacute (a misnomer) effect, i.e., an effect that arises (or not) from just a few days worth of exposure to a sufficiently high acetonitrile concentration. Death may be delayed some, per haps by at most a few weeks, but the damage which results in death will have been done (or not) in just a few days worth of exposure. Thus, I disagree strongly with the assertion that it is reasonable to anticipate that acetonitrile would cause mortality as a result of chronic exposure. First and foremost is the fact that survival was not affected adversely by chronic inhalation exposure of mice (rats) to airborne acetonitrile concentrations up to 200 (400) ppm in the NTP study (1996). This maximum exposure level for mice of 200 ppm acetonitrile, which lasted 6 hours per day, 5 days per week, for up to 2 years, corresponds to a cumulative acetonitrile exposure of approximately 26,071 ppm

- days. In the related subchronic study (NTP, 1996) that was cond ucted in part to establish the doses for the chronic study, significant mortality was observed in mice exposed to 800 ppm acetonitrile for as little as 3 weeks, and all mice exposed to 1600 ppm died within 3 weeks of exposure onset. These exposures corres pond to cumulative acetonitrile exposure of only 3,00033 and 6000 ppm

- days, respectively. Thus, if there were in fact a toxicity mechanism operating that caused mortality as a consequence of cumulative acetonitrile exposure, then increased mortality should have been readily apparent in the NTP chronic oncogenicity study, not only in the mice exposed to 200 ppm (26,071 ppm

- days), but also in the mice exposed to 100 ppm (13,036 ppm

- days) and 50 ppm (6,518 ppm

- days). Yet, there were no effects on clinical sig ns, body weight, survival, complete histopathology, hematology, or targeted organ weights in any of the exposed groups of either mice (or rats) in the NTP (1996) study. In my opinion, the assertion that acetonitrile should be reasonably anticipated to cau se mortality as a result of chronic exposure has no scientific support whatsoever.

EPA Response III

- B

- CH3

- Q2

- 11: The EPA believes there is not enough evidence to make the statement that “This (mortality in the NTP (1996) studies) is an effect that arises as a consequence of cyanide

- induced toxicity, which is itself an acute or subacute (a misnomer) effect, i.e., an effect that arises (or not) from just a few days worth of exposure to a sufficiently high acetonitrile concentration.” The mode of action for acetonitrile toxicity following chronic exposure is unknown. Modes

- of

action have been proposed and supported by the scientific literature as potentially contributing to acetonitrile toxicity. Furthermore, the drastically varied patterns of death between the 800 ppm and 1600 ppm subchronic mouse study suggests there may be multiple modes of action dependant on dose. As stated previously, mortality in the 800 ppm treatment group of the NTP (1996)13

- week mouse study extended throughout the entire 13

- week s with the deaths on days 20, 21, 45, 69, 89. This pattern of mortality is not “delayed some, perhaps by at most a few weeks,” and is not consistent with the conclusion that damage from the first few days of exposure led to later term mortality. In addi tion, the doses used in the NTP (1996) 2

- year study were 4

- fold lower than the LOAEL for mortality in the NTP (1996) 13

- week study. This combined with the recognized steep dose

- response curve for acetonitrile supports the conclusion that the doses used in the NTP (1996) 2

- year study were not sufficient to cause mortality. The lack of mortality in the lower dose levels of the NTP (1996) 2

- year study does not preclude mortality due to chronic exposure at higher dose levels or for longer durations. As deta iled in Comment III

- B

- CH3

- Q1

- 17.(d). by Commenter Five

, “The role of metabolism is crucial in determining the acute toxicity of ingested cyanide. Both the total amount administered and the rate of absorption are important (U.S. EPA 1992) because acute

tox icity results from rapid ingestion of high doses of cyanide at a rate that exceeds the body’s capacity for metabolism (detoxification). In contrast, slow absorption of the same or greater total oral load of cyanide may allow for complete metabolism. Unmet abolized cyanide can be both acutely and chronically toxic, although cyanide is not known to accumulate in the body.” Therefore, the decreased rate of exposure, and not solely the increased total amount administered, in the NTP (1996) 2

- year study is a cr itical component that may affect the observed mortality.

Comment III

- B

- CH3

- Q2

- 12(a): Commenter Five U.S. EPA is asking peer reviewers whether the existing science supports a conclusion that acetonitrile can be reasonably anticipated to cause mortality as a result of chronic exposure. Based on a critical weight

- of

- evidence evaluation of available data on cyanide and acetonitrile, my answer to this question is No.34 The reasoning that U.S. EPA presents in support of its position that chronic or repeated

- dose exposure to acetonitrile causes mortality is not scientifically defensible. (See specific subsectio ns)

First, the data do not support this interpretation. Among repeated

- dose studies, only one subchronic study showed mortality; the deaths were clustered at the beginning of the 13

- week experiment (discussed in more detail above).

EPA Response III

- B

- CH3

- Q2

- 12(a): Mortality in the 800ppm treatment group of the NTP (1996) 13

- week mouse study extended throughout the entire 13

- weeks with the deaths on days 20, 21, 45, 69, 89. This pattern of mortality should not be classified as “clustered at the beginn ing of the 13

- week experiment”. This pattern of delayed mortality observed in the 800 ppm treatment group is distinct from that seen in higher dose group (1600ppm) resulting in rapid mortality due to acute exposure (See Figure 3

- 2). These data support th e conclusion that extended repeat exposures to acetonitrile, at levels below those that result in mortality due to acute exposure, may cause mortality.

Comment III

- B

- CH3

- Q2

- 12(b): Second, based on what is known about the toxicokinetics of acetonitrile a nd cyanide, and their modes of toxic action, mortality induced by chronic acetonitrile or cyanide exposure is biologically implausible.

Why? The mode of toxic action for cyanide

- associated mortality is acute. The dose

- response is steep but mortality occurs at high dose rates. A total cyanide dose load at lower daily concentrations over a longer period of time does not produce mortality. The mode of toxic action for cyanide

- associated mortality is also well characterized. Inhibition of cellular respiration by oxidative phosphorylation of cytochrome c oxidase, the terminal electron transporter in the mitochondrial respiratory chain, resul ts in lack of energy production. If the inhibition is complete or near

- complete, asphyxiation is rapid and irreversible, and is followed by death. The delay in acetonitrile conversion to cyanide may result in a delay in onset of symptoms but the response is still acute.

Chronic exposure to acetonitrile would result in cyanide

- induced toxicity associated with increased tissue burdens of thiocyanate, which is the metabolic detoxification product of cyanide. The conversion of acetonitrile

- derived cyanide t o thiocyanate is catalyzed by the sulfurtransferase enzyme, rhodanase. Elevated concentrations of thiocyanate are thyrotoxic. No evidence of thiocyanate

- induced thyrotoxicity was observed in any of the NTP or other subchronic studies.

Cyanide and aceton itrile are not known to accumulate in body tissues. If adverse effects were to be associated with the bioaccumulation of cyanide, then a concentration x time (C x T) effect would be anticipated. The clustering of deaths in the early weeks of the NTP (199 6) subchronic study does not suggest bioaccumulation. Rather, the data indicate that the acute MTD was approximated or reached at the acetonitrile exposure concentrations at which the deaths occurred. Had this been a chronic effect, then mortality would h ave continued to occur at later times during the study, as “continued generation of cyanide from the existing body burden of acetonitrile, coupled with cyanide generated from new exposures, results in cyanide levels that exceed the capacity of enzymes to m etabolize cyanide to thiocyanate” (Chapter 3, p. 23). This mode of toxic action is biologically implausible, given the lack of mortality at later times in the35 study or at lower doses (cumulative toxicity) and the absence of neurotoxicity symptoms in surviv ing animals (after the first week). Chronic tissue effects of repeated cyanide exposure, in addition to those of thiocyanate, include liver, kidney and reproductive organ toxicity and are associated with oxygen deprivation but are not lethal (ATSDR 1999). The absence of mortality in the NTP (1996) chronic bioassays and the Hazelton (1983) subchronic studies also demonstrates a lack of consistency and concordance of acetonitrile findings across similar studies. It also shows the absence of a C x T effect.

Thus, using a weight

- of

- evidence approach, it is scientifically indefensible and biologically implausible to conclude that acetonitrile can reasonably be anticipated to cause mortality as a result of chronic exposure.

EPA Response III

- B

- CH3

- Q2

- 12(b): Cyan ide

- associated mortality can be caused by acute exposures to acetonitrile. Further, the EPA agrees with the commenter’s assessment and description of a mode of action of acute acetonitrile toxicity. However, the EPA does not feel that this abrogates all concern of mortality due to chronic exposure. The toxicokinetics of chronic exposure to acetonitrile has not been well studied. Currently there is little evidence to suggest that the mode of action leading to mortality due to acute or chronic exposure is the same. In response to the reviewer’s comments regarding thiocyanate

- induced thyrotoxicity, the EPA has expanded the discussion of thyrotoxicity in the Technical Review of Acetonitrile (Methyl Cyanide). See response to Comment III

- B

- CH3

- Q1

- 6. A mod e of action showing bioaccumulation of acetonitrile, cyanide, or thiocyanate requires significant additional details (i.e. PBPK modeling) that are not currently available within the scientific literature for proper support. Therefore, the discussions of p ossible acetonitrile modes of toxic action have been revised in the Technical Review of Acetonitrile (Methyl Cyanide). In response to this and other reviewers’ comments, the EPA has reevaluated its position regarding the accumulation of acetonitrile, cyan ide, and thiocyanate and has revised the mode of action discussions. As stated by the reviewer, “Had this been a chronic effect, then mortality would have continued to occur at later times during the study”. Based on the deaths in the 800 ppm
treatme nt group of the NTP (1996) 13

- week mouse study, which occurred between the third and thirteenth week of the study with no acute mortality within the first 20 days of exposure, reasonable evidence exists to support the conclusion that long term repeated exp osures to acetonitrile caused mortality.

C. Specific Observations. Comment III

- C

- 1: Commenter Two Page 29, line10

- 11. This states that the conversion of acetonitrile to cyanide “is delayed”. That is not what the literature says. Conversion of acetonitril e to cyanide is slow such that it takes some time for toxic amounts to build up. That terminology is used elsewhere in the document, it is inaccurate, and is very confusing. There are data to indicate that co

- administration of acetone with acetonitrile d elays the formation of cyanide from acetonitrile (Freeman and Hayes, 1985) which is a different matter.

EPA Response III

- C

- 1: Section 3.8., Potential Modes of Action, of the Technical Review of Acetonitrile (Methyl Cyanide) has been modified to reflect th at the rate of cyanide formation from acetonitrile is not36 altered

, but rather it is slower than the rate of cyanide release from other nitriles.

“

Acetonitrile is metabolized to inorganic cyanide but the conversion occurs slowly compared to other nitriles (Hartung, 1982) ...”

Comment III

- C

- 2: Commenter Two Page 35, lines 20

- 23. This is nonsense. What is meant that the body can no longer successfully counter the toxic effects of cyanide? What happens is that cytochrome oxidase is inhibited to the point th at cellular respiration can no longer accommodate the metabolic needs of critical tissues

– most specifically the brain.

EPA Response III

- C

- 2: This paragraph has been deleted. The discussion of cytochrome oxidase inhibition has been incorporated into Sec tion 3.8., Potential Modes of Action, in the Technical Review of Acetonitrile (Methyl Cyanide)

.

Comment III

- C

- 3: Commenter Two There is also an issue of whether cyanide does not lead to apoptosis in brain cells. If that is massive enough, it could preclude recovery. I recommend that the authors' explore this question in the cyanide literature. It has been used to induce apoptosis in some in vitro systems. I would try to provide references except that would require a far ranging literature search that I am not being paid to explore.

EPA Response III

- C

- 3: The m ode of action section of the Technical Review of Acetonitrile (Methyl Cyanide) has been expanded to include a discussion of how cyanide may affect cellular respiration. However, the mode of action of cyanide, while informative, is not required in identifying hazard following exposure to acetonitrile. The information requested by this commenter is beyond the scope of this document.

Comment III

- C

- 4: Commenter Two Page 41, lines 10

- 24. This discussion is a critical in to the attribution of teratogenic effects of acetonitrile to cyanide. Could this be done in a more quantitative fashion? For example, is it possible to estimate the systemic cyanide concentrations that are produced by acetonit rile with those that are produced by direct treatment with cyanide? This requires some exploration of the cyanide literature.

Page 42, lines 14ff. Again, this discussion would be more convincing if a quantitative approach could be taken among different nitriles. Preferably it would be related to systemic concentrations of cyanide that would be achieved by different treatments. Barring that, one could compare doses in terms of cyanide equivalents (since inhalation doses are essentially in molar terms, i t is easier to do with ppm in air than with mg/kg per day).

EPA Response III

- C

- 4: A comparison of nitriles on a quantitative basis using systemic cyanide concentrations as a standardizing tool would be useful in the evaluation of acetonitrile toxicity. U nfortunately, there is a lack of information required for this type of analysis. This would also be further complicated by the varying rates of metabolism from nitrile to cyanide and from cyanide to thiocyanate among species. Therefore, this type of PBPK modeling analysis is beyond the scope of the37 Technical Review of Acetonitrile (Methyl Cyanide).

Comment III

- C

- 5: Commenter Two Page 44, lines 7

- 10. This needs to be stated more clearly or at least discussed more fully. The implication is that a microso mal system degrades cyanide that is administered directly, but not the cyanide produced from metabolism of acetonitrile. That is not a very satisfying dataset considering the emphasis placed in this document on the conversion of acetonitrile to cyanide as accounting for the toxicity of acetonitrile.

EPA Response III

- C

- 5: The Technical Review of Acetonitrile (Methyl Cyanide) reports the findings of the Saillenfait and Sabate (2000) study report. This study reports that toxicity associated with acetonitrile exposure was found to be very low, nearly 3 orders

- of

- magnitude less potent than the highest aliphatic nitrile tested. Furthermore, the study showed the acetonitrile toxi city to be unaltered by microsomal enhancement. This does not suggest that the microsomal system “degrades cyanide that is administered directly, but not the cyanide produced from metabolism of acetonitrile.” Rather, it suggests the time frame of exposur e (46 hours) was not adequate to allow acetonitrile metabolism to cyanide and the very low toxicity level is a result of acetonitrile directly. This has been more clearly detailed in the Technical Review of Acetonitrile (Methyl Cyanide).

Section 3.5.1. “ In vitro studies performed by Saillenfait and Sabaté (2000), conceptuses were explanted from uteri of 10

- day pregnant dams and cultured with medium containing aliphatic nitriles with or without hepatic microsomes and NADPH. After 46 hours of culture, via ble embryos (embryos with a beating heart) were examined for growth and morphological development. The yolk sac was assessed for its vascularization and circulation and its diameter was recorded, as were the crown

- rump length, head length, and number of s omite pairs of the developing embryo. All of the nitriles tested, including acetonitrile, produced concentration

dependent decreases in growth and differentiation, including decreased yolk sac diameter,

crown

- rump length, head length, and number of somite s, and increases in the incidences of morphologically abnormal embryos, including defects in prosencephalon, mesencephalon, eye, maxillary process, auditory system, olfactory system, and flexion (Saillenfait and Sabaté, 2000). The addition of the biotrans formation system to the culture medium did not change acetonitrile

induced embryotoxicity, while the percentage of sodium cyanide

- treated embryos with defects dropped from 100% without the metabolic system to 0% with microsomes and NADPH. ”

Comment III

- C

- 6: Commenter Three p. 28, lines 3

- 6: Disposition study with 2

- 14 C

- acetonitrile (Ahmed et al., 1992). The summary statement of this study could be misleading. The technical review authors are building the argument that cumulative tissue acetonitrile will be metabolized to cyanide, the mediator of toxicity. One question pertinent to cumulative effects of repeat dose is whether a significant fraction of previous dose(s) of parent acetonitrile is/are retained in target tissue at time of subsequent dail y dose. The Technical Review sentence describing Ahmed et al. (1992) could be interpreted in this context to support widespread distribution of the parent molecule and tissue retention, since time of measurement is not specified. However, techniques used in the Ahmed et al 1992 study detected radioactivity due to non

- volatile metabolites from the methyl 14 C of the radiolabeled acetonitrile. Further, methods used by Ahmed et al. (1992) distinguished radioactivity that was covalently bound. Their report i ncludes immediate times after pulse dosing with tracer acetonitrile, but also 24 and 48 h points, which would appear to support 48 h retention of acetonitrile. However, this is more likely due to formaldehyde adducts. The38 wording in the Technical Review is the same as in the EPA (1999) report, where it is constructed to emphasize that disposition in stomach can arise from a systemic source after inhalation exposure. That wording may give incorrect emphasis to the present context.

EPA Response III

- C

- 6: T he EPA agrees that the use of this reference does not fully support the retention of acetonitrile and subsequent conversion to the mediator of toxicity, cyanide. As such the EPA has altered this section, eliminating the reliance on this reference.

Comm ent III

- C

- 7: Commenter Four There is simply no way that chronic mortality can be demonstrated in subchronic studies or those of shorter duration. There is no evidence to support convincingly the assertion that mortality should be a reasonably anticipated consequence of chronic acetonitrile exposure. All of the mortality that has been observed to date in subchronic and shorter duration studies can be explained readily in terms of the acute toxicity that is associated with the metabolism of acetonitrile to cyanide.

EPA Response III

- C

- 7: The EPA does not believe that all the toxicity observed in the subchronic NTP 13

- week studies can be explained in terms of acute toxicity. The justification for this conclusion has been documented in the Technical Review of Acetonitrile (Methyl Cyanide) and in response to Comments III

- B

- CH3

- Q2

- 12(a and b).

Comment III

- C

- 8: Commenter Four Section 3.1, p 25, lines 9

- 12: The phrase “uncertainty as to the significance of certain results” sounds very mysterious. The specific concerns ought to be identified explicitly.

EPA Response III

- C

- 8: The conclusion section (Section 3.11) of the Technical Review of Acetonitrile (Methyl Cyanide) has been revised to reflect the specific concerns that introduce the uncertainty, preventing a definitive conclusion regarding the carcinogenic potential of acetonitrile for humans.

“ Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), the available data provide inadequate information to assess the carcinogenic potential of aceton itrile. The data from a NTP 2

- year cancer bioassay indicate that the carcinogenic potential of acetonitrile in the rat and mouse is low (NTP, 1996). However, because of uncertainty regarding the significance of results due to dose selection in the NTP 2

- year bioassay, the carcinogenic potential of acetonitrile for humans following inhalation, oral or dermal exposure cannot be determined. ”

Comment III

- C

- 9: Commenter Four Section 3.1, p 29, lines 5

- 6: Lower affinity is the same as higher Km, but lower rate is the same as lower Vmax. This should be made clear. Also, the assertion that humans are thus less able than rats to detoxify cyanide is speculative. Under what specific conditions, such as high dose, or low dose, might this be true? Can one be certa in that rhodanese is not an inducible enzyme, perhaps more inducible in human than rodents?

EPA Response III

- C

- 9: The Km and Vmax have been clarified in section 3.2.3., Metabolism.39 “Rhodanese has also been found in the respiratory epithelium of human nasal tissue; however, Lewis et al. (1991) showed that the rhodanese in human nasal tissue had a lower affinity (higher Km) for cyanide and a lower rate (lower Vmax) for cyanide metabolism than rh odanese from rat nasal tissue. K(m) is the concentration of substrate which permits the enzyme to achieve half V(max)(the maximum rate of the reaction )

. ”

Rhodanese has been shown to be inducible in humans in vitro. (Lewis 1991). This is also reported in Section 3.2.3., Metabolism.

“Lewis et al. (1991) further reported an induction of rhodanese activity and an inability to saturate rhodanese enzymatic activity at high potassium cyanide concentrations using human nasal tissue in vitro.”

Although speculati ve, EPA believes it is acceptable to include the interpretation that humans may be less able to detoxify acetonitrile based on the data available. Should additional data become available regarding the levels to which rhodanese is inducible, it would also be included in the weight

- of

- evidence. The conclusion has also been revised based on a balanced evaluation:

“Due to the dynamic nature of rhodanese activity it is not possible to directly compare the human and rat capacity or rate of cyanide conversion t o thiocyanate via rhodanese.”

Comment III

- C

- 10: Commenter Five p. 30

- 31. Section 3.3.1. The discussion about why U.S. EPA (1999) did not apply an uncertainty factor for subchronic to chronic exposure duration is not clear or rational.

EPA Response II I

- C

- 10: The application of uncertainty factors, although necessary for developing RfC and RfD values, are not relevant to this hazard assessment. Therefore, this discussion has been removed from the Technical Review of Acetonitrile (Methyl Cyanide)

.

Comm ent III

- C

- 11: Commenter Five p. 34, lines 1

- 4. This is an example where lack of rigor is demonstrated. Mortality in the Mast et al. (1994) study was 1/33 at 181 mg/kg/day in pregnant females, 1/10 and 2/10 in pregnant and

non

- pregnant females at 543 mg/k g/day. Assuming the control rates are 0 (and control rates are not given in most study summaries including tables and need to be added), these mortality rates have neither statistical nor toxicological significance! In contrast, in Saillenfait et al. (19 93), a lethality incidence of 8/20 dams in the highest dose group (what were the incidences in the other dose groups) would be both statistically and toxicologically significant. So there is no divergence or discrepancy in the findings between the studies . Mast et al. did not observe significant treatment

- associated mortality whereas at higher doses, Saillenfait et al. did.

This discussion is also repeated on p. 42

- 43 comparing the two studies.

EPA Response III

- C

- 11: The discussion of these two studies has been revised to reflect this comment.40 IV. RESPONSE TO CHEM ISTRY and ENVIRONMENTAL FATE & ECOLOGICAL TOXICITY PEER REVIEW COMMENTS

A. General Impressions

IV

- A

- 1 : Commenter Six The overall contents of the Technical Review of Acetonitrile are acceptable. However, after reviewing the document, it was not clear what the overall conclusions were with regards to the status of acetonitrile and reporting requirements of the EPCRA. In addition, several areas either need clarification or additional documentation. Specifically, the review indicates that most of the acetonitrile produced in the United States is released into the environment by deep well injection.

The section discussing the potential for migration from on

- site injection wells to groundwater and the persistence of acetonitrile in groundwater should be expanded.

EPA Response IV

- A

- 1:

The purpose of the report is to provide a hazard assessment on acetonitrile. A conclusion on the findings of the assessment and EPCRA reporting requirements is beyond the scope of this report.

The persistence of acetonitrile in groundwater has been expanded. However, there is very limited information concerning the environmental fa te of acetonitrile under biotic anoxic conditions or abiotic conditions. In general, it is very stable under abiotic conditions. No changes to the text will be done concerning the potential migration of acetonitrile to groundwater from off

- site injection w ells. This is beyond the sco pe of this hazard assessment. Revisions to the text relevant to the potential subterranean transport and persistence of acetonitrile in groundwater are provided below:

“ Section 2.6.1 Environmental Transport

... A soil partitioning coefficient (K oc ) value of 5 has been calculated for acetonitrile using its experimental log K ow value of

- 0.34 (Hansch et al., 1995) and a regression

- derived equation (HSDB, 2002; Lyman et al., 1990). Based on this K oc value, aceton itrile is not expected to bind to soil or sediments, but will be highly mobile in soil (HSDB, 2002; Swann et al., 1983). Acetonitrile may leach from soil to groundwater due to its high mobility. Deep

- well injection containment failures are described among the sources of groundwater contamination in the U.S. EPA Ground Water Handbook (U.S. EPA, 1987). The potential for dispersion and transport by groundwater would be high for acetonitrile accidentally released into an aquifer due to its high water solubili ty and relatively low affinity for soils. As discussed in the section 2.6.3 of this report, acetonitrile in subterranean environments may be subject to biotic degradation. Due to its high solubility in water, once in contact with underground water , acetoni trile will readily disperse throughout the water body. The volatilization of acetonitrile from a contaminated ground water body is dependent upon multiple factors including available air head space above the groundwater body, air exchange rates, and gro undwater p H, temperature and velocity. Acetonitrile released in vapor form from subterranean sources, including groundwater, may permeate through the ground and enter into the atmosphere depending on the geology, temperature and moisture content of the la nd mass above its release zone. In instances where acetonitrile contaminated groundwater makes it way to surface water, it potentially can volatilize into the atmosphere, the predicted volatilization rates stated above for a river and lake body of water.”41 “2.6.3.3 Anae robic Biodegradation Studies: Lim ited literature was identified concerning the anaerobic biodegradation of acetonitri le. In the one recent study, Li et al., (2007), had sewage sludge acclimated aerobically for 2 months using acetonitrile as the sole source of carbon and nitrogen. It is important to note that the conditions this study was done are likely representative of anaerobic digestion within a sewage treatment plant and might not be applicable to anaerobic conditions found in the envi ronment, in particular conditions relevant to underground injection wells or subterranean environments. The acclimated mixed culture was used to access degradation rates of acetonitrile at initia l concentrations of 1 and 2 g/L under both aerobic and anoxi c conditions in a syntheti c mineral medium salt solution . With acetonitrile at an initial concentration of 1 g/L, complete degradation was observed within 20 hours under anoxic conditions. The parallel study done by Li et al. (2007) under aerobic conditi ons resulted in a faste r degradation rate of 10 hours for complete degradation for an initial concentration of 1g/L. This is consistent with the findings of Boethling et al. (1995) which observed for various organic chemicals classes anaerobic biodegradat ion rates were generally slower than aerobic biodegradation rates, with the exception of some chemical classes (e.g., nitroaromatic, halogenated hydrocarbons, but not nitriles)

. At a higher concentration of 2 g/L acetonitrile, observed anaerobic degradati on was significantly slower with complete degradation being observed within 212 hours (versus 20 hours). Acetamide, acetic acid, and ammonia were detected during the anaerobic biodegradation process which suggests that these products may be possible under anaerobic conditions (Li et al., 2007). Another anaerobic study utilizing sewage sludge was conducted by Nagel et al., (1995). An inoculum obtained from low

- solids anaerobic digesters at the Denver Municipal Waste Water Reclamat ion Plant in Denver, C olorado, demonstrated an 83% reduction of acetonitrile on a dry municipal solid waste feedstock mixed with a liquid nutrient solution. The 83% reducti on of acetonitrile was observed after a 90

- day incubation period at 37 o C using an initial acetonitrile

co ncentration of 50 mg/L. ”

Comment IV

- A

- 2: Commenter Six The review also indicates that in mammalian systems acetonitrile is metabolized to cyanide (the toxic metabolite). It is not clear if this pathway does (or does not) exist in ecological receptors. This issue should be clarified.

EPA Response IV

- A

- 2 : Concerning ecolo gical receptors/ aquatic species

- no literature was readily identified on

m echanism(s) of acetonitrile toxicity. Vertebrate biotransformation toxicity mechanisms can be found in section 3.2

- Kinetics and Metabolism in the report. No chan ges will be made based upon this comment.

Comment IV

- A

- 3 : Commenter Six The review also indicates that acetonitrile is “not expected to present a significant concern” for ecological receptors because the acute toxicity is greater than or equal to 100 mg/L. The justification for using this value as a determinant for minimal to xicity should be included in the text. While it is understood that 100 mg/L is a large dose, this should be explained in relevance to other toxins or regulatory standards.

EPA Response IV

- A

- 3 : An elaboration on toxicity exposure levels and the degree o f concern for adverse environmental impact is beyond the intent of this hazard assessment. The text in question has been revised and is presented below. A reference has been placed in the text which relates acute toxic response to42 level s of toxicity.

“4.1 Summary Based upon a review of the limited acute and chronic exposure information available for aquatic species (algae, invertebrates, and vertebrates), adverse environmental effects due to acetonitrile can only be anticipated at exposures to elevate d concentrations . This conclusion is based on observed acute toxicity values for aquatic organisms are greater than or equal to 100 mg/L indicating relatively low acute aquatic toxicity (Table 4

- 2) ( see for example: http://www.epa.gov/oppefed1/ecorisk_der s/toera_analysis_eco.htm#Ecotox ). Acute toxicity values for freshwater fish and aquatic invertebrates ran ged from 100 to 3600 mg/L. The 96 hour LC 50

/EC 50 values for fish and green algae ranged from 100 to 3454 mg/L. Furthermore, experimental chronic tox icity values for green and blue

- green algae, freshwater invertebrates, freshwater fish embryos, and duckweed ranged from 160 to 7300 mg/L, indicating relatively low chronic toxicity.”

Comment IV

- A

- 4: Commenter Seven The overall impression is of an adequ ate summary of available information on the physicochemical and toxicological behavior of methyl cyanide. There are, however, some unanswered questions that make it difficult to use this document to fully judge this chemical’s potential to impact humans o r the environment and where any such impacts may occur. The description of biotic and abiotic degradation processes is not clear whether these processes differ in different media or what degradation byproducts might be expected in different media (if any)

.
EPA Response IV

- A

- 4 :

The text has been clarified to indicate what general media we are referring to for abiotic and biotic degradation. Most of the information available for biotic degradation is based on studies designed for sewage treatment plants versus the general environment although there may be some relevancy. Where there is information on degradation by

- products, it also is presented

. Relevant portions of the revised report in response to this comment are provided below.

“

2.6.2. Abiotic Degradation. The abiotic degradation of acetonitrile released in the environment is expected to be very slow. Acetonitrile does not undergo direct photodegradation (HSDB, 2002). The main abiotic degradation processes are considered to be reaction with hydroxyl radicals in the atmosphere. Experimental data for acetonitrile reactions in the atmosphere are presented by Atkinson (1994) as second

- order rate coefficients. Half

- life estimates for the acetonitrile reaction with the hydroxyl radi cal in air vary from months to over a year, depending on variations in OH radical concentrations and other factors (EPIWIN,(U.S.EPA, 2000a)). The National Library of Medicine’s Hazardous Substance Data Base (HSDB, 2002) reports an estimated half

- life of 6 20 days based upon an experimental rate constant for the reaction of acetonitrile with hydroxyl radicals of 2.63E

- 14 cm3/molecu le

- sec and an atmospheric concentration of 5X10+5 hydroxyl radicals per cu cm. Limited literature was available addressing the a tmospheric reaction products of acetonitrile. The reaction between acetonitrile and atmospheric hydroxyl radicals is expected to proceed through hydrogen abstraction or addition to the triple bond (Atkinson, 1994; Graedel et al., 1986). Based on other hy drogen abstraction reactions involving hydrocarbon side chains, oxygenated species are expected but no literature was identified43 stating specific a biotic degradation products. However, photochemical smog chamber studies show that acetonitrile is unreacti ve towards photochemically generated free radicals (Dimitriades and Joshi, 1977). Abiotic hydrolysis of acetonitrile is extremely slow under environmental conditions (Brown et al., 1975). HSDB (2002) reports a rate constant for the base

- catalyzed hydrol ysis of acetonitrile of 5.8E

- 3/M

- hr and a corresponding half

- life at pH 7 of 150,000 years. As stated earlier in this repor t, in 2009 some 17.1 million pounds of acetonitrile were reported to the TRI as being burned on

- site for energy recovery by the manuf acturers (U.S.EPA, 2011). During combustion in air the decomposition of acetonitrile initiates at 760 °C . At 950 °C (with a 2 sec residence time) 99.99% of the acetonitrile will have decomposed (Britt, 2002). The ignition of acetonitrile in excess oxygen results in the production of CO, CO2, H2O, N2 and NO as the major combustion products. The combustion of acetonitr ile at temperatures below 900 °C and low oxygen concentrations can result in the production of HCN (Britt, 2002). ”

“

2.6.3.2 Aerobic Biodegrad ation Studies. In review of the studies available on the aerobic biodegradation of acetonitrile, several of the s tudies have been conducted using acclimated microorganisms in sludge or sewage media, conditions and may not be reflective of environmental conditions of interest. As stated previously, experimentally observed biotic degradation rates are dependent upon se veral factors including media, temperature, available oxygen, acetonitrile concentration, specific microorgani sms involved and the degree of acclimation. For acetonitrile, the Chemicals Evaluation Research Institute (CERI, 2003) reports an average of 32% degradation observed in 21 days based upon biochemical oxygen demand (BOD) testing (OECD Test Method 301C). In a study by Urano and Kato (1986b) that used activated sludge as a microbial inoculum, the lag period of acetonitrile degradation in an

aqueous me dia was about 1 day, after which the compound, at an initial concentration of 100 ppm, was degraded by 17% after 3 days and 40% after 10 days based upon the BOD. Acclimated mixed microbial cultures isolated by an enrichment culture technique provided a 58% degradation of 100 ppm acetonitrile in an aqueous solution in 5 days (BOD test) (Babeu and Vaishnav, 1987). Earlier studies reported that the biodegradation of acetonitrile using activated sludge or sewage proceed slowly with unacclimated microorganisms in comparison to acclimated microorganisms (Kalmykova and Rogovskaya, 1978; Ludzack et al., 1958; Placak and Ruchhoft, 1947). Ludzak et al. (1961) reported the results of a 28

- day biodegradation study
that used a semi

- continuous feed of previously acclima ted activated sludge. Under the conditions of the briefly described study, 82

- 94% of the theoretical biochemical oxygen demand (BOD) was achieved when an initial concentration of 139 ppm of acetonitrile was introduced. A graphical representation in the L udzack et al. paper indicated that acetonitrile degradation proceeded more slowly prior to acclimation of the sludge. Acetonitrile at an initial concentration of 490 ppm reached 0

- 17% of the theoretical BOD after 24 hours in a screening test using an acti vated sludge inoculum that was apparently unacclimated (Placak and Ruchhoft, 1947). The National Institute of Technology and Evaluation (NITE, 2002) reports that 84% degradation of acetonitrile from an initial concentration of 100 ppm was observed in 28 d ays using an activated sludge inoculum (OECD Test Method 301C). The biodegradability of acetonitrile was also observed with river water; the 12

- day44 ThOD (theoretical oxygen demand) test measured the generation of CO 2 from Ohio River water spiked with a cetonitrile at concentrations ranging from 0.1

- 25 mg/L. After 12 days, a total generation of CO 2 was observed equivalent to 40% of the ThOD estimated for acetonitrile (Ludzack et al., 1959). Acclimation of the microorganisms was examined by re

- dosing; th e degradation was 5 times faster after acclimation and was also 4 times faster at 20° C than at 5° C (Ludzack et al., 1958; Ludzack and Ettinger, 1960). In a more recent study, Li et al.(2007), studied the degradation of higher concentration of
acetonit rile by a mixed culture of microorganisms obtained from raw activated sewage sludge. It is important to note that the conditions this study was done under are likely representative of anaerobic digestion within a sewage treatment plant and might not be app licable to anaerobic conditions found in the environment. The experimental medium used was a synthetic mineral salt solution. The microorganisms used in the study were acclimated with acetonitrile. As analyzed by gas chromatographic analysis for acetoni trile, complete degradation of acetonitrile was realized in about 10 hours for initial acetonitrile concentrations of 1g per liter, and 75 hours for initial concentrations of 5g per liter acetonitrile. At 10 g per liter acetonitrile, even a greater reducti on in the degradation rate was observed with only 75% degradation after 186 hours (Li et al., 2007). Concerning media type and its impact on acetonitrile degradation, based upon studies conducted by Takemoto et al., (1981) the biodegradation of acetonitri le is expected to be slower in seawater than in freshwater. ”

2.6.3.3 A naerobic Biodegradation Studies. Limited litera ture was identified concerning the anaerobic biodegradation of acetonitr ile. In the one recent study, Li et al., (2007), had sewage slu dge acclimated aerobically for 2 months using acetonitrile as the sole source of carbon and nitrogen. It is important to note that the conditions this study was done are likely representative of anaerobic digestion within a sewage treatment plant and migh t not be applicable to anaerobic conditions found in the environment, in particular conditions relevant to underground injection wells or subterranean environments. The acclimated mixed culture was used to access degradation rates of acetonitrile at initia l concentrations of 1 and 2 g/L under both aerobic and anoxic conditions in a syntheti c mineral medium salt solution . With acetonitrile at an initial concentration of 1 g/L, complete degradation was observed within 20 hours under anoxic conditions. The parallel study done by Li et al. (2007) under aerobic conditions resulted in a faste r degradation rate of 10 hours f or complete degradation for an initial concentration of 1g/L. This is consistent with the findings of Boethling et al. (1995) which observed for various organic chemicals classes anaerobic biodegradation rates were generally slower than aerobic biodegradation rates, with the exception of some chemical classes (e.g., nitroaromatic, halogenated hydrocarbons, but not nitriles) . At a higher concen tration of 2 g/L acetonitrile, observed anaerobic degradation was significantly slower with complete degradation being observed within 212 hours (versus 20 hours). Acetamide, acetic acid, and ammonia were detected during the anaerobic biodegradation proce ss which suggests that these products may be possible under anaerobic conditions (Li et al., 2007). Another anaerobic study utilizing sewage sludge was conducted by Nagel et al., (1995). An inoculum obtained from low

- solids anaerobic digesters at the Denver Municipal Waste Water45 Reclamation Plant in Denver, Colorado, demonstrated an 83% reduction of acetonitrile on a dry municipal solid waste feedstock mixed with a liquid nutrient solution. The 83% reductio n of acetonitrile was observed after a 90

- day incubation period at 37 o C using an initial acetonitrile concentration of 50 mg/L. ”

2.6.5. Toxic Transformation Products. As noted in section 2.6.3, biodegradation is the most significant degradation process for acetonitrile in the environment. The maj or transformation products of acetonitrile in the environment are expected to be acetic acid and ammonia (Graham et al., 2000). Although some acetamide may be generated as a part of the two

- step biodegradation process noted in section 2.6.3, it is expecte d to biodegrade rapidly resulting in the formation of acetic acid and ammonia (Graham et al., 2000; Li et al., 2007; Nawaz et al., 1989 Urano and Kato, 1986a,b). Abiotic hydrolysis of acetonitrile is extremely slow under environmental conditions (Brown et al., 1975). HSDB reports a rate constant for the base

- catalyzed hydrolysis of acetonitrile of 5.8E

- 3/M

- hr and a corresponding half

- life at pH 7 of 150,000 years. Based on the reaction pathway for the abiotic hydrolysis of nitriles, the hydrolysis products of acetonitrile are expected to be acetamide, acetic acid, and ammonia (Smith and March, 2001). Acetamide will degrade under abiotic conditions, in particular in reaction with hydroxyl radicals. The National Library Of Medicine’s Hazardous Substance Data Base (HSDB, 2010) reports an experimental rate constant for the reaction of acetamide with hydroxyl radicals of
2.1E

- 12 cm3/molecule

- sec and a corresponding half

- life of 7.6 days. Hydrolysis rates for acetamide are much slower. Using reported rate const ants for the acid

- and base

- catalyzed hydrolysis of acetamide of 0.030/M

- hr and 0.17/M

- hr, respectively, a corresponding half

- life at pH 7 of 3,440 years was calculated. (HSDB, 2010).
C oncerning the biodegradation of acetamide , Urano and Kato (1986b) us ing activated sludge (30 mg/l) as a microbial inoculum and acetamide at an initial concentration of 100mg/l (100 ppm) in an aqueous medium at 20 °C, observed 50% degradation after approxim ately 4 days based upon the BOD

. In an earlier study, reported acet amide incubated with activated sludge resulted in a 29.6

- 45.7% of the theoretical BOD after 24 hours (Malaney et al., 1962 as reported in HSDB, 2010) In the study by Li et al., (2007), the observed acetamide formed as a part of the biodegradation of acet onitrile under aerobic conditions peaked at 1800 mg/L about 8 hours after the initial exposure of acetonitrile to acclimated bacteria. Within 30 hours, concent ration levels of acetamide were reduced to non

- detectable levels. As reported earlier in this report, some 17.1 mill ion pounds of acetonitrile are burned

on

- site for energy recovery by the manufacturers (U.S.EPA, 2011). During combustion in air the decomposition of acetonitrile initiates at 760 °C . At 950 °C (with a 2 sec residence time)
99.99% o f the acetonitrile will have decomposed (Britt P. 2002). The ignition of acetonitrile in excess oxygen results in the production of CO, CO2, H2O, N2 and NO as the major combu stion products (Britt P. 2002) . The combustion of acetonitrile at temperatures b elow 900 °C and low oxygen concentrations can result in the production of hydrogen cyanide (Britt P. 2002). ”

Comment IV

- A

- 5 : Commenter Seven46 Use of the EPIWIN model to assess the distribution of methyl cyanide following release was good, but more discuss ion of its transport and fate in groundwater (an important disposal media) is needed.

EPA Response IV

- A

- 5 : Information concerning the transport and fate of ACN in groundwater is limited and mainly based on predictive physical

- chemical properties. The section has been revised for clarity. Please see the EPA response to Comment IV

- A

- 1 above for specific changes in the text to address this comment.

Comment IV

- A

- 6 : Commenter Seven The review of what is known about the ecological hazards posed by this chemical suffers from the lack of available information on chronic effects or on population

- level responses.

EPA Response IV

- A

- 6 : This is correct. No information is available concerning population effects do to chronic
exposure. No changes to the report will be made based upon this comment

.

Comment IV

- A

- 7 : Commenter Eight The January 15, 2008 Draft Technical Review of Acetonitrile is a very clear and balanced document. It is well

- written with no area lacking clarity and coverage.

The only slight confusion that I had was associated with th e large amounts of acetonitrile injected underground. Given this major input to the environment and the mention of possible groundwater contamination on page 11, I was left wondering why there was not mor e discussion of exposure via groundwater.

EPA Response IV

- A

- 7: Information concer ning the transport and fate of ACN in groundwater is limited and mainly based on predictive physical

- chemical properties. The section has been revised for clarity. Please s ee the EPA response to Comment IV

- A

- 1 above for specific changes in the text concerning the fate of acetonitrile relevant to groundwater contamination. Actual exposure concentrations relative to people or the environment based upon groundwater contaminati on scenarios are beyond the scope of this technical report con cerning the hazard of acetonitri le.

Comment IV

- A

- 8 : Commenter Eight Also the efficiency of the other major disposal method, on

- site incineration, might be described

more.

EPA Response IV

- A

- 8 : Additional information on the incineration of acetonitrile has been provided in the revised report. The newly provided information is presented below from section 2.6.5. Toxic Transformation Products of the report

.

“

As reported earlier in this report, s ome 17.1 mill ion pounds of acetonitrile are burned on

- site for energy recovery by the manufacturers (U.S.EPA, 2011). During combustion in air the decomposition of acetonitrile initiates at 760 °C . At 950 °C (with a 2 sec residence time) 99.99% of the acet onitrile will have decomposed (Britt P. 2002). The ignition of acetonitrile in47 excess oxygen results in the production of CO, CO2, H2O, N2 and NO as the major comb ustion products (Britt P. 2002) . The combustion of acetonitrile at temperatures below 900 °C and low oxygen concentrations can result in the production of hydrogen cyanide (Britt P. 2002). ”

A. Response to Charge Questions

Comment IV

- B

- Ch2

- Q 1

- 1 : Commenter Six Overall, the chemical and physical properties are clearly described. As stated above, additional details regarding the fate of acetonitrile from underground injection wells would be valuable to the review.

EPA Response IV

- B

- Ch2

- Q 1

- 1 : No experimental i nformation was located in the literature concerning the fate of acetonitrile in underground injection wells. Limited information on the degradation of acetonitrile by bacteria under anaerobic conditions may have some relevancy to the fate of acetonitrile in underground injection wells. This information has been presented in section 2.6.3.3

. General information of the potential transport of acetonitrile underground has been added to section 2.6.1 and is also duplicated below. The revised text can be foun d in EPA’s response to Comment IV

- A

- 1 above.

Comment IV

- B

- Ch2

- Q 1

- 2 : Commenter Six Section 2.6.5, Toxic Transformation Products, is brief and could be improved by expanding the discussion. Additional editorial comments are included in the section on specific comments (below).

EPA Response IV

- B

- Ch2

- Q 1

- 2 : Section 2.6.5, Toxic Transform ation Products, had been revised/ expanded . The revision can be found in the EPA response to Comment 1V

- 4, section “2.6.5. Toxic Transformation Products.”

Comment IV

- B

- Ch2

- Q 1

- 3: Commenter Seven Given their importance in estimating transport and fate, corroborating references (if available) for the values of KOW, Henry’s Law, and hydroxyl reaction would be helpful (some are given in the text but not in the table).

EPA Response IV

- B

- Ch2

- Q 1

- 3 : The a dditional references have been inserted into the table

, which is duplicated below.

Table 2

- 1, Physical and Chemical Properties of Acetonitrile (CAS Reg. No. 75

- 05

- 8)

Property Value a Conditions Reference Molecular Formulae C 2 H 3 N Molecular Weight 41.05 Chapter Two, Chemistry and Environmental Fate Charge Question 1. Has an objective (accurate, clear, complete, and unbiased) and technically accurate description of acetonitrile been provided in terms of chemical and physical properties?48 Freezing point (°C)

- 45.7

- McKinney and DeVito, 1998 Boiling point (°C) 81.6

- McKinney and DeVito, 1998 Vapor pressure (mm Hg) 88.8 25°C Boublik et al., 1984 Water solubility (mg/L) 1x10 6 25°C Riddick et al., 1986 Log K ow - 0.34

- Hansch et al., 1995 Henry’s Law constant (atm

m 3 /mol) 3.45x10 - 5 25°C Gaffney et al., 1987

Dissociation constant, pKa

- 4.30

- Perrin, 1965 Hydroxyl radical reaction rate constant (cm 3 /molec

sec) 2.63x10 - 14 25°C Atkinson, 1994

Density (g/cm 3 ) 0.786 20°C McKinney and DeVito, 1998 Vapor density 1.42 Air = 1 Clayton and Clayton, 1982 K oc 5 (HSDB, 2002; Lyman et al., 1990)

Comment IV

- B

- Ch2

- Q 1

- 4: Commenter Seven Page 53, Table 4

- 1. This table reproduces many of the values cited in Table 2

- 1 but with more complete source citations. It appears possible to combine these tables into Table 2

- 1 and simply cite this table in Chapter 4.

EPA Response IV

- B

- Ch2

- Q 1

- 4 : The two tables will be maintained as is. The table 4

- 1 specifically identifies which values were used in the operation of the EPIWIN software.

Comment IV

- B

- Ch2

- Q2

- 1 : Commenter Six The report objectively addresses the fate of acetonitrile in soil and groundwater. To a more limited extent, fate and transport in the atmosphere is also included. Since approximately 90% of acetonitrile is disposed of in on

- site injection wells, this section should be expanded. The discussion should include information regarding the potential for release of contaminants from inject ion wells into groundwater and the fate and transport of contaminants if they are released into groundwater.

EPA Response IV

- B

- Ch2

- Q2

- 1 : No changes to the text will be done concerning the potential migration of acetonitrile to groundwater from off

- site injection wells. This is beyond the scope of this hazard assessment. The transport and persistence of acetonitrile in groundwater will be e xpanded. However, there is Chapter Two, Che mistry and Environmental Fate Charge Question 2. Does the report objectively address the fate of the chemical in the environment including both transport between environmental compartments (atmosphere, aquatic and terrestrial) and transformation (e.g., degradation), including both abiotic and biotic processes where relevant?49 very limited information concerning the persistence of acetonitrile under biotic anoxic conditions or abiotic conditions in the environmental. Information concerning the transport and fate of ACN in groundwater is limi ted and t heoretical based upon physical

- chemical properties. The section has been revised for clarity. Revisions to the report relevant to the potential migration and persistence of acetonitrile in groundwater can be found in the EPA response to Comment IV

A

- 1, section s “ 2.6.1 Environmental Transpor t” and “2.6.3.3 Anaerobic Biodegradation Studies

”

.

Comment IV

- B

- Ch2

- Q2

- 2 : Commenter Six Also, the text indicates that 30 million tons of acetonitrile are burned on

- site each year. Are data available to determine the efficiency of these methods? What levels of acetonitrile may be released into the atmosphere as a result of incomplete combustion (or are their other toxic by

products of combustion)?

EPA Response IV

- B

- Ch2

- Q2

- 2 :
Additional information on the incinera tion of acetonitrile including the products formed during incineration has been added to the report and is duplicated below. See the EPA response to Comment IV

- A

- 8

.

Comment IV

- B

- Ch2

- Q2

- 3 : Commenter Seven Page 10, line 27. The volatilization pathway would only be accessible to methyl cyanide disposed of on the soil or water surface. Yet it appears (from Lines 22

- 25) that the bulk of releases are to groundwater or to air following combustion. Is there any information on methyl cyanide ’ s transport and fate in groundwater?

EPA Response IV

- B

- Ch2

- Q2

- 3 : See EPA r esponse s to Comment IV

- B

- Ch2

- Q2

- 1 and Comment IV

- B

- Ch2

- Q2

- 2

.
Comment IV

- B

- Ch2

- Q2

- 4 : Commenter Seven It is there any information on whether the combustion of methyl cyanide produces toxic by

products? (I would think not but some information to this effect, if available, would be helpful.)

EPA Response IV

- B

- Ch2

- Q2

- 4 : See EPA r esponse to Comment IV

- B

- Ch2

- Q2

- 2

.

Comment IV

- B

- Ch2

- Q2

- 5 : Commenter Seven Page 11, Line 2 et seq . What are the degradation byproducts of methyl cyanide? Do these byproducts differ with respect to media and degradation pathway? If such information is available, it would helpful in assessing the nature and extent of any p roblems caused by methyl cyanide ’ s breakdown in the environment. It would be helpful to summarize what is known about degradation by pathway and byproduct into a simple table.

EPA Response IV

- B

- Ch2

- Q2

- 5 : In response to the peer reviewers comment, Section 2.6.5, Toxic Transformation Products, had been revised/ expanded. For revised text see the EPA response to Comment II

- 1

- 2.

Comment IV

- B

- Ch2

- Q2

- 6 : Commenter Seven Page 14, Section 2.6.1. Given that large amounts of methyl cyanide are disposed of via underground injection, the seeming lack of information on its transport and fate in groundwater50 is troublesome. There are scattered mentions (e.g., Page 11, Line 8 and Page 15, Line 13) of its potential to contaminate groundwater but no detailed exposition of this process or its consequences. Even if such information is not available, some mention should be made of groundwater as a separate media and of methyl cyanide ’ s possible transport and fate in this media.

EPA Response IV

- B

- Ch2

- Q2

- 6 : See EPA response to Comment IV

- B

- Ch2

- Q2

- 1

Comment IV

- B

- Ch2

- Q2

- 7 : Commenter Seven Page 14, Line 15. Please specify these “various” waste streams.

EPA response Comment IV

- B

- Ch2

- Q2

- 7 : When reading the phrase “various waste streams” in context with the text, it is understood one could be referring to land, water or air. No changes will be made to the text based upon this
comment.

Comment IV

- B

- Ch2

- Q2

- 8 : Commenter Seven Page 15, Section 2.6.2. Except for its cryptic last sentence (Page 16, Line 1), this section makes it appear that abiotic degradation only occurs in air. Is this true? And what are the presumed byproducts of such degradation?

EPA R esponse IV

- B

- Ch2

- Q2

- 8 : Abiotic degradation of acetonitrile is extremely slow under environmental conditions

. (Brown et al., 1975) and in general would be limited to air given the fact the estimated hydolysis ha lf

- life rate is 150,000 years. The section of the report on abiot ic degradation has been expanded in response to the peer reviewer’s comment and is duplicated in the EPA response to Comment IV

A

- 4, section “2.6.2. Abiotic Degradation.”

Comment IV

- B

- Ch2

- Q2

- 9 : Commenter Seven Page 16, Line 11. The “...most significant degradation process...” Please clarify whether this refers to all such processes or just the biotic.

EPA Response IV

- B

- Ch2

- Q2

- 9 The paragraph containing the sentence of concern has been revised for clarity, indicating that biodegradation is the most significant process for acetonitrile degradation. The revised paragraph is provided below.

“ 2.6.3. Biotic Degradation. The most significant degradation process for acetonitrile is biodegradation. The biological capability for acetonitrile degradation a ppears to be widespread over several genera of prokaryotes (organisms that lack a cell nucleus or any other m embrane

bound organelles), and eukaryotes (organisms whose cells are internally organized into complex structures enclosed within membranes) . Expe rimentally observed degradation rates vary based upon several factors including media type, temperatur e, acetonitrile concentration , available oxygen, specific microorganisms involved and the degr ee of acclimation. Within this section additional informa tion is provided on specific microo rganisms capable of degrading acetonitrile along with studies assessing the degradation of acetonitrile under aerobic and anaerobic conditions. It is important to note that many of the biodegradation results provided be low are from studies that used test conditions likely representative of aerobic or anaerobic digestion51 within a sewage treatment plant. These conditions are not necessarily reflective to relevant environmental conditions, in particular those conditions tha t are associated with underground injection wells or subterranean environments.”

Comment IV

- B

- Ch2

- Q2

- 10 : Commenter Seven Page 16, Line 22. The compound is degraded into what?

EPA Response IV

- B

- Ch2

- Q2

- 10 : Text in sections 2.6.3.1 and 2.6.5 contain information concerning the biodegradation products of acetonitrile. The relevant tex t is provided below.

“ 2.6.3.1 Mechanisms of Biotic Degradation While mechanisms for the microbial degradation of acetonitrile have been proposed, they have been elucidat ed using pure microorganism strains and/or acetonitrile as the sole carbon source. Whether or not these mechanisms occur in the environment is unknown. A single

- step mechanism for the direct intracellular conversion of acetonitrile to acetic acid and am monia via the enzyme nitrilase (See Figure 2

- 1) has been proposed (Chapatwala et al., 1992; Li et al., 2007; Nawaz et al., 1989).

Figure 2

- 1

A two

- step enzymatic hydrolysis mechanism for the aerobic biodegradation of acetonitrile has also been proposed (see Figure 2

- 2) (Dadd et al., 2000; Rezende et al., 1999; Linardi et al., 1996; Babu et al., 1995; Nawaz and Chapatwala, 1991; Nawaz et al., 1 989 ,Chapatwala et al., 1992; Li et al., 2007). In this mechanism, acetonitrile undergoes enzymatic degradation to form acetamide, which then readily biodegrades to form acetic acid and ammonia.

Figure 2

- 2

Since acetamide is not formed in the single

- step mechanism and is expected to be degraded in the two

- step process, acetic acid and ammonia are expected to be the predominant end products of the aerobic biodegradation of acetamide (Graham et al., 2000; Li et al., 2007; Nawaz et al., 1989) ”

.

“ 2.6. 5. Toxic Transformation Products. As noted in section 2.6.3, biodegradation is the most significant degradation process for acetonitrile in the environment. The major transformation products of acetonitrile in the environment are expected to be acetic acid and ammonia (Graham et al., 2000 ). Although some acetamide may be generated as a part of the two

- step biodegradation process noted in section 2.6.3, it is expected to biodegrade rapidly resulting in the formation of acetic acid and ammonia (Graham et al., 2000; Li et al., 2007; Nawaz et al., 1989 Urano and Kato, 1986a,b).”

Comment IV

- B

- Ch2

- Q2

- 11 : Commenter Seven Page 17, Line 15. Please define anaerobic “consortium.” There is also no mention between C N H 3 C H 2 O

N i t r i l a s e H 3 C O H O N H 3 +

C N H 3 C H 2 O

N i t r i l e h y d r a t a s e H 3 C N H 2 O H 2 O

A m i d a s e H 3 C O H O N H 3 +52 Lines 14 and 23 of the environmental media in which these experiments were conducted.

EPA Response IV

- B

- Ch2

- Q2

- 11 : Anaerobic consortium is self explanatory ( a mix of the available bacterium present in the media). Specifics on the microorganisms composing the anaerobic consortium were not provided in Nagel (1995). The inno culum was obtained from the low

- solids anaerobic digesters at the Denver Municipal Waste Water Reclamation Plant in Denver, Colorado. Th e following has been added to the technical report.

2.6.3.3 Anaerobic Biodegradation Studies “... An inoculum obtained from low

- so lids anaerobic digesters at the Denver Municipal Waste Water Reclamation Plant in Denver, Colorado, demonstrated an 83% reduction of acetonitrile on a dry municipal solid waste feedstock mixed with a liquid nutrient solution. The 83% reducti on of acetoni trile was observed after a 90

- day incubation period at 37 o C using an initial acetonitrile concentration of 50 mg/L. ”

Comment IV

- B

- Ch2

- Q2

- 12 : Commenter Seven Page 17, Line 17. When you say anaerobic degradation results can be “applied” to groundwater, do you mean by extrapolation of tests in other media or because test were actually conducted in groundwater?

EPA Response IV

- B

- Ch2

- Q2

- 12 : The sentence of concern, Page 17, Line 17, (see below), has been deleted from the revised report after consideration of the lack of information available to predict anaerobic degradation of acetonitrile under the relevant environmental conditions.

Dele ted

--- “These estimates may be applied to sediments or to soils and groundwater beyond aerated zones.”

Comment IV

- B

- Ch2

- Q2

- 13 : Commenter Seven Given (per Page 18, Lines 3

- 4) that groundwater is a source of methyl cyanide contamination, it would seem import ant to provide as much information as is available about its behavior in this media.

EPA Response IV

- B

- Ch2

- Q2

- 13 The report does not state that ground water is a source of methyl cyanide contamination. The persistence of acetonitrile in groundwater will be expanded

. Please see the response to Comment IV

- B

- Ch2

- Q2

- 5.

Comment IV

- B

- Ch2

- Q2

- 14 : Commenter Seven Page 18, Section 2.6.4. Using EPIWIN v 3.20 and the physicochemical parameters listed in Table 2

- 1, I was able to reproduce the half

- life, but not the mass amount, estimates given in Table 2

- 2. For example, my estimate for air was 11.8% rather than the 51.7% given in Table 2

- 2. Either an error was made or not all of the assumptions used to produce 51.7% and the other mass amount values were clearly stated. Given the importance of the EPIWIN results to understanding the fate of this chemical, all of the inputs/assumptions used for this model should be disclosed and listed (possibly in a separate appendix), that replication of results is more straigh tforward.

EPA Response IV

- B

- Ch2

- Q2

- 14 : The model was rerun using values for releases analogous to those being reported as a part of the53 TRI. The model inputs will be stated in the report. The regenerated results based upon TRI reported releases for 200 9 are in the following table taken from the revised report. Note that the masses placed in the model were the TRI values reported for total air, total land and surface water releases; releases to underground injection wells were not a part of this analysi s. When running the fugacity model, the advection was turned off by placing advection times at 10,000 hours.

Compartment Mass amount (percent) Half

- life Values (hours) Air 79.8 1.54x10 3
Water 16.7 360 Soil 3.5 720 Sediment 0.0329 3.24x10 3 Source: EPI Suite, (U.S. EPA, 2011b)

Comment IV

- B

- Ch2

- Q2

- 15 : Commenter Seven Page 18, Line 9. The EPIWIN model can release 1000 kg/hr to all three compartments simultaneously or one at a time. Which of these release scenarios was followed? Was an inve stigation made of what affect releasing to only one compartment at a time would have on the environmental distribution of methyl cyanide? In other words, are all release points equal or do they produce differing outcomes or is Table 2

- 2 always the same re gardless of release point?

EPA Response IV

- B

- Ch2

- Q2

- 15 : Different relative releases can have a moderate impact on the predicted environm ental distribution percentages masses. However, for this report, the model was rerun using release s reflective of what was reported to the TRI for 200 9

. See the response to Comment IV

- B

- Ch2

- Q2

- 14 above for the actual results.

Comment IV

- B

- Ch2

- Q2

- 16 : Commenter Seven Page 18, Lines 18

- 21. These are redundant with Page 10, Lines 22

- 25.

EPA Response IV

- B

- Ch2

- Q2

- 16 : That is correct. Some of the same information in the main body of the chapter has been placed in the chapter summary. No changes to the report will be done based upon this comment.

Comment IV

- B

- Ch2

- Q2

- 17 : Commenter Seven Page 18, Line 10

- 11. This sentence is unclear. Please clarify whether these estimated half

- lives those estimated internally by EPIWIN or user

- entered?

EPA Response IV

- B

- Ch2

- Q2

- 17 : The text will be clarified to indicate the half

- life values used were generated by the EPIWIN software. Please note that the f ugacity model data has been revised based upon the overall TRI reported releases to soil, air and water for TRI reporting year 200 9

. For further details see the response to Comment IV

- B

- Ch2

- Q2

- 14

.

Comment IV

- B

- Ch2

- Q2

- 18 : Commenter Seven Page 18, Lines 16

- 24. The vast majority of releases occur in injection wells, which have shown the potential to contaminate groundwater. Yet the EPIWIN model does not include groundwater as one of its environmental compartments. As has been said previou sly, this leaves open the54 question of the transport and eventual fate of methyl cyanide in groundwater.

EPA Response IV

- B

- Ch2

- Q2

- 18 : No information is available that indicate s that acetonitrile is leaking out of underground injection wells. To create pot ential scenarios of underground injection well failures along with a fugacity model for environmental concentrations based upon the scenario is beyond the scope of this hazard assessment report. EPA’s response to Comment IV

- B

- Ch2

- Q2

- 1 does provide report revisions relevant to this comment in terms of the transpo rt and fate of acetonitrile in subterranean environments.

Comment IV

- B

- Ch2

- Q2

- 19 : Commenter Seven Page 18, Line 22. Is there a reference for the statement “...can be anticipated to be significantly slower...”?

EPA Response IV

- B

- Ch2

- Q2

- 19 : Based upon further review of the literature, the term “significantly” has been replaced with “considerably” in the revised text. References for the statement have been provided in the revised report a nd are associated with the text in sections 2.6.3.2 and 2.6.3.3. . The revised section is provided below

.
Section 2.6.4 Environmental Transport and Distribution “... Based upon the information presented in section 2.6.3.2 and 2.6.3.3 of this report, it can b e anticipated that the actual biodegradation of acetonitrile in underground injection wells where high concentrations of the chemical and anaerobic conditions exist , will be considerably slower than observed biodegradation rates under aerobic conditions fo r relatively low concentrations of the chemi cal. ”

Comment IV

- B

- Ch2

- Q2

- 20 : Commenter Seven Page 19, Line 8 lists the toxic byproducts as acetic acid and ammonia

- is this always the case? What about cyanide?

EPA Response IV

- B

- Ch2

- Q2

- 20 : Acetonitirile is extremely stable under environmental conditions and in general biotic degradation is the major mechani sm of decomposition. Hydrogen cyanide was not reported in the literature reviewed as a b acterial decomposition product h ydrogen cyanide is recognized as a decomposition product of acetonitrile in incomplete combustion processes conducted at temperatures below the ignition point of acetonitrile (Britt, 2002). The report has been expanded to discuss the incineration (combustions) of acetonitrile and the decomposition products formed. Text from the revised report is provided in the response to Comment IV

- B

- Ch2

- Q 1

- 2.

Comment IV

- B

- Ch2

- Q2

- 21 : Commenter Eight Yes. A minor issue has already been mentioned above. Except for brief comments and a few sentences on page 18, there was not much detail about the transport and transformation of acetonitrile in groundwater, a major location for its disposal.

EPA Response IV

- B

- Ch2

- Q2

- 21 : Please note that groundwater was not identified as a disposal location for acetonitrile. The report has been expanded to provide additional information on the transport and transformation of acetonitrile in groundwater (anaerobic conditions) . Please see the response s to Comments IV

- B

Ch2

- Q2

- 1 and IV

- A

- 1.55 Comment IV

- B

- Ch2

- Q2

- 22 : Commenter Eight Section 2.6.4. is a sou nd description of the modeling. A Monte Carlo

- based exercise that included uncertainties would provide more insight about the environmental distribution under a range of conditions or realistic scenar ios.

EPA Response IV

- B

- Ch2

- Q2

- 22 : It is beyond the scope of this report to provide environmental distribution information for a range of conditions related to realistic scenarios. The environmental distribution section of the report has been being revis ed based upon the overall relative release percentages (air, la nd, and water) reported to TRI. For details see the response to Comment IV

- B

- Ch2

- Q2

- 14 above.

Comment IV

- B

- Ch4

- Q 1

- 1: Commenter Six The information on ecological toxicity has been objectively described. I am not aware of any additional studies that should be included. The text could be more clearly presented, specific comments are provided below. EPA Response IV

- B

- Ch4

- Q1

- 1 : No response to this general comment.

Comment IV

- B

- Ch4

- Q1

- 2 : Commenter Seven There are two concerns with this chapter : (a) an exclusive focus on lower trophic level aquatic and terrestrial species with no discussion of terrestrial vertebrates and (b) the lack of robust available information on the chronic toxicity of methyl cyanide , particularly in vertebrate species. Ecological receptors include more than plants, invertebrates, and fish. Was there no information available on methyl cyanide effects in terrestrial vertebrates? If this is the case, it would be helpful to make a clea r statement to this effect in Section 4.1.

Although somewhat problematic, would it not have been possible to draw some tentative conclusions about terrestrial vertebrate responses in the wild from the rat and mouse laboratory data presented in Chapter 3?

EPA Response IV

- B

- Ch4

- Q1

- 2 : In keeping with strong precedent for ecotoxicological investigations, data are not reported here on laboratory mouse, rat, hamster, and rabbit studies that are normally performed to assess potential human health impacts. Thes e data are reported in the Human Health section. All data on additional vertebrates (e.g., fish) that was located can be found in the Ecological Hazard Assessment section.

No changes to the report will be made based on this comment.

Comment IV

- B

- Ch4

- Q1

- 3

- : Commenter Seven Page 51, Line 15. Please add “or biomagnify” after “bioaccumulate” to distinguish these two different processes. Also on Page 57, Line 11. Chapter Four, Ecological Hazard Assessment 1. Has the available information on the ecological toxicity of acetonitrile been objectively described? Are there any additional studies that should be included?56 EPA Response IV

- B

- Ch4

- Q1

- 3 : The recommendation to add ‘biomagnify’ was not suppor ted by available information. No changes to the report will be made based on this comment.

Comment IV

- B

- Ch4

- Q1

- 4: Commenter Seven Page 51, Section 4.1. There is no mention of whether there is any information available on the mechanism of toxicity of methyl cyanide in various ecological receptors. If no information is available, then it would be helpful to make a clear statement to this effect in Section 4.1. Couldn’t what is known in this regard about cyanide be used to make some informed judgments about methyl cyanide?

EPA Response IV

- B

- Ch4

- Q1

- 4 : An elaboration on the mechanism of toxicity to various ecological receptors is beyond the scope of this report. However, some information relevant to the toxicity mechanism in vert ebrates is provided in s ection 3.2, “Kinetics and Metabolism.” No changes to the report will be made based upon this comment.

Comment IV

- B

- Ch4

- Q1

- 5: Commenter Seven Page 53. Separate tables for acute and chronic data would be helpful.

EPA Response IV

- B

- Ch4

- Q1

- 5 : Acute and chronic toxicity sections of the table are distinct. No changes to the report will be made based upon this comment.

Comment IV

- B

- Ch4

- Q1

- 6: Commenter Seven Page 53, Table 4

- 2. It is not clear whether footnotes “b” and “c” apply to all or just selected

s tudies listed [It would also help if the font size of the footnotes was increased to, say, 10 pt]. Also, is the “d” in the “Value” column supposed to be a footnote?

EPA Response IV

- B

- Ch4

- Q1

- 6 : Editorial changes have been made to the table. We were unable to increase the footnote designations to font size 10 do to limitations in the software. Footnote “b” applies to all studies unless otherwise noted by a footnote specifying concerns over water hardness

. Footnote “c” presents the reminder of “mg/L = ppm.” Footnote “d” has been updated and moved.

Comment IV

- B

- Ch4

- Q1

- 7: Commenter Seven Page 55, Section 4.2.2. The chronic toxicity testing was performed on embryos of a non

standard, fairly robust, test species. It has been sh own (for cyanide) that juveniles and adults, and not embryos, are the more sensitive life stages. The text also notes several other weaknesses in the very limited studies on chronic toxicity in fish. The ECOSAR estimated chronic values are lower by a fac tor of 2 to 10 than those from these deficient studies.

EPA Response IV

- B

- Ch4

- Q1

- 7 : No changes to the report based upon this comment. The experimental study data , even with the specific issues with the data as noted, is preferred over the model data.

Comment IV

- B

- Ch4

- Q1

- 8: Commenter Eight It is my opinion that the ecological toxicity was objectively described. In addition, the earlier chapter on health effects clearly presented useful insight germane to the ecological effects discussion. A few additional references might be useful. They ar e given below in Point 13 of my57 Specific Observations.

It must be noted (Specific Observations 14

- 17 below) that the toxicity data that the EPA had available was not high quality information. Given the widespread and high use of this compound, I found thi s very surprising. However, I did not find much more information during a brief journal search.

EPA Response IV

- B

- Ch4

- Q1

- 8 : The recommended references have been reviewed. Inform ation provided was not chemical specific for acetonitrile. No changes will be made to the report based upon these references.

Comment IV

- B

- Ch4

- Q2

- 1 : Commenter Six The conclusion appears to be supported with respect to ecological receptors. Supporting information is needed for the statement that “acute toxicity va lues . . . greater than or equal to 100 mg/L indicating relatively low acute aquatic toxicity.” While I agree with this conclusion, documentation (either by regulatory guidelines or comparison with values for aquatic toxicity for other chemicals) is neede d

.
EPA Response IV

- B

- Ch4

- Q2

- 1 : The te xt in question has been revised and is presented below. Conte xt to the phrase “indicating relatively low acute aquatic toxicit y” is provided and a reference cited in the report.

“ Based upon a review of the limited acute and chronic exposure information available for aquatic species (algae, invertebrates, and vertebrates), adverse environmental effects due to acetonitrile can only be anticipated at exposures to elevated concentrations. This conclusion is based on o bserved acute toxicity values for aquatic organisms are greater than or equal to 100 mg/L indicating relatively low acute aquatic toxicity (Table 4

- 2) ( see for example: http://www.epa.gov/oppefed1/ecorisk_ders/toera_analysis_eco.htm#Ecotox ). ”

Comment IV

- B

- Ch4

- Q2

- 2 : Commenter Six In addition, minimal information exists with regards to terrestrial toxicity, population effects, or general effects on the environment. Thus, the conclusion (in Section 4.1) should be qualified that it primarily addresses ecolog ical receptors in aquatic environments.

EPA Response IV

- B

- Ch4

- Q2

- 2 : Vertebrate biotransformation toxicity mechanisms can be found in section 3.2 Kinetics and Metabolism in the report. No changes to the report will be made based on this comment.

Comment IV

- B

- Ch4

- Q2

- 3 : Commenter Seven No, for two primary reasons. First, it does not appear possible to determine if there is a concern without some information on typical environmental concentrations for comparison with these toxicity values.

EPA Re sponse IV

- B

- Ch4

- Q2

- 3 : Providing a risk assessment is out of the scope of this ecological hazard assessment. No changes Chapter Four, Ecological Hazard Assessment 2. Is the conclusion presented in section 4.1, “Acetonitrile does not present a concern for adverse effects on th e environment” supported by the data/information presented?58 to the report will be made based upon this comment.

Comment IV

- B

- Ch4

- Q2

- 4 : Commenter Seven Second, the already noted weaknesses in chr onic toxicity information make it particularly difficult to assess population

- level effects from, for example, impairment of reproductive ability due to chronic exposures to low levels (e.g., from a groundwater seep to surface water).

EPA Response IV

- B

- Ch 4

- Q2

- 4 : It is recognized that there is limited information in the area of chronic toxicity. No information was identified for population

- level effects. The conclusi on statement will be qualified as follows:

“Based upon a review of the limited acute and chronic exposure information available for aquatic species (algae, invertebrates, and vertebrates), adverse environmental effects due to acetonitrile can only be anticipated at exposures to elevated concentrations . This conclusion is based on observed ac ute toxicity values for aquatic organisms are greater than or equal to 100 mg/L indicating relatively low acute aquatic toxicity (Table 4

- 2) ( see for example: http://www.epa.gov/oppefed1/ecorisk_ders/toera_analysis_eco.htm#Ecotox ). Acute toxicity values f or freshwater fish and aquatic invertebrates ranged from 100 to 3600 mg/L. The 96 hour LC 50

/EC 50 values for fish and green algae ranged from 100 to 3454 mg/L. Furthermore, experimental chronic toxicity values for green and blue

- green algae, freshwater in vertebrates, freshwater fish embryos, and duckweed ranged from 160 to 7300 mg/L, indicating relatively low chronic toxicity.”

Comment IV

- B

- Ch4

- Q2

- 5 : Commenter Seven All of this suggests that available information is insufficient to make unequivocal stat ements about the chronic toxicity of methyl cyanide . This also limits this technical document’s ability to support broad, supportable statements about a lack of concern. Because what is of “concern” is typically largely a policy determination, it is not clear why a technical review has to make this determination?

EPA Response IV

- B

- Ch4

- Q2

- 5 The comment is well taken and the relevant text has been revised as follows:

“

Based upon a review of the limited acute and chronic toxicity data available for aquatic species (algae, invertebrates, and vertebrates), adverse environmental effects due to acetonitrile can only be anticipated at relatively high exposure concentrations. This conclusion is based on observed acute toxicity values for aquatic organisms that are greater than or equal to 100 mg/L; indicating relatively low acute aquatic toxicity (Table 4

- 2) ( see for example: http://www.epa.gov/oppefed1/ecorisk_ders/toera_analysis_eco.htm#Ecotox ). Acute toxicity values for freshwater fish and aquatic inve rtebrates ranged from 100 to 3600 mg/L. The 96 hour LC 50 values for fish and green algae ranged from 100 to 3454 mg/L.

Experimental chronic toxicity values for green and blue

- green algae, freshwater invertebrates, freshwater fish embryos, and duckweed ranged from 160 to 7300 mg/L indicating relatively low chronic toxicity. Experimental chronic toxicity studies on earthworms reported 100% survival at exposure concentrations of 500 μ g/g soil and 200 μ L/g soil. No data were available to assess chronic to xicity to estuarine/marine fish or invertebrates. Acetonitrile is not expected to bioaccumulate in aquatic organisms (BCF is 3.2 based on log K ow :

- 0.34).”59 And the following footnote has been added:

1 Many of the test concentrations were reported as nom inal and thus table values could potentially underestimate toxicity as they are likely higher than the actual exposure concentrations. The EPIWIN model was therefore used to calculate a volatilization 1⁄2 life for lakes which was then used to estimate aceto nitrile concentrations remaining in the static exposure systems for the nominal 100 mg/L treatments. The model estimated 1⁄2 life of 7.5 daysdays indicates that approximately 6 9 mg/L would be expected to remain at the end of the 96 hour static exposures. Th e 100 mg/L was chosen as it was the lowest effect concentration observed. See section 2.6.1 Environmental Transport for the details of the Model Lake estimation.

Comment IV

- B

- Ch4

- Q2

- 6 : Commenter Eight Yes. This seems a reasonable conclusion from the facts presented. Acetonitrile does not appear to be very toxic but the toxicity data are not high quality information. Acetonitrile does not have any properties suggesting that it would bioaccumulate to any lev el of concern. My only minor reservation is described below in Point 2 of my Specific Observations. Although the acetonitrile has low toxicity as expressed as effect concentrations, it is released from diverse sources in large
amounts. The amounts and w idespread nature of its release suggests the need for extra diligence to possible ecological effects under various exposure scenarios despite its low toxicity.

EPA Response IV

- B

- Ch4

- Q2

- 6 : As mentioned above , an ecological risk assessment is beyond the sc ope of this hazard
assessment

. No changes will be made to the report based upon this comment.



### C. Specific Observations

Comment IV

- C

- 1 : Commenter Six Section 1.2 Conclusions: Pg. 2; Lines 3

- 6: The introductory statement to this section is very long and somewhat confusing. Suggest the following: “The EPA has concluded that it is reasonable to anticipate mortality in animals chronically exposed to acetonitrile. This conclusio n is based on observations of mortality in acute, short

- term and subchronic repeated exposure studies in animals. In addition, acetonitrile has been shown to have a steep dose

- response curve; and, the mode of action for acetonitrile toxicity (i.e., conver sion to cyanide) also supports this conclusion.”

EPA Response IV

- C

- 1 : The recommended edits have been taken into consideration. A revised copy of the text is provided below.

“ In review of t he potential human toxicity of acetonitrile, based on the available data, and given the severity of the effect, mortality, EPA concludes that there is sufficient evidence to support a concern for moderately high toxicity from long

- term repeated exposure. ”

Comment IV

- C

- 2 : Commenter Six In addition, the conclusion s should be clearly stated and perhaps bulleted at the introduction of this section. For example:

• Acetonitrile has been found to produce mortality in acute, short

- term and subchronic animal studies;60 • Due to it’s mode of action and steep dose

- response cur ve, it is reasonable to assume that mortality would also occur under chronic conditions;

• In ecological studies, acetonitrile has been found to have minimal toxicity (i.e., concentrations of greater than 100 mg/L are required to produce mortality in aquati c studies);

• Acclimated microbes have been found to be capable of rapidly degrading acetonitrile under aerobic conditions; however, under anaerobic conditions or with unacclimated microbes, acetonitrile may persist in the environment.

EPA Response IV

- C

- 2 : The style of the report along with the complexity of the findings, does not lend to a set of discrete on

- line statements capturing all significant findings. No changes have been made to the report based upon this comment.

Comment IV

- C

- 3 : Commenter Six Pg. 2; Line 9: Revise text to read “. . . oxygen needed for cellular respiration. The lack of oxygen in cells leads to cytotoxic anoxia (Albaum et al., 1946).”

EPA Response IV

- C

- 3 : The text in question has been removed from the Executive Summary chapter of the report; the recommended edit is not applicable to the revised document.

Comment IV

- C

- 4 : Commenter Six Pg. 2; Line 12: The text states that “similar clinical signs have been observed in humans”. Are these case reports involving humans exposed to cyanide or acetonitrile? This should be clarified in the text.

EPA Response IV

- C

- 4 : The text in question has been removed from the Executive Summary chapter of the report; the recommended edit is not applicable to the revised document.

Comment IV

- C

- 5 : Commenter Six Pg. 2; Line 15: Delete “animal” (before repeat

- dose toxicity).

EPA Response IV

- C

- 5 : The text in question has been removed from the Executive Summary chapter of the report; the recommended edit is not applicable to the revised document.

Comment IV

- C

- 6 : Commenter Six Pg. 2; Line 18: Delete “the” (before 13

- week inhalat ion studies).

EPA Response IV

- C

- 6 : The text in question has been removed from the Executive Summary chapter of the report; the recommended edit is not applicable to the revised document.

Comment IV

- C

- 7 : Commenter Six Pg. 2; Line 20: Delete “a delayed period of time (weeks)” and replace with “several weeks.”61 EPA Response IV

- C

- 7 : The text in question has been removed from the Executive Summary chapter of the report; the recommended edit is not applicable to the revised document.

Section 1.3 Chemist ry and Environmental Fate:

Comment IV

- C

- 8: Commenter Six Pg. 3; Line 17: The text describing volatilization of acetonitrile from a river should include the parameters (temperature and wind speed) that were used in developing this estimate.

EPA Response IV

- C

- 8 : The recommended edits have been taken into consideration. A revised copy of the text is provided below.

“Section 2.6.1...Based on a Henry’s Law constant of 3.45x10 - 5 atm

- m 3 /mol (Lyman et al., 1990; Gaffney et al., 1987) using the EPIWIN estimation software (U.S. EPA, 2009a), the volatilization half

- lives of acetonitrile are calculated to be 0.5 days for a model river (assuming a wind velocity of 5m/sec, water depth of 1 meter, current velocity of 1 m/sec, temp at 25 °C ) and 7 .5 days f or a model lake (assuming a wind velocity of 0.5m/sec, water depth of 1 meter, current velocity of 0.05 m/sec, temp at 2 5 °C ).”

Comment IV

- C

- 9 : Commenter Six Pg. 3; Line 19: Delete “land” and replace with “soil.”

EPA Response IV

- C

- 9 : The recommended edit has been made to the report.

Comment IV

- C

- 10 : Commenter Six Pg. 3; Lines 20

- 21: Revise text to read

– “sorption coefficient and high water solubility it is predicted that acetonitrile will migrate rapidly through permeable soils.”

EPA Response IV

- C

- 10 : The recommended edit has been made to the report.

Comment IV

- C

- 11 : Commenter Six Pg. 4; Line 5: Insert “acetonitrile in the presence of” after “Half

- lives for.”

EPA Response IV

- C

- 11 : The report has been revised in this area to read as follows:

“ The most significant degradation process for acetonitrile is biodegradation; neither hydrolysis nor photodegradation is expected to be an important degradation process for acetonitrile (Brown et al., 1975, Atkinson, 1994). Observed biodegradation rates of acetonitr ile have been reported at 58% in 5 days (Babeu and Vaishnav (1987)), and 40% after 10 days (Urano and Kato (1986 a,b)). ”

Comment IV

- C

- 12 : Commenter Six Pg. 4; Line 7: Revise text to read “for non

- acclimated or anaerobic bacteria” (assuming this is correct?).62 EPA Response IV

- C

- 12 : In further review of the studies available, most all produced relevant to sewage treatment plant conditions, the text has been revised as follows:

“ Section 1. 2 Chemistry and Environmental Fate

... The most significant degradation process for acetonitrile is biodegradation; neither hydrolysis nor photodegradation is expected to be an important degradation process for acetonitrile (Brown et al., 1975, Atkinson, 1994). Observed biodegradation rates of acetontirile have been reported at 58% in 5 days (Babeu and Vaishnav (1987)), and 40% after 10 days (Urano and Kato (1986 a,b)). The potential for anaerobic biodegradation has been indicated by studies conducted by Li et al., (2007) although studies

were not conducted under natural environmental conditions but conditions more so pertinent to sewage treatment plants. Based upon the information presented in sections 2.6.3.2 and 2.6.3.3 of this report, it can be anticipated that the actual biodegradation of acetonitrile in underground injection wells where high concentrations of the chemical are present and anaerobic or oxygen limiting conditions are likely to exist, will be considerably slower than observed biodegradation rates under aerobic conditions for r elatively low concentrations of the chemical. ”

Comments IV

- C

- 13 : Commenter Six

Section 1.4 Human Health Hazard Assessment Summary:

Comments IV

- C

- 13

a. Pg. 4; Line 19: Revise text to read “oxygen needed for cellular respiration. The reduction of oxygen levels in cells m ay result in cytotoxic anoxia.”

b. Pg. 4; Line 20: Insert “in mammalian systems” after “converted to cyanide.”

c. Pg. 4; Line 23: Delete “then” (before “it may be partially”).

d. Pg. 4; Line 24: Place a period (.) after Hartung, 198 2 citation; delete “resulting” and revise text to read “This process results in a greater retention time for cyanide.”

e. Pg. 5; Line 1: Delete “occurs” and replace with “does not occur until several.”

EPA Response to Comments IV

- C

- 13 a - e: The texts in question, as well as the whole section on kinetics and metablosim, have been removed from the Executive Summary chapter of the report.

Comments IV

- C

- 14 : Commenter Six Pg. 5; Lines 2

- 5: Delete text “Following repeated . . .metabolize cyanide to thiocyanate.” Replace with “Following repeated exposure to acetonitrile, it is likely that cyanide will accumulate in the body due to the generation of cyanide from the existing body burden as well as new exposures to acetonitrile. This accumulation may r esult in cyanide levels in tissues that exceed the capacity of enzymes to metabolize cyanide to isocyanate.”

EPA Respon s e IV

- C

- 14 : The text in question, as well as the whole section on kinetics and metabolism , has been removed from the Executive Summary chapter of the report.

Comment IV

- C

- 15 : Commenter Six63 Pg. 5; Line 6

- 7: Revise text to read “The accumulation of toxic concentrations of cyanide and the metabolic rate at which lethal cyanide concentrations are reached will largely depend on the balance . . .”

EPA Response IV

- C

- 15 : The text in question has been removed from the Executive Summary chapter of the report.

Comment IV

- C

- 16 : Commenter Six Pg. 6; Line 8: There is almost a 10

- fold difference between mice and rats for LD50. Is there any metabolic difference between these organisms that can be attributed to this difference? And, if so, what is the relevance of this difference between rats and mice for acetonitrile toxicity in humans? [Note, it is also interesting that acetonitr ile is 10

- fold more toxic in rats; and, male rats appear to be the sensitive species with regards to acetonitrile carcinogenicity].

EPA Response IV

- C

- 16 : Toxicokinetic data demonstrating the metabolic difference between rats and mice and the relationshi p between metabolic differences and the LD50s for rats and mice is unavailable for acetonitrile.

Comment IV

- C

- 17 a : Commenter Six P g. 6; Lines 13

- 14: The text indicates that there is an “overall lack of genotoxic potential” for acetonitrile in cell culture bioassays. This may be an incorrect conclusion. Typically, negative results in cell culture bioassays reflect an insensitivity of the assay more than a potential lack of response in mammalian systems. Acetoni trile did produce a positive response in several cell culture bioassays (SCE in CHO cells, gene conversions in S. cerevisiae , and chromosome aberrations in CHO cells). This should be more clearly stated in the summary.

EPA Response IV

- C

- 17a : In considera tion of this comment, the text stating an “overall lack of genotoxic potential” for acetonitrile was removed from the document.

Comment IV

- C

- 17b: Commenter Six Pg. 7; Lines 20

- 21: Revise text to read “This conclusion is based on the observation that th e minimum acute toxicity values for aquatic organisms is greater than or equal to 100 mg/L. In comparison with other chemicals, acetonitrile has a relatively low aquatic toxicity.”

EPA Response IV

- C

- 1 7 b : The text in question has been revised as follows:

“ Based upon a review of the limited acute and chronic toxicity data available for aquatic species (algae, invertebrates, and vertebrates), adverse environmental effects due to acetonitrile can only be anticipated at relatively high exposure conce ntrations. This conclusion is based on observed acute toxicity values for aquatic organisms that are greater than or equal to 100 mg/L, indicating relatively low acute aquatic toxicity (Table 4

- 2) ( see for example: http://www.epa.gov/oppefed1/ecorisk_ders /toera_analysis_eco.htm#Ecotox ). ”

Comments IV

- C

- 18 : Commenter Six Pg. 8; Line 4: Delete “experimental.”

EPA Response IV

- C

- 18 :64 The word experimental was not deleted since it distinguishes data from experimental studies from studies of estimated data.

2. CHEMISTRY AND ENVIRONMENTAL FATE

2.1 SUMMARY Comment IV

- C

- 19 : Commenter Six Pg. 10; Lines 13

- 21: Information included in the technical summary should be included in this section; and, the text on production values should be revised to match the technical summary (which is more clear). I suggest the following revisions:

“Acetonitrile is produced as a by

- product of acrylonitrile production. The principal uses of acetonitrile include: as a solvent in hydrocarbon extraction processes, as a specia lty solvent, as a reaction solvent in the production of synthetic pharmaceuticals, as a solvent for the extraction of fatty acids, and as a solvent in analytical instrumentation (Lewis 2001; Budavari et al., 1996; WHO 1993). The estimated US production of acetonitrile has varied over the past decade between 10 million and 100 million pounds per year (US EPA 2002). Of the millions of pounds produced each year, a commercial market exists for only half of the inventory. The US production capacity for aceton itrile in 1994 was estimated to be 46 million pounds (McKinney and DeVito, 1998).”

EPA Response IV

- C

- 19 : The recommended editorial changes noted above have been taken in consideration. The revised section s of the report in question follows:

Section 2.1

... ” Acetonitrile is produced as a by

- product of acrylonitrile production. The principal uses of acetonitrile include: as a solvent in hydrocarbon extraction processes, as a specialty solvent, as a reaction solvent in the production of synthetic pharmaceutic als, as a solvent for the extraction of fatty acids, and as a solvent in analytical instrumentation (Lewis 2001; Budavari et al., 1996; WHO 1993). In 2006 the aggregated U.S. productio ns levels reported to EPA were betwe en 50 to less than 100 million po unds per year (U.S. EPA, 2011A). Of the millions of pounds of acetonitrile produced each year, a commercial market exists for about half of the inventory. In 2009, releases of acetonitrile reported to the Toxic Release Inventory (U.S. EPA, 2011) totaled 1 7.7 million pounds, of which 17.4 million pounds were disposed to on

- site in underground injection wells. An additional 17.1 million pounds were burned on

- site for energy recovery by the manufacturers. ”

Section 2

. 4.... “ In 2006 the aggregated U.S. productio ns levels reported to EPA were betwe en 50 to less than 100 million pounds per year (U.S. EPA, 2011 a ). Of the millions of pounds of acetonitrile produced each year, a commercial market exists for about half of the inventory. In 2009, releases of a cetonitrile reported to the Toxic Release Inventory (U.S. EPA, 2011) totaled 17.7 million pounds, of which 17.4 million pounds were disposed to on

- site in underground injection wells. An additional 17.1 million pounds were burned on

- site for energy recove ry by the manufacturers. ”

Comment IV

- C

- 2 0 : Commenter Six Pg. 10; Lines 24

- 25: The text indicates that 30 million pounds of acetonitrile were burned on

site for energy production. Are there any estimates of the efficiency of the combustion procedures? Or, how much acetonitrile (or other combustion by

- products) may be released by65 this process?

EPA Response IV

- C

- 2 0 : The following text has been added to the report in an effort to address information know n on the combustion of acetonitrile:

Chapter 2.6.5 Toxic Transformation Products “... During combustion in air the decomposition of acetonitrile initiates at 760 °C . At 950 °C (with a 2 sec residence time) 99.99% of the acetonitrile will have decomposed (Britt, 2002). The ignition of acetonitrile in excess oxygen results in the production of CO, CO2, H2O, N2 and NO as the major combustion products. The combustion of acetonitrile at temperatures below 900 °C and low oxygen concentrations can result in the production of HCN (Britt, 2002).”

Comment IV

- C

- 21 : Commenter Six Pg. 10; Line 26: Insert “most of” before “the chemical will volatilize”

EPA Response IV

- C

- 21 : The recommended editorial change noted above ha s been placed in the technical report.

Comment IV

- C

- 22 : Commenter Six Pg. 11; Line 1: Insert “predicted to be” before “the environmental compartment”

EPA Response IV

- C

- 22 : The recommended editorial change noted above has been placed in the technical report.

Comment I IV

- C

- 23 : Commenter Six Pg. 11; Line 2: Delete “distribute” and replace with “partition”

EPA Response IV

- C

- 23 : The recommended editorial change noted above has been placed in the technical repo rt.

Comment IV

- C

- 24 : Commenter Six Pg. 11; Line 12: Insert “from” before “weeks to months”

EPA Response IV

- C

- 24 The sentence in question has been edited out of the revised report.

Comment IV

- C

- 25 : Commenter Six Pg. 12; Line 12: Insert “an” before “oxidation catalyst”

EPA Response IV

- C

- 25 : The recommended editorial change noted above has been placed in the technical report.

Comment IV

- C

- 26 : Commenter Six Pg. 12; Line 15

- 16: This reaction should be shown in it’s complete form (or as stated in the text: the reaction produces acrylonitrile with acetonitrile and hydrogen cyanide as principal by

products).66 EPA Response IV

- C

- 26 : The recommended editorial change noted above has been placed in the technical report.

Comment IV

- C

- 27 : Commenter Six Pg. 14; Line 14: Revise text to read “The production and wide use of acetonitrile as a solvent.”

EPA Response IV

- C

- 27 : The recommended editorial change noted above has been placed in the technical report.

Comment IV

- C

- 28 : Commenter Six
P g. 15; Line 2: What temperature was used in estimating the half

- life in a model river?

EPA Response IV

- C

- 28 : Values used in the model have been placed in the report. Below is the report revision of the area of concern.

“ Based on a Henry’s Law constant of 3.45x10 - 5 atm

- m 3 /mol (Lyman et al ., 1990; Gaffney et al., 1987) using the EPIWIN estimation software (U.S. E PA, 2009a), the volatilization half

- lives of acetonitrile are calculated to be 0.5 days for a model river (assuming a wind velocity of 5m/sec, water depth of 1 meter, current velocity of 1 m/sec, temp at 25 °C ) and 7 .5 days for a model lake (assuming a wind velocity of 0.5m/sec, water depth of 1 meter, current velocity of 0.05 m/sec, temp at 25 °C ) ”

.

Comment IV

- C

- 29 : Commenter Six Pg. 16; Lines 1

- 2: Rather than indicate hydrolysis is “extremely slow,” can a half

- life be provided and products of abiotic hydrolysis identified?

EPA Response IV

- C

- 29 : Yes. The text has been expanded to provide a predicted hydrolysis half

- life values. See the text below extracted from the revised report.

Section 2.6.2. Abiotic Degradation “... Abiotic hydrolysis of acetonitrile is extremely slow under environmental conditions (Brown et al., 1975). HSDB (2002) reports a rate constant for the base

- catalyzed hydrolysis of acetonitrile of 5.8E

- 3/M

- hr and a corresponding half

- life at pH 7 of 150,000 years. ”

Comment IV

- C

- 30 : Commenter Six Pg. 16; Line 13: Insert “enzyme” before “transforms the nitriles.”

EPA Response IV

- C

- 30 : The text in question has been edited out of the revised report.

Comment IV

- C

- 31 : Commenter Six Pg. 16; Lines 19

- 20: Revise text to read “Activated sludge and sewage have been found to contain populations of mixed microorganisms that are capable of transforming organic and inorganic substances into less toxic forms.”67 EPA Response IV

- C

- 31 : The text in que stion has been edited out of the revised report.

Comment IV

- C

- 32 : Commenter Six Pg. 16; Lines 22

- 23: Delete “degraded with” and replace with “exhibited”, and delete “More dated studies” and replace with “Several older studies have.”

EPA Response IV

- C

- 32 : The term “degraded with” was replaced with the word “exhibited;” “More dated studies” replaced with “Earlier studies” in the revised report.

Comment IV

- C

- 33 : Commenter Six Pgs. 16

- 17; Lines 24

- 1: Revise text to read “proceeded slowly with unacclimated microorganisms.”

EPA Response IV

- C

- 33 : The recommended editorial change noted above has been placed in the revised technical report.

Comment IV

- C

- 34 : Commenter Six Pg. 17; Lines4

- 5: Delete “develop a strain” and replace with “enrich populations”, and on following line, revise text to read “cultures achieved a rate of degradation of 58% for acetonitrile in water in 5 days (BOD test).”

EPA Response IV

- C

- 34 : The text in question has been edited out of the revised report.

Comment IV

- C

- 35 : Commenter Six Pg. 17; Line 8: Insert “concentrations ranging from” before “0.1

- 25 mg/L.”

EPA Response IV

- C

- 35 : The recommended editorial change noted above has been placed in the revised technical report.

Comment IV

- C

- 36 : Commenter Six Pg. 17; Lines 23

- 24: Revise text to read “anaerobic degradation is roughly four times slower than aerobic degradation”, and on following line, delete “beyond aerated” and replace with “under anoxic conditions.”

EPA Response IV

- C

- 36 : The phrase in question has b een edited out of the report. “Beyond aerated” has been replaced with “under anoxic conditions.”

Comment IV

- C

- 37 : Commenter Six Pg. 18; Line 1: Revise text to read “Due to large differences in temperature, pressure, and other conditions these estimated degradation rates for acetonitrile cannot be applied to underground injection.”

EPA Response IV

- C

- 37 :68 The text in question has been edited out of the revised technical report.

Comment IV

- C

- 38 : Commenter Six Pg. 18; Lines 4

- 5: Because such a large volume of the waste product is disposed in these wells, this section should be expanded to discuss more specifically the solubility of acetonitrile and the potential for migration from the injection zone to aquifers . It may be adequate to indicate that since acetonitrile is highly soluble in water any material discharged into an aquifer from deep

well injection is likely to be transported with groundwater migration. It should also be indicated that the environmenta l conditions (i.e., temperature, nutrient concentration and microbial populations) in groundwater are not conducive to the degradation of any material that does reach the aquifer.

EPA Response IV

- C

- 38 : See EPA Response to Comment IV

- A

- 1.

Comment IV

- C

- 39 : Commenter Six Pg. 18; Lines 19

- 20: It is preferable to use the same units in discussing TRI and injection, suggest that the TRI release for 2003 be rounded off to 20.8 million pounds.

EPA Response IV

- C

- 39 : The recommended editorial change noted above has been placed in the revised technical report.

Comment IV

- C

- 40 : Commenter Six Pg. 18; Lines 21

- 23: Revise text to read “It is anticipated that the rate of acetonitrile degradation following release from an underground injection well will be very slow. Chemical concentrations from an underground release are likely to be relatively high; and, anaerobic or oxygen limiting conditions are likely to exist. Optimal conditions for acetonitrile degradation are generally assumed to require aerobic condit ions and relatively low chemical concentrations.”

EPA Response IV

- C

- 40 : The section in question has been revised as follows:

“2.6.4 Environmental Distribution Assessment ... Based upon the information presented in section 2.6.3.2 and 2.6.3.3 of this report, it can be anticipated that the actual biodegradation of acetonitrile in underground injection wells, where high concentrations of the chemical and anaerobic or oxygen limit ing conditions are likely to exist

, will be considerably slower than observed biodegradation rates under aerobic conditions for relatively low concentrations of the chemical. ”

Comment IV

- C

- 41 : Commenter Six Pg. 19; Section 2.6.5 Toxic Transformation Products. Additional explanation is needed for this statement. Are these transformation products from aerobic degradation? Are there no pathways which would result in the release of cyanide to the environment?

EPA Response IV

- C

- 41 : See EPA Response to Comment IV

- B

- Ch2

- Q1

- 2

.

Comment IV

- C

- 42 : Commenter Six69 Pg. 51; Line 1: Revise text to read “Acetonitrile is not expected to present a significant risk for adverse effects in ecological receptors.”

EPA Response I IV

- C

- 42 : In response to the comment the text has been changed as follows:

“ Based upon a review of the limited acute and chronic exposure information available for aquatic species (algae, invertebrates, and vertebrates), adverse environmental effects due to acetonitrile can only be anticipated at exposures to elevated concentrations. This conclusion is based on observed acute toxicity values for aquatic organisms are greater than or equal to 100 mg/L indicating relatively low acute aquatic toxicity (Table 4

- 2) (Brooks, et al., 197 3 ). ”

Comment IV

- C

- 43 : Commenter Six Pg. 51; Line 11: Delete “ranged” and replace with “are also relatively low, ranging from 160 to 7300 mg/L.”

EPA Response IV

- C

- 43 : Current language in report does capture the author ’ s position. No changes to the report will be made based upon this comment.

Comment IV

- C

- 44 : Commenter Six Pg. 51; Line 24: Revise text to read “found for acetonitrile. Limited data . . .”

EPA Response IV

- C

- 44 : The recommended edit will be made to the report.

Comment IV

- C

- 45 : Commenter Six Table 4.2

– You would have to assume if you have footnotes for a table from “a

- v” that something is not quite right! This table is very difficult to follow or interpret. I believe it would be easier to follow if broken into Table 4.2, Acute Toxicity and Table 4.3 Chronic Toxicity.

EPA Response IV

- C

- 45 : A sing l e table to capture the ecotoxicity values will remain. A larger 10 point font will be put in place for the footnotes.

Comment IV

- C

- 46 : Commenter Six Also, since the conclusions rely heavily on predicted values, it would help the reader if some example of predictability were provided; i.e., compare toxicity values derived under experimental conditions with those predicted using the ECOSAR model. While it is assumed th at the model provides a conservative estimate of toxicity values, this should be demonstrated in the text.

EPA Response IV

- C

- 46 : For the purposes of this report we focus on model data when experimental data is not available. Comparison of model

- generat ed data with experimental results is beyond the scope of this report. No changes to the report will be made based on this comment.

Comment IV

- C

- 47 : Commenter Six70 Finally, the conclusions for ecological receptors would be easier to follow if a summary table were included that provided ranges of values for acute, chronic, aquatic, and terrestrial toxicity.

EPA Response IV

- C

- 47 : The tables provided are sufficient to capture the observed variability. No changes in the report will be made based on this comment.

Comment IV

- C

- 48 : Commenter Seven Chapter One, Integrated Technical Summary:

Page 2, Section 1.2. This section appears to focus exclusively on potential adverse health outcomes in humans, while there is a separate section (Section 1.5) for poten tial ecological impacts. It would be better to have one comprehensive conclusion section, encompassing both human and ecological effects, for the whole report, rather than leave the impression that its conclusions are based exclusively on human impacts.

EPA Response IV

- C

- 48 : No changes to the document will be made based upon this editorial comment. Without increasing the redundancy of the document any further, a separate all encompassing conclusion was not drafted. The conclusions of each assessment type are captured in each of the relative sections of the Executive Sum mary chapter.

Comment IV

- C

-- 49 : Commenter Seven Page 7, Lines 8

- 9. It is not clear how you can assert that this chemical does not pose a concern for the environment without some knowledge of its levels in the environment. If there is information on known environmental levels, it does not appear to have been presented in this report.

EPA Response IV

- C

- 49 : Providing a risk assessment is out of the scope of this ecological hazard assessment. The actually language in question in the text has been changed and can be found in the response to comment IV

- B

- Ch4

- Q2

- 5

.

Comment IV

- C

-- 50 : Commenter Seven Page 7, Lines 19

- 20. The information presented on chronic ecological outcomes is not robust enough to support the statement that methyl cyanide “...does not pose a concern for ... adverse effects on the environment...” The statement “This conclusion is based on ...” with reference to acute effects does not follow from the previous sentence. It is also questionable whether acute outcomes are a reasonable basis for conclusions about chronic outcomes. Chronic ecological outcomes could be of importance in instances where, for example, groundwater leakage to surface water provides a continual, low

- level source of methyl cyanide to aquatic receptors (c.f., Pa ge 15, Line 13 and Page 18, Lines 4

- 5).

EPA Response IV

- C

- 50 : In response to this comment the paragraph in questions has been revised in the report as follows:

Section 1.4.... ” Based upon a review of the limited acute and chronic toxicity data available for aquatic species (algae, invertebrates, and vertebrates), adverse environmental effects due to acetonitrile can only be anticipated at relatively high exposure concentrations. This conclusion71 is based on observed acute toxicity values for aquatic organisms that are greater than or equal to 100 mg/L, indicating relatively low acute aquatic toxicity (Table 4

- 2) ( see for example: http://www.epa.gov/oppefed1/ecorisk_ders/toera_anal ysis_eco.htm#Ecotox ). Acute toxicity values for freshwater fish and aquatic invertebrates ranged from 100 to 3600 mg/L. The 96 hour LC 50 values for fish and green algae ranged from 100 to 3454 mg/L (Table 4

- 2).

Experimental chronic toxicity values for green and blue

- green algae, freshwater invertebrates, freshwater fish embryos, and duckweed ranged from 160 to 7300 mg/L indicating relatively low chronic toxicity. Experimental chronic toxicity studies on earthwor ms reported 100% survival at exposure concentrations of 500 μ g/g soil and 200 μ L/g soil. No data were available to assess chronic toxicity to estuarine/marine fish or invertebrates. Acetonitrile is not expected to bioaccumulate in aquatic organisms (BCF is 3.2 based on log K ow :

- 0.34)

Available studies on the toxicity of acetonitrile to terrestrial species are limited to experimental chronic toxicity values for earthworms. These studies reported 100% survival at exposure concentrations of 500 μ g/g soil for 14 days and 200 μ L/g dry soil for 15 days (Phillips et al., 1993 and Robidoux et al., 2000, respectively). The phytotoxicity of acetonitrile has been assessed experimentally in duckweed. Chronic toxicity values for duckweed ranged from 368 to 1342 m g/L (Tong and Hongjun, 1997; 2001), indicating relatively low chronic toxicity. No data on population effects were located. ”

Comment IV

- C

- 51 : Commenter Seven Specific comments and observations for Chapters 2 and 4 appear above under the relevant chapter.

EPA Response IV

- C

- 51 : Comment does not require a response.

Comment IV

- C

-- 52 : Commenter Eight Minor technical suggestions:

Page 1, Line 10. Please spell out INEOS at its first use.

EPA Response IV

- C

- 52 : “INEOS” is the name of the corporation. No changes to the report will be made based upon this
comment.

Comment IV

- C

- 53 : Commenter Eight Page 1, Lines 18

- 25. The goal of an assessment is to sufficiently understand the risk of specified effects to gauge what level of regulatory attention is warrante d. Although the other qualities of acetonitrile suggest relatively low risk, the sheer magnitude of its annual release and its introduction underground via injection wells suggests that an elevated level of diligence might be warranted in this decision pro cess. The amounts released from diverse sources increases the probability of an adverse effect relative to an equivalent chemical that is released in small amounts by only a limited number of users.

EPA Response IV

- C

- 53 : The goal of this “hazard ” assessment is not to provide a risk assessment but a hazard assessment based upon the observed or predicted toxicity (human and ecological) of the chemical. Based72 upon this hazard assessment, a decision will be rendered by EPA on whether to hon o r the req uest of the petitioner to remove acetonitrile from the list of chemicals required for reporting under the TRI Program. No changes to the report will be made based upon this comment.

Comment IV

- C

- 54 : Commenter Eight Page 13, Lines 13

- 14. One sentence para graph. Perhaps this sentence could be moved to the second paragraph of Section 2.1?

EPA Response IV

- C

- 54 : This paragraph of the repo r t has been expanded as follows:

Section 2.4 Production Methods: “...Acetonitrile is also a naturally occurring substance. Small quantities are present in in coal tar (Budavari, et. al., 1996). It is also formed during the burning of biomass and released to the atmosphere (Karl et al., 2004; Kondo et al., 2004; Sanhueza et al., 2003).”

Comments IV

- C

- 55 : Commenter Eight Page 16, Line 24. Perhaps the word “sluggishly” is too vague for this technical description?

EPA Response IV

- C

- 55 : The te xt has been revised removing the term “sluggishly

. ” The revised text relevant to this comment can be found in section 2.6.3.2 Aerobic Biodegradation Studie s and now reads as follows:

“ Earlier studies reported that the biodegradation of acetonitrile using activated sludge or sewage proceed s slowly with unacclimated microorganisms in comparison to acclimated microorganisms (Kalmykova and Rogovskaya, 1978; Ludzack et al., 1958; Placak and Ruchhoft, 1947). ”

Comment IV

- C

- 56 : Commenter Eight Page 17, Line 4. Change “strain” to “culture”?

EPA Response IV

- C

- 56 : The recommended edit has been made to the report.

Comment IV

- C

- 57 : Commenter Eight Page 1 7, Line 23. Change to read “... anaerobic degradation rate is roughly...”?

EPA Response IV

- C

- 57 : The sentence relevant to the comment has been deleted from the revised report.

Comments IV

- C

- 58 : Commenter Eight Page 18, Lines 7

- 15. A Monte Carlo

- based model that incorporated uncertainty in estimates would enhance this presentation.

EPA Response IV

- C

- 58 : It is beyond the scope of this report to provide environmental distribution information for a range73 of conditions related to possible environmental scenarios. The environmental distribution section of the report is being updated using a fugacity level I II model along with TRI reported releases to air, land and wate r. For further details see the EPA response to c omment IV

- B

- Ch2

- Q2

- 14

.

Comments IV

- C

- 59 : Commenter Eight Page 18, Line 20. Change to “... were disposed on

- site in ...”?

EPA Response IV

- C

- 56 : The sentence in question has been revised as follows:

“ Independent of the release scenario and model estimates reported above, it needs to be reiterated that of the millions of pounds of acetonitrile produced each year, the vast majority of releases are r eported by manufacturers as being disposed of on

- site in underground injection wells ”

.

Comment IV

- C

- 60 : Commenter Eight Page 24, 1 st full paragraph (and elsewhere). A figure might help the reader understand these details better.

EPA Response IV

- C

- 60 : Figure 3

- 2, “Exposure

- response array for toxicological studies of acetonitrile” has been added to the report to help the reader understand key information provided by the studies.

Comment IV

- C

- 61 : Commenter Eight Page 24, Lines 16

- 18. Consider shifting the clause at the end to the beginning of the sentence.

EPA Response IV

- C

- 61 : The text in question has been removed from the Executive Summary chapter of the report; the recommended edit is not applicable to the revised document.

Comment IV

- C

- 62 : Commenter Eight Page 39, Section 3.3.3. The (undefined) power associated with analysis of these data is likely very low because the number of observed exposed females was so low. I wonder about the value of any discussion of these data which might be used to weakly imply low risk.

EPA Response IV

- C

- 62 EPA believes that the limitations of the study in question are appropriately caveated by the existing statement "However, the small number of cases and other limitations (e.g., self

- reported exposure) preclude any definitive conclusions from this study."

Comment IV

- C

- 63 : Commenter Eight Page 51, Line 6. Here and elsewhere the phrase “the fact that” could easily be omitted.

EPA Response IV

- C

- 63 : The recommended edit has been made in some sec tions of the report.

Comment IV

- C

- 64 : Commenter Eight74 The following references might also be helpful: Chen, C

- Y, S

- L Chen and ER Christensen. 2005. Individual and combined toxicity of nitriles and aldehydes to Raphidocelis subcapitata. Environ. Toxicol. Chem. 24(5): 1067

- 1073. Chen, C

- Y and C

- F Huang. 1996. Toxicity of organic mixtures containing cyanogenic toxicants. Environ. Toxicol. Chem. 15(9): 1464

- 1469. Grogan, J, SC DeVito, RS Pearlman and KR Korzekwa. 1992 Modeling cyanide release from nitriles: prediction of cytochrome P450 mediated acute nitrile toxicity. Chem. Res. Toxicol. 5: 548

- 552.

EPA Response IV

- C

- 64 : The recommended references have been reviewed. Information provided was not chemical specific for aceton itrile. No changes will be made to the report based upon these references.

Comment IV

- C

- 65 : Commenter Eight Issues related to the Tong publications: I found the citation for Tong and Hongiun (2001) to be Zhang, T. and H. Jin. 2001. Aquatic toxicity of acetonitrile and its water quality criteria for the protection of aquatic life in China. Inter. J. Environ. Pollut. 15(5): 568

- 575. Perhaps, the first names might incorrectly be inverted in the draft EPA report or by the journal editor? The bottom of the first page of the journal article indicates Zhang and Jin is the correct way to cite the article. However, this individual’s other publications suggest that Tong is his proper name.

EPA Response IV

- C

- 65 : Agree. Journal article suggests Zhang and Jin (2001) is correct. However, Bull. Environ. Contam. Toxicol. shows a previous publication as Tong et al. (1996). The citations have been changed to reflect the way they appear in the origin al references.

Comment IV

- C

- 66 : Commenter Eight Relative to Tong and Hongiun (2001), note that, in addition to the shortcomings mentioned in the draft report about this paper, it appears that nominal concentrations were used. Measured concentrations are much better. Also the applied trimmed Spearman

- Karber LC50 estimates are not as good as parametric methods. There are other issues with this publication relative to methods and statistical analyses. So, I agree with the concern stated on page 56, line 9 about these studies.

EPA Response IV

- C

- 66 : The trimmed Spearman

- Karber estimates were appropriate for non

- normally distributed data. Based upon the comment in reference to the nominal concentrations, the text has been modified with as follows :

“ Fourth, the study reported that four pollutants were tested, but presented results for only two. The study reported water hardness outside of the preferred range of 50 to 250 mg/L as CaCO 3
. Fifth, it appears that nominal concentrations were used. Prefer ence is given to actual measured concentrations

. ”

Comment IV

- C

- 67 : Commenter Eight Relative to Tong et al. (1996), the least powerful option, the Steel’s many

- one test was used75 because the data failed tests for normality. Yet, there is no discussion of c onventional transformations that might have resulted in normal data. The associated NOEC/LOEC/ACR values are likely not of sufficient quality to give them much weight in this EPA draft report.

EPA Response IV

- C

- 67 : While the nonparametric tests may have been overly conse rvative and thus less powerful, the endpoint values reported by Tong et al. (1996) are not at the low end and thus do not influence the overall assessment to a great degree.

Comment IV

- C

- 6 8 : Commenter Eight There are also statistical compromises in Barahona

- Gomariz et al. (1994) that produce considerable uncertainty in my mind about the estimated effect metrics. The applied methods were not very powerful, and power influences the effect metric estimation very much.

EPA Response IV

- C

- 68 : We agree. But the data is presented for informational purposes only in the t able.

Comment IV

- C

- 69 : Commenter Eight The Henderson et al. (1961, or 1960?) paper uses a “static with no renewal” design that was standard in the 1960s but would be co nsidered unacceptable today. I suspect that the acetonitrile concentrations dropped from the nominal concentrations during the test and, as a result, the effect metrics are expressed in concentrations higher than what they should be.

EPA Response IV

- C

- 6 9 : Agree, b ut its hard to reject data on this basis. See EPA response to Comment IV

- C

- 70.

Comment IV

- C

- 70 : Commenter Eight Generally, the Ewell et al. (1986) paper used a rudimentary extrapolation to estimate LC50 values for the tested compounds because they did not have enough partial kills. The lack of high concentration treatments resulted in a <100 mg/L estimate for what seemed to be a static test. Nominal concentrations were used instead of measured concentrations.

EPA Response IV

- C

- 70 : Based on this comment, the text has been modified as follows:

“ Based upon a review of the limited acute and chronic toxicity data available for aquatic species (algae, invertebrates, and vertebrates), adverse environmental effects due to acetonitrile can only be anticipated at relatively high exposure concentrations. This conclusion is based on observed acute toxicity values for aqu atic organisms that are greater than or equal to 100 mg/L; indicating relatively low acute aquatic toxicity (Table 4

- 2) ( see for example: http://www.epa.gov/oppefed1/ecorisk_ders/toera_analysis_eco.htm#Ecotox ) 1 . Acute toxicity values for freshwater fish a nd aquatic invertebrates ranged from 100 to 3600 mg/L. The 96 hour LC 50 values for fish and green algae ranged from 100 to 3454 mg/L.

Experimental chronic toxicity values for green and blue

- green algae, freshwater invertebrates, freshwater fish embryos, and duckweed ranged from 160 to 7300 mg/L indicating relatively low chronic toxicity. Experimental chronic toxicity studies on earthworms reported 100% survival at exposure concentrations of 500 μ g/g soil and 200 μ L

/g soil. No data were76 available to assess chronic toxicity to estuarine/marine fish or invertebrates. Acetonitrile is not expected to bioaccumulate in aquatic organisms (BCF is 3.2 based on log K ow :

- 0.34.”

And the following footnote has been added:

1 Many of the test concentrations were reported as nominal and thus table values could potentially underestimate toxicity as they are likely higher than the actual exposure concentrations. The EPIWIN model was therefore used to calculate a volatilization 1⁄2 life for lakes

, which was then used to estimate acetonitrile concentrations remaining in the static exposure systems for the nominal 100 mg/L treatments. The model estimated 1⁄2 life of 7.5 days indicates that approximately 6 9 mg/L would be expected to rema in at the end of the 96 hour static exposures. The 100 mg/L was chosen as it was the lowest effect concentration observed. See section 2.6.1 Environmental Transport for the details of the Model Lake estimation.77 

## External Peer Review of EPA’s Draft Technical Review of Acetonitrile (Methyl Cyanide)

Prepared for: Nicole C. Paquette, Ph.D. U.S. Environmental Protection Agency

Office of Environmental Information 1200 Pennsylvania Avenue, N. W. ( 2842T ) Washington, D

. C

. 20460

Prepared by:

Versar, Inc. 6850 Versar Center Springfield, VA 22151

Contract No. EP

- C

- 07

- 025 Task Order 8

June 30, 2008External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

i TABLE OF CONTENTS

I. INTRODUCTION

................................ ................................ ................................ ................. 1

II. CHARGE TO THE PEER REVIEWERS

................................ ................................ ............. 3

III. RESONSE TO HUMAN HEALTH CHARGE

................................ ................................ ... 5 A. General Impression..................................................................

....... .5 B. Response to Charge Questions......................................................

... .8 C. Specific Observations.......................................................................................... 27

IV. RESPONSE TO ECOTOXICOLOG Y CHARGE

................................................ 32 A. General Impressions..................................................................... 32 B. Response to Charge Questions............................................................33 C. Specific Observations.................................................................... 40

APPENDICES:

Appendix A: Peer Review Comments from Commenter One.

................................ .......... A

- 1 Appendix B: Peer Review Comments from Commenter Two

................................ .......... B

- 1 Appendix C: Peer Review Comments from Commenter Six.

................................ ........... C

- 1 Appendix D: Peer Review Comments from Commenter Seven

................................ ........ D

- 1 Appendix E: Peer Review Comments from Commenter Three

................................ .......... E

- 1 Appendix F: P eer Review Comments from Commenter Eight.

................................ ......... F

- 1 Appendix G: Peer Review Comments from Commenter Four

................................ .......... G

- 1 Appendix H: Peer Review Comments from Commenter Five

................................ .......... H

- 1External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

1 I. INTRODUCTION

On June 28, 2002, BP Chemicals, Inc. (BP) petitioned the U.S. Environmental Protection Agency (EPA) to remove acetonitrile (methyl cyanide) from the list of chemicals subject to the reporting requirements of Section 313 of the Emergency Planning and Community Right

- to

Know Act (EPCRA) of 1986. Section 313 of EPCRA requires certain facilities manufacturing, processing or otherw ise using listed toxic chemicals to report their annual releases. The EPCRA Section 313 Toxic Release Inventory (TRI) list was originally derived by combining lists of approximately 300 toxic chemicals and 20 chemical categories. In November 1994, another 286 chemicals and chemical categories were added to the list of toxic chemicals under EPCRA Section 313. Chemicals that meet the statutory criteria described in EPCRA Section 313 (d)(2)(A)

- (C) are subject to reporting. As specified by the statute, a chemic al may be added to the list if any of the following criteria are met:

(A) The chemical is known to cause or can reasonably be anticipated to cause significant adverse acute human health effects at concentration levels that are reasonably likely to exist beyon d facility site boundaries as a result of continuous, or frequently recurring, releases.

(B) The chemical is known to cause or can reasonably be anticipated to cause in humans

– (i) cancer or teratogenic effects, or (ii) serious or irreversible

– (I) reproductive dysfunctions (II) neurological disorders (III) heritable genetic mutations, or (IV) other chronic health effects

(C) The chemical is known to cause or can reasonably be anticipated to cause, because of

- (i) its toxicity (ii) its toxicity and persistence in the environment, or (iii) its toxicity and tendency to bioaccumulate in the environment, a significant adverse effect on the environment of sufficient seriousness, in the judgment of the Administrator, to warrant reporting under this section.

In response to the petition, the EPA’s Of fice of Information Analysis and Access (OIAA)

c onducted a full hazard assessment of acetonitrile. Eight senior scientists were selected to peer review this draft Technical Review of Acetonitrile (Methyl Cyanide) , having expertise in either human health ha zard assessment or environmental fate and ecological assessment. Reviewers

were asked to thoroughly review and comment on the content and scope of the draft report, respond to a series of charge questions, and assess whether the report reflects the current state

- of

the

- science.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

2External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

3 II. CHARGE TO THE PEER REVIEWERS

The key issue of the 2002 BP petition is the petitioner’s assertion that the toxic effects of acetonitrile

, as evidenced in the National Toxicology Program (NTP) 1996 study, were mortalities resulting solely from acute exposure and

, thus

, were entirely an acute toxic effect. The EPA does recognize that acetonitrile demonstrates the potential for acute toxicity resulting in death. However, based upon the review of animal test studies including the NTP 1996 study, EPA believes that there is sufficient evidence to support a concern for potential death following repeated exposures to acetonitrile. Repeated exposure studies in animals indicate that acetonitrile can cause death in a nimals under short

- term and subchronic exposure conditions as well as acute exposure regimes. EPA is asking peer reviewers whether the existing science supports a conclusion that acetonitrile can also be reasonably anticipated to cause mortality as a resul t of chronic exposure. The EPCRA section 313 listing criteria are different for acute versus chronic effects; therefore, EPA must assess acute and chronic effects differently in order to determine if they meet the listing criteria. An effect could be classified as both acute and chronic if it occurs under both types of exposure.

In response to the petition, the EPA’s Office of Information Analysis and Access (OIAA) conducted a full hazard assessment of acetonitrile. The assessment is documented in the draft Technical Review of Acetonitrile (Methyl Cyanide)

. The draft technical report provides an overall review and consideration of both human health and environmental hazards. It assesses the scientific soundness of the readily available data/studies inc luding an assessment of the severity of the effect(s) and the dose or concentration levels at which the effect(s) were observed. In the assessment of human h ealth hazards, the IRIS (1999) Toxicological Review of Acetonitrile was the predominant source of i nformation. The EPA/OIAA draft technical report contains several chapters including chemistry and environmental fate, human health hazards, and ecological toxicity. An “Integrated Technical Summary” chapter is placed up front in order to serve as an execut ive summary for the report. EPA’s technical review of the mortality reported in the 1996 NTP study along with a review of other human health and environmental hazards and the environmental fate of acetonitrile are provided in the Technical Review of Aceton itrile (Methyl Cyanide) report.

Human health reviewers are asked to review Chapter 3, the Human Health Hazard Assessment, and relevant sections of Chapter One, the Integrated Technical Summary. Ecotoxicology reviewers are asked to review Chapters Two and Four (Chemistry and Environmental Fate, and Ecological Hazard Assessment, respectively ), and relevant sections of Chapter One. S pecific charge questions for this assignment are presented below.

Charge Questions for Human Health Toxicologists

Chapter Th ree, Human Health Hazard Assessment Please note that the following EPA IRIS definitions (IRIS glossary http://www.epa.gov/iris/gloss8.htm ) for acute, short

- term, subchronic, and chronic are in effect for this hazard assessment of acetonitrile (listed below in alphabetical order).

Acute Exposure: Exposure by the oral, dermal, or inhalation route for 24 hours or less.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

4 Acute Toxicity: Any poisonous effect produced within a short period of time following an exposure, usually 24 to 96 hours. Chronic Effect: An effect that occurs as a result of repeated or long term (chronic) exposures. Chronic Exposure: Repeated exposure by the oral, dermal, or inhalation route for more than approximately 10% of the life span in humans (more than approximately 90 days to 2 year s in typically used laboratory animal species). Chronic Study: A toxicity study designed to measure the (toxic) effects of chronic exposure to a chemical. Chronic Toxicity: The capacity of a substance to cause adverse human health effects as a result of ch ronic exposure. Short Term Exposure : Repeated exposure by the oral, dermal, or inhalation route for more than 24 hours, up to 30 days. Subchronic Exposure: Repeated exposure by the oral, dermal, or inhalation route for more than 30 days, up to approximatel y 10% of the life span in humans (more than 30 days up to approximately 90 days in typically used laboratory animal species).

1. Has the available information supporting the hazard assessment of acetonitrile been objectively described? Are there any addit ional studies that should be included?

2. The U.S. EPA has determined that mortality is the critical endpoint for acetonitrile as described in the IRIS “Toxicological Review of Acetonitrile” (1999). Mortality has been observed in a 13

- week subchronic study as well as in developmental and reproductive toxicity studies. Mortality was not observed in a 2

- year chronic study conducted at doses below those that caused mortality in the 13

- week subchronic study. Based on the totality of available evidence, par ticularly the 13

- week and 2

- year studies, is it reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure? Please explain.

Charge Questions for Ecotoxicologists

Chapter Two, Chemistry and Environmental Fate 1. Has an objective (accurate, clear, complete, and unbiased) and technically accurate d escription of acetonitrile been provided in terms of chemical and physical properties?

2. Does the report objectively address the fate of the chemical in the environment includi ng both transport between environmental compartments (atmosphere, aquatic and terrestrial) and transformation (e.g., degradation), including both abiotic and biotic processes where relevant?

Chapter Four, Ecological Hazard Assessment 1. Has the available information on the ecological toxicity of acetonitrile been objectively described? Are there any additional studies that should be included?

2. Is the conclusion presented in section 4.1, “Acetonitrile does not present a concern for adverse effects on the environment” supported by the data/information presented?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

5 III. RESPONSE TO HUMAN HEALTH CHARGE

A. General Impressions

Commenter One

The document “Technical Review of Acetonitrile,” CAS registry #75

- 05

- 8, is well written. The inform ation in chapter three on “Human Health Hazard Assessment” of acetonitrile (ACN) is well described and clearly presented. Chapter O ne, “The integrated technical summary”, clearly summarizes the interpretation of the technical data available to the authors on the chemistry, environmental occurrences, and health hazards of ACN.

Chapter T hree describes in some details the studies available on ACN

- induced human and animal acute, sub

- acute, and chronic toxicities. However, the data available are relatively old information that is derived from accidental human ingestion of ACN and relatively few idealized animal studies in mice rats and rabbits. Also, the interpretations of the results are based on somewhat obsolete mechanistic information.

Therefore, the overall conclusion that “ACN

- induced acute, sub

- acute and chronic toxicities and subsequent mortality is due to the amount of un

- detoxified cyanide moieties alone” is not valid because of the current understanding of the molecular mechanisms of actions of cyanide ions and other reactive metabolites of ACN.

Commenter Two

The intent of this technical report is not clear. It is stated that it is related to the review of a petition to remove acetonitrile from the list of chemicals subject to reporting requir ements. A major concern is how consistent this document is with the rationales that underlie other regulatory activities of the Agency. This document does make reference to IRIS reviews of acetonitrile and the analysis appears consistent with what is pro vided in the IRIS document. The present document makes the conclusion that the effects of acetonitrile are related to its conversion to cyanide. The conclusions of this document are not consistent with the Office of Ground Water and Drinking Water's (OGW DW) Consumer Factsheet on Cyanide, where effects of cyanide on thyroid function and neurological effects which are related to its conversion to thiocyanate. Unfortunately, OGWDW has no transparent link to the criteria document that supported the cyanide r egulation. It is my recollection that the concern over thyroid effects of cyanide largely relied on human data related to exposure to cyanogenic compounds (i.e. similar to acetonitrile) in the diet that lead to inhibited thyroid hormone synthesis. This l iterature needs to be revisited and an explicit determination made as to its applicability to the problem the present document is supposed to address. I recognize that many of the papers that address this issue are old and may well be confounded with othe r socioeconomic factors. Nevertheless, they need to be explicitly considered in this document because of the huge emphasis that is placed upon the interpretation that the toxicity of acetonitrile is attributable to its conversion to cyanide.

The docume nt was generally well written and the review of data that has been directly associatedExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

6 with studies of acetonitrile were largely accurate. Below, there are further comments on whether the document adequately explored potential thyroid effects of this chem ical.

Commenter Three

This Technical Review of Acetonitrile is a well written document and organized such that information necessary to address this reviewer’s charge question was readily accessed. Statements summarizing cogent information from primar y resources are clearly written and accurately represent relevant study results with minimal extraneous detail. The conclusion reached by the reviewers that mortality can be reasonably anticipated as an effect of chronic acetonitrile exposure is based upo n application of generally accepted principles of toxicology. This conclusion is, in this reviewer’s opinion, unquestionably sound for rodents in standard test protocols. Extrapolation of this conclusion to humans is not as definitively supported since

s imilarity of mode of action of lethality from acute and chronic exposures is not evidenced with review of direct experimental data. Instead, the review presents a persuasive argument for human relevance based upon similar acetonitrile toxicokinetics, slow rate of metabolism to reactive metabolite cyanide and recycling of detoxication product thiocyanat e in both animals and humans.

The reviewers have made a compelling case for acceptance of mortality as a toxic endpoint of chronic exposure for this chemical. Since CNS regulation of respiration is so critical for viability of the organism, it is feasible that its dysfunction could cause rapid onset of mortality in the apparent absence of preceding clinical symptoms and histopathological lesions detec table with techniques typical of standard toxicology test protocols. The argument that mortality is not an endpoint of chronic toxicity is based upon an inherent contradiction imposed operationally by the design guidelines for chronic studies specifying a voidance of lethality as a criterion for dose selection. An exception seems appropriate in this case.

One citation error and one study summary statement that could be misleading were found in the review; these are detailed in section III, items a and b, of these comments. Also, I would recommend o n first occurrence of description of the cyanide molecular defect (p. 2, line 5) , use of terminology “ chemical reduction of oxygen” to distinguish from “reduction” meaning loss of quantity. Also, semicolons and commas are used inconsistently to separate items of a series in the last column of Table 3.1.

Commenter Four

Chapter 3 of the draft document is comprised of four main sections: 3.1 Summary, 3.2 Kinetics and Metabolism, 3.3 Effects of Repeated Exposure, and 3.4 Effects of Acute Exposure. These sections are generally well

- written, logically organized, and easy to follow. They also appear to be reasonably comprehensive in their coverage of the relevant scientific information. In addition to the chapter’s primary focus on mortality as the critical health endpoint in both single and repeat dose studies, studies of the potential for acetonitrile to cause carcinogenic, genotoxic, reproductive and developmental, as well as neurotoxic effects were identified, r eviewed, and summarized. This reader was left, however, with the distinct impression that certain data pertaining to acetonitrile pharmacokinetics and mortality were not simply being reported in an objective,External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

7 unbiased manner, but rather, were being marshal ed to support the Agency’s principal assertion regarding acetonitrile that “it is reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure”, without appropriate and balanced consideration of the alternatives. While th is lack of balance is discussed in greater detail below, I believe this Agency position is altogether unjustified by the available information. In my opinion, the data presented and summarized in this draft chapter point convincingly toward mortality aris ing solely from intense (high concentration) acetonitrile exposures of quite short durations (from hours to at most a few weeks). Thus, if excessive mortality was to be seen in a chronic study, and it has not, it would be reasonably anticipated to occur d uring and as a consequence of the first few weeks of acetonitrile exposure. This view is consistent with what is known of the kinetics of acetonitrile (and cyanide) uptake, distribution, metabolism, and elimination in laboratory animals and humans.

Comme nter Five

The health assessment chapter is repetitive and does not provide a thorough, integrated hazard characterization with a weight

- of

- evidence analysis. It also is not sufficiently rigorous, complete and transparent to meet current scientific standa rds and regulatory guidance.

Table 3

- 1 is misleading. First, no statistical significance is reported. A single death or even two deaths in a dose group would not be considered an effects level even if higher rates of mortality occur at higher doses because the incidence is too low t o differ significantly from control rates and could have occurred by chance. Second, in developmental studies, all fetotoxic or reproductively toxic effects occurred at maternally toxic doses and thus acetonitrile is not a fetal/embryo toxicant. Third, some studies showing no treatment effect reported in the text are not summarized in this table (e.g., Hazelton 1983). I discuss these issues under the Charge Questions.

The conclusions are not supported by the data.

Human and animal data should be p resented separately.

Human relevance is inadequately characterized.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

8 B. Response to Charge Questions

Commenter One

The available information that resulted from some limited, older studies has been adequately described in the document. The interpretations presented in the document are based on the interpretations of the results that are presented by the authors of original studies and or from previous U

. S

. EPA documents.

To my knowledge, there are no new studies on ACN adverse activities. There are, however, several recent studies that describe the molecular mechanisms of toxicities of the cyanide moieties and how cyanide actually interferes with mitochondrial respiratory chain mechanisms. Also, there are modern studies that describe the molecular mechanisms of other ACN metabolites suc h as formaldehyde (see below).

Commenter Two

I did not identify any studies of acetonitrile, itself, in the literature that were of importance that were not included in this review. The information that is discussed appears to be objectively described. The toxicological data on acetonitrile are pret ty sparse. The major studies have been performed via inhalation exposure, although there are some reproductive and developmental data that have been obtained using gavage.

While the data are sparse, this review does not make adequate use of the availab le data to form the conclusions of the report. One example is the handling of the genotoxicity data. A second was the failure to utilize the limited metabolism data to support their hypothesis that the lethal effects (and other effects) of acetonitrile w ere attributable to the generation of cyanide as a metabolite. A third example, addressed further in the response to the next question, is that potential effects of acetonitrile on thyroid function were not adequately discussed and evaluated in this review

.
The data appear to clearly establish that acetonitrile produced chromosome aberrations and aneuploidy in several test systems. The document treats these as “only marginal effects”, which is a prejudicial editorial comment made without discussing why th e data were marginal. The sentence talks about “potential” of acetonitrile being demonstrated. If the results were statistically significant, one should conclude that it produced the effect. It is entirely possible that these data can be dismissed by a more thorough discussion of the data, including questions of consistency among tests measuring the same endpoint, that is, comparing the doses used in positive vs. negative effects in tests measuring the same endpoint. That discussion should come

to a j udgment based on the data as a whole. As this section now stands, it comes to no Chapter Three, Human Health Hazard Assessment 1. Has the available information supporting the hazard assessment of acetonitrile been objectively descr ibed? Are there any additional studies that should be included?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

9 conclusions and largely leaves the issue open.

The metabolism and pharmacokinetic data on acetonitrile and its metabolites are limited, but there are sufficient details avai lable to allow more defensible conclusions to be made related whether mortality is the key event. Some of elements of this discussion are provided on page 29 of the document, but the discussion of the data is interspersed with a series of speculative

comm ents that detract from the proper use of these data (e.g. “overwhelming enzymatic detoxifying mechanisms” and “reconversion of thiocyanate to cyanide”). These statements should not be made without specifically discussing how those two things might occur o r have been shown to occur. I would strongly suggest the inclusion of a diagram that lays out the metabolism of acetonitrile to cyanide and thiocyanate. In that diagram, all the important routes of elimination of acetonitrile and these two metabolites sh ould be estimated. That will mean some exploration of the cyanide and thiocyanate literature to obtain the important quantitative data (hopefully in humans as well as rats and/or mice).

As pointed out, the half

- life of acetonitrile in humans poisoned wi th acetonitrile was 32 hours while that of cyanide is about 15 hours. Therefore, the slow conversion of acetonitrile will not allow substantive amounts of cyanide to accumulate at low doses. Thus, it would appear that the “detoxification mechanism” allud ed to is rhodanese conversion of cyanide to thiocyanate. The authors need to explore the kinetics of this latter conversion to determine what systemic concentrations of cyanide will begin to saturate this enzyme. Until that occurs, there will not be a sh arp accumulation of cyanide. The idea that the reconversion of thiocyanate back to cyanide can contribute to this build

- up also needs to be supported. As written, the implication is that the elimination of thiocyanate is slower than that of cyanide, but limited quantitative data are provided to support this contention (i.e. simply stated that thiocyanate is not entirely eliminated between doses). This needs to be confirmed, not simply be a suggestion by an author (line 1, page 30). To get accumulation c yanide, it needs to be established from the literature that this is the main route of cyanide elimination and that the relative rate of cyanide generation is more rapid than its elimination by rhodanese. One way to slow this conversion is to build up leve ls of thiocyanate. However, that is simply a question approaching equilibrium concentrations of cyanide and thiocyanate in the systemic circulation. It does not mean that there is a net conversion of thiocyanate conversion back to cyanide, but that they are at equilibrium (or more properly in a steady state that is controlled by the rate of thiocyanate elimination).

The question of whether acetonitrile would be likely to produce inhibition of iodide uptake and thus decrease thyroid hormone synthesis will depend upon the systemic concentrations at which this steady

- state establishes itself with continued treatment.

Commenter Three

A comprehensive review of studies pertinent to the hazard assessment of acetonitrile has been presented. The Technical Revie w includes all relevant studies on health effects of acetonitrile that this reviewer identified through independent searching of NTP, EPA and Medline databases. Results are summarized objectively except with a possible misleading context for one mention of Ahmed et al. (1992) that I have detailed in my Specific Observations, item b. No additional studies were identified that would impact on my overall opinion. However, I believe addition information on clinical symptoms from the reviewed studies should be presentedExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

10 and explicitly used to provide some evidence for equivalence of mode of action of lethality from acute, short term and subchronic exposure to acetonitrile. The rationale for calling for this additional material is detailed below in paragraph 4 o f Section II.2. Summary of clinical symptoms from the Argus 1984 study, however, should be qualified by disclosure of the Study Director’s (Dr. A.M Hoberman) observation, expressed Apr 1, 2002, to Mr. D.E. Strother, that gastrointestinal irritation and d ecreased food consumption were coincident adverse effects.

The Technical Review is weighted in presenting support for the concept that a biological dose of reactive metabolite cyanide at target tissue can be achieved with low dose repeat exposure of

ace tonitrile that is equivalent to that associated with high dose acute exposure causal to lethality. This argument is made based upon acetonitrile metabolism, rates of conversion and clearance/recycling of toxic metabolite cyanide and cumulative functional e ffects. Brief mention should be given to alternative or complementary mechanisms, such as an expansion of the
comment on thyroid toxicity from thiocyan a te (p. 31, lines 12

– 15). If the authors believe these mechanisms are not relevant, their reasoning s hould be given.

Commenter Four

Most of the information presented in the draft chapter has been objectively described. However, there are specific areas where there seems to be a lack of balance. In Section 3.2, Kinetics and Metabolism, for example, th e lower measured affinity for cyanide (higher Km) of rhodanese from human nasal tissues and lower measured maximal rate (smaller Vmax) of cyanide metabolism, relative to that in rat nasal tissues, were highlighted as key results from the study by Lewis et al. (1991) (see p 29 lines 1

- 8). A comment follows immediately that humans are thus “less able [than rats] to detoxify cyanide to thiocyanate,” with the implication that humans would thus be at greater risk of cyanide

- induced toxicity.

However, the draft chapter neglects to point out that Lewis et al. were unable to saturate the human rhodanese enzyme despite the use of potassium cyanide concentrations of 500 mM and greater. In fact, Lewis et al. reported that the rhodanese enzyme exhib ited activation, not saturation, in one of their samples, consistent with previous work by Jarabak and Westley (1974) (cited in Lewis et al., 1991). This suggests that a simple Michaelis

- Menten model of cyanide metabo lism fails to adequately describe the actual dose

- response relationship obtained by Lewis et al., particularly at high concentrations (see their Figure 2)! Such enzyme induction in human nasal tissues and, possibly, liver would also, presumably, make humans less sensitive to cyanide
exposure than rats at least at sufficiently high concentrations where the rat metabolic response is saturated but the human response is not.

Lewis et al. (1991) also provide highly relevant discussion regarding the high “maximum detoxication” capacity for cyanide metabolism observed in the rat nasal cavity: “This capacity is sufficient to completely metabolize a maximum of 2800 ppm inhaled hydrogen cyanide ...” This high detoxication capacity may explain in part the reduced sensitivity of rats, compared to mice, to cyanide

- induced toxicity that was observed in animals exposed to high acetonitrile concentrations in the NTP subchronic inhalation study (NTP 1996). Section 3.2 also fails to mention that CYP2E1, the P450 enzyme responsible for the metabolism of acetonitri le to cyanide, is an enzyme that is inducible by a variety of substrates, including ethanol, c.f., Lu and Cederbaum, Free Radic Riol Med 2008, 4 4(5)723

- 38 . This property couldExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

11 lead to significantly higher rates of cyanide production from inhaled acetonitr ile, as well as shortening of the half

- life of acetonitrile in the body.

Section 3.2 of the draft ends with two highly speculative paragraphs. They assert that the slow metabolism of acetonitrile to cyanide, species differences in the tissue distribution of rhodanese, and the slow elimination rate of the thiocyanate, at least in some species, when taken together, strongly suggest that acetonitrile from one day’s dose is not totally eliminated before the next day’s dose in repeat dose studies (in what speci es?, at what doses?, after how many doses?), leading to an accumulation of both acetonitrile and cyanide “to the point of overwhelming detoxifying mechanisms .” However, I am aware of no direct evidence that this cascade of events occurs during chronic exp osure to acetonitrile, thus producing excess mortality as a chronic endpoint, as is stated in Section 3.3 of the draft document.

I was also surprised by the primarily qualitative nature of the discussion of acetonitrile pharmacokinetics. This is a topi c that cries out for quantitative treatment with a PBPK model, yet a brief search of the literature using PubMed failed to identify any recent work in this area. Perhaps this is a significant data gap that needs to be filled with additional research.

Com menter Five

There is a lack of objectivity in the health hazard assessment. The hypothesis or postulated mode of lethality is presented in the first paragraph of the summary. Data are subsequently presented in a manner which supports this position, witho ut providing a thoughtful discussion and integration of all available data and consideration of alternatives.

Although new data are not available, existing data are not framed in accordance with the currently

- accepted standards of hazard characterizat ion. In particular, insufficient information is presented on mode of toxic action and the human relevance of animal studies, and a weight

- of

evidence narrative is incomplete. There is also a reasonable lack of objectivity, as indicated by the almost

- comp lete focus on mortality with little discussion of other health outcomes (e.g., liver, lung, forestomach, blood), incomplete presentation of toxicokinetics and mode

- of

- action data, and lack of rigor in assessing and interpreting findings from individual stu dies in accordance with current standards and regulatory guidance.

There is a developmental toxicity study discussed on p. 18 of the NTP (1996) report on the toxicology and carcinogenesis of acetonitrile, in which cyanide and acetonitrile were measured in Sprague

- Dawley rats exposed to acetonitrile at concentrations of 100, 400 or 1200 ppm for 6 hours/day on gestation days 6

- 19. The only reported effects were “a few” maternal deaths at 1200 ppm and one at 400 ppm. Analysis indicated “significant exposure

- related concentrations of acetonitrile in the blood of all exposed groups and the presence of cyanide in the blood of the 1200 ppm group (cyanide was detected at a very low level in one 400 ppm rat at each time point but “could not be quantified”

– presum ably because it was too low). And: “The concentration of cyanide in the blood of 1200 ppm rats declined from approximately 2 mg/mL on GD 8 to approximately 0.8 mg/mL on GD 18, while acetonitrile concentration remained essentially constant during the same period...due to induction of rhodanese.” These data do not support the hypothesis that cyanide bioaccumulates and are consistent with other data that also do not indicate bioaccumulation. This study should be added, as should others referenced inExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

12 WHO/EHC ( 1993), which address blood levels of acetonitrile, cyanide, and/or thiocyanate.

References which might be useful for a more integrated assessment of acetonitrile toxicity include the following documents.

WHO. 2004. World Health Organization. Concise International Chemical Assessment Document (CICAD): 61. Hydrogen Cyanide and Cyanides: Human Health Aspects. Geneva, Switzerland.

Way J.L. 1984. Cyanide intoxication and its mechanism of antagonism. Ann Rev Pharmacol and Toxicol. 24:451

- 481.

Sylves ter, M. and C. Sander. 1990. Immunohistochemical localization of rhodanese. Histochem. J. 22(4):197

- 200.

Melnick, R.L., Roycroft, J.H., Chou, B.J., Regan HA and Miller. RA 1990. Inhalation toxicology of isoprene in F344 rats and B6C3F2 mice. Environ . Health Perspect. 86:93

- 98.

U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. [ See section on writing a weight

of

- evidence hazard narrative, using a modified set of Bradford Hill (1965) criteria]

Boobis AR

, Doe JE

, Heinrich

- Hirsch B

, Meek ME

, Munn S

, Ruchirawat M

, Schlatter J

, Seed J

, Vickers C

. 2008. IPCS framework for analyzing the relevance of a n oncancer mode of action for humans. Crit Rev Toxicol. 38 (2):87

- 96

.
Hill AB. The environment and disease: association or causation? Proc Royal Soc Med 1965; 58:295

- 300.

NAS 2005 . Health Effects of Perchlorate Ingestion. National Research Council. Washington, D.C. [ Thyrotoxicity induced by ions which competitively inhibit iodide uptake in the thyroid gland

– as thiocyanate does

– is thoroughly discussed in this report .]

1. Tox icokinetic and mode of toxic action data are incompletely summarized

.
Data which are omitted or inadequately summarized include the following:

(a ). Acetonitrile is more slowly metabolized to cyanide than cyanide salts or some other nitriles (WHO/EHC 1993; Hartung 1982). Therefore, during acetonitrile exposure, the body/tissue burden of acetonitrile is also increased. Both human case reports of po isoned individuals and animal studies show that acetonitrile is distributed fairly uniformly throughout the body (EHC 1993).

(b) Acetonitrile is also excreted unchanged in urine and in expired air. Therefore, the clinical and subclinical effects are l ikely to be a result of the combined effects of the parent molecule and its cyanide metabolite (Hartung 1982; EHC 1993). Further, animal studies do not indicate that either acetonitrile or cyanide has the potential for bioaccumulation (EHC 1993; ATSDR 199 9).

(c) Released cyanide is metabolized to thiocyanate by rhodanese, a sulfur transferase enzyme,External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

13 which is readily excreted in the urine. Rhodanese is an inducible enzyme and levels of rhodanese increase in response to increasing blood and tissue cyan ide concentrations. Sites with the greatest abundance of rhodanese are located to maximize conversion of cyanide to thiocyanate following both oral and inhalation exposure (Sylvester and Sander, 1990 ). Detoxificati on of cyanide by rhodanese exhibits zero

- order kinetics relative to cyanide; the concentration of sulfur

- containing donor molecules is the rate

- limiting factor.

(d) The role of metabolism is crucial in determining the acute toxicity of ingested cyanide. Both the total amount administered and the rate of absorption are important (U.S. EPA 1992) because acute toxicity results from rapid ingestion of high doses of cyanide at a rate that exceeds the body’s capacity for metabolism (detoxi fication). In contrast, slow absorption of the same or greater total oral load of cyanide may allow for complete metabolism. Unmetabolized cyanide can be both acutely and chronically toxic, although cyanide is not known to accumulate in the body.

The p rincipal features of the acute toxicity profile for cyanide are its severe toxicity, including lethality, by all routes of administration, with a steep rate

- dependent dose

– effect curve. The toxic effects of cyanide ion in humans and animals are generally similar and are believed to result from inactivation of cytochrome oxidase and inhibition of cellular respiration during the terminal reaction of the electron transport chain. This prevents the formation of adenosine triphosphate (ATP) via oxidative phosp horylation. Although acute oral doses of cyanide cause cardiovascular, respiratory, and neurophysiological changes, the brain appears to be the organ most sensitive to acute cyanide toxicity (WHO 2004). Death from cyanide poisoning is believed to result from central nervous system depression, subsequent to inhibition of brain cytochrome oxidase activity (WHO 2004).

Thiocyanate is not acutely toxic; approximately 60

- 80% of an ingested dose of inorganic cyanide is converted to thiocyanate, the majority of which is rapidly excreted in the urine. However, elevated long

- term ingested cyanide can result in sustained serum levels of thiocyanate leading to inhibition of iodide uptake at the level of the sodium

- iodide transporter in the thyroid gland, subsequent thyrotoxicity and resulting neurotoxicity (iodide deficiency

- associated neuropathy and mental retardation).

U.S. EPA’s hazard assessment suggests that: “With repeated exposure to acetonitrile, it is possible that continued generation of cyanide from the existing body burden of acetonitrile, coupled with cyanide generated from new exposures, results in cyanide levels that exceed the capacity of enzymes to metabolize cyanide to thiocyanate.”

However, if this is the case, then thiocyanate levels in both uri ne and blood would be expected to be significantly elevated and abnormalities in the thyroid gland (e.g., goiter) and thyroid hormone homeostasis (i.e., increased TSH and/or T4) would be expected to occur. Chronic exposure to cyanide, resulting in excess ive production of thiocyanate, can interfere with iodine uptake by the thyroid gland, leading to enlargement of the thyroid (e.g., goiter) and abnormal levels of thyroid hormones (increased TSH and/or T4). The most prominent health hazard of chronic expos ure to elevated levels of cyanide is thyrotoxicity resulting in neurotoxicity. There is no evidence of either thyroidal or neurotoxic effects that could be attributed to prolonged or repeated cyanide exposure in either the subchronic or chronic NTP aceton itrile studies.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

14 The potential effects of thiocyanate, which are associated with chronic cyanide toxicity, are not discussed in this assessment. The only thyroid

- related finding was noted in the subchronic rat study by NTP (1996) and was reported as a s tatistically significant increase in T3 in the absence of any changes in T4 or TSH. This is puzzling. T3 is the bioactive thyroid hormone and is converted from T4 in situ by specific iodinase enzymes (NAS 2005). A change in iodide uptake in the thyroid gland because of the competitive inhibition of thiocyanate usually results in an increase in TSH and/or a change in T4. A change only in T3 might indicate an increased energy or metabolic tissue demand, as could occur with cyanide

- induced oxygen deprivati on, which is met by increased conversion of stored T4 to T3.

(e ) The results from both the subchronic and chronic studies do not support the following assumptions :

“With repeated exposure to acetonitrile, it is possible that continued generation of cyanide from the existing body burden of acetonitrile, coupled with cyanide generated from new exposures, results in cyanide levels that exceed the capacity of enzymes to metabolize cyanide to thiocyanate” and

“As some deaths from acetonitrile did not occ ur immediately, but instead occurred after longer exposure, the results suggest that certain key biologic processes may be gradually overwhelmed or gradually degraded in function such that the body can no longer successfully counter the toxic effects of cy anide generated from acetonitrile” (p.35).

Based on the findings, the above statements are not biologically plausible scenarios. If there had been cumulative toxicity resulting from repeated

- dosing, then the results should indicate a temporal relationshi p between effect and exposure

- duration. If Concentration x Time (C x T) effects are invoked as a possible explanation for the mortality findings, an increase in mortality with increasing exposure duration should have also been observed. This did not occu r. The animal deaths in the subchronic study were concentrated in weeks 1

- 3; a gradual degradation in function or an overwhelming of key biologic processes would predict that mortality would increase in subsequent exposure weeks, especially in the higher dose groups. Similarly, mortality would be expected in the 2

- year chronic study at lower concentrations.

It is understandable that U.S. EPA is concerned about possible mortality associated with a cyanide

- containing compound because mortality is a very frank, irreversible effect and cyanide has a steep acute concentration

- response curve. Lethality is well known as an acutely toxic response. However, it is the dose rate that determines the acute lethality of cyanide. The weight

- of

- evidence shows that a lower dose rate for a longer period of time is detoxified and it is only when the detoxification system is acutely overwhelmed that lethality occurs. The data on mortality in the subchronic studies, in conjunction with the significant amount of knowledg e on the mode of toxic action, suggest that the observed findings were due to acute toxicity. In this context, the MTD was exceeded in the higher doses. Repeated dosing (6 hours/day, 5 days per week

– note that it was not continuous) is better construed as a series of acute toxicity doses with regard to lethality as an endpoint.

The pattern of responses under very high atmospheric concentrations of acetonitrile for a 6

- hourExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

15 period is strongly supportive of an acutely toxic response. The clustering o f deaths at the beginning of the study, in conjunction with an absence of mortality in the chronic study and in the Hazelton (1983) subchronic studies, indicates that there is no CxT response. What is known about the acute and chronic modes of toxic actio n of cyanide provide further evidence in support of assigning mortality to acute, rather than chronic, toxicity.

With regard to U.S. EPA’s statement that mortality was not observed in the chronic studies because exposure concentrations were significantl y lower than those in the subchronic studies, I note that the acetonitrile concentrations in the chronic study are still orders of magnitude greater than relevant environmental concentrations. This is also a tautological argument.

(f) The pattern of dosing would also predict mortality as an acutely to xic response, not as a chronic one.

The dosing regime was 6 hours/day, 5 days/week for 13 weeks. Thus, animals were not exposed for 18 hours a day. There was also a non

- exposure period of approxima tely 60 hours over the weekend (Friday evening to Monday morning). Given what is known about cyanide, its toxicity and modes of toxic action and detoxification, cyanide levels would have been significantly reduced during non

- dosing periods. Mortality is thus an acute response. Chronic effects, if they occurred, would be manifested in organ toxicity, such as liver toxicity, and would be associated with repeated oxygen deprivation in tissues.

2 . Human relevance is not adequately addressed

.

Human relevance is noted in only one sentence and framed as a default statement (i.e., in the absence of data to the contrary, animal studies are assumed to be relevant to humans). The state

- of

- the

- science has progressed significantly since the times when this t ype of sentence would be considered sufficient for inter

- species extrapolation (see Boobis et al. 2008 cited above). Human relevance topic should be expanded to indicate that there are sufficient data to indicate whether there are sufficient data to concl ude that the mode of toxic action of acetonitrile and cyanide are qualitatively similar among species, including humans. How well are the quantitative differences among species (including absorption and metabolism) described? Further, as implied on p. 3 6, lines 7

- 10, if effects observed in animals are only likely to occur at very high acetonitrile atmospheric exposures in humans (which are orders of magnitude above environmentally relevant concentrations), is there human relevance?

3

. The weight

- of

- evidence narrative does not consider all data and is incomplete

.
This hazard assessment does not meet current quality standards for integration and synthesis of all available data. U.S. EPA (2005) provides an updated framework for a weight

- of

- ev idence narrative evaluation, based on the Bradford Hill criteria for causality in epidemiology (Hill 1965).

Briefly, in a weight

- of

- evidence narrative, all data is considered simultaneously and evaluated for (1) strength of association; (2) consistency of association; (3) specificity of association; (4)External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

16 temporality of association; (5) dose

- response or “biologica l gradient”; (6) biological plausibility; (7) coherence; and (8) other factors.

4. There is a lack of rigor in assessing and interpreting individual study findings

a. The health hazard assessment focuses on mortality and does not give sufficient cons ideration to other potentially adverse health effects, or lack thereof (see below).

b. No statistical significance evaluation has been conducted on any of the reported studies. As a result, the LOAEL/NOAELs of various studies are overestimates

.

The stat istical significance of cancer findings is presented and discussed. A major omission is lack of consideration of statistical significance of noncancer findings. Current guidance mandates an evaluation of the statistical significance of all findings.

D ose groups in which one or two animals die are not effect

- levels because as compared to concurrent (or historical) controls, the increase in mortality would not significantly differ from chance. This increase would not be either statistically or toxicologi cally significant. Although mortality is a frank effect, the death of one or two animals in a dose group is usually considered a random event which can also occur in control animals, even if there is a significant increase in mortality incidence at higher doses. Therefore, a dose level in which 1 or 2 deaths occur does not constitute an effects level.

In the NTP (1996) 13

- week rat studies, the mortality incidence for males was 0/10 in each of the 0, 100, 200 and 400 ppm groups, 1/10 in the 800 group ( one death occurring in week 1), and 6/10 in the 1600 group (four deaths occurring during week 1, one death occurring during week 2, one death occurring during week 4). For females, the mortality incidence was 0/10 in each of the 0, 100, 200, 400 and 800 p pm groups, and 3/10 in the 1600 ppm group (one death occurring during week 1, two deaths occurring during week 2). In the 13

- week mouse studies, the mortality incidence in males was 0/10 in each of the 0, 100, 200 and 400 ppm groups, 1/10 in the 800 ppm g roup (death occurring sometime during study weeks 6

- 13), and 10/10 in the 1600 ppm group (two deaths occurring during week 1, eight deaths occurring during week 3). In females, the mortality incidence was 0/10 for the 0, 100 and 200 ppm groups, 1/10 in t he 400 ppm group (occurring during week 2), 4/10 in the 800 ppm group (one occurring during week 2, two occurring during week 3, and one occurring during weeks 6

- 13), and 10/10 in the 1600 ppm group (four occurring during week 1, six occurring during week 3).

Inspection of these data for statistical significance suggests that (1) the data would be statistically positive in a trend test; and (2) pairwise comparisons would indicate a significant effects level of 1600 ppm for rats and 800 ppm for mice, not 80 0 ppm for rats and 400 ppm for mice.

c. The pattern of results indicate that at the higher exposures , the maximum tolerated dose (MTD) was exceeded.

The temporal pattern of deaths would suggest to objective reviewers that the MTD had been exceeded. Thi s rationale also accounts for why NTP ( 1996 ) reduced the highest exposure concentration for the chronic bioassay to 400 ppm for rats and 200 ppm for mice.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

17 The MTD may be defined as the maximum daily dose which induces signs of toxicity but not lethality o r significant morbidity. The 100% mortality incidence in mice and the 30

- 60% mortality incidence in rats at an exposure concentration of 1600 ppm demonstrates that the MTD was exceeded. The 10

- 30% mortality incidence in mice and the 10% mortality inciden ce in rats at 800 ppm indicate that this exposure concentration is near the MTD and the deaths are associated with individual variability in susceptibility.

Although concern for acetonitrile is based on its metabolism to cyanide which has a steep
concent ration

- response curve, cyanide

- induced lethality is an acute response and has a steep concentration

- response slope. For cyanide or cyanide

- containing compounds, high

- dose mortality is associated with acute toxicity so that the MTD is also acute. For an a cutely toxic end point, a 6

- hour daily dosing regime for 5 days/week is toxicologically equivalent to a series of repeated acute

- dosing periods.

Lower exposures to cyanide induce a range of effects associated with tissue oxygen deprivation or no effect s. Although peak cyanide concentrations following acetonitrile exposure are delayed (due to low metabolic rate for conversion to cyanide), there is no evidence to suggest that cyanide bioaccumulates.

All but two of the deaths in both rats and mice at the two highest exposure concentrations occurred during the first three weeks of the study. This pattern is not unusual for a range of compounds in which the MTD has been exceeded. Typically, mortality occurring early in a repeated

- dose study results in a reduction of the doses/concentrations during the study to a level at which lethality does not occur.

d. A fuller discussion of adverse findings, including mortality, is needed for completeness, comprehensiveness and transparency.

NTP (1996) reports that that (1) the majority of deaths occurred during the first two weeks of exposure; and (2) the spectrum of lesions reported in animals dying early was similar to those encountered in early

- death animals in other NTP studies (in additi on to being lesions consistent with previous descriptions of acetonitrile toxicity). Thus, it is reasonable to assume that these early deaths were associated with overdosing. Overdosing with a range of compounds typically results in clinical signs of neu rotoxicity which may be followed by death.

NTP (1996) also notes that (1) the character of the forestomach lesions (i.e., hyperplasia with and without inflammation, focal ulceration) observed in mice surviving to the end of the study has also been seen in mice in other NTP inhalation studies (Melnick et al., 1990), and (2) this suggests that the effect may not be specifically related to acetonitrile exposure but rather to “some other factor associated with the conditions of exposure” (p.55). The most li kely explanation for this histopathology is incidental ingestion which would be increased by grooming

- and

- swallowing behavior during whole body exposure.

It is useful to note that acetonitrile is water

- soluble and is also irritating to the skin and the

r espiratory tract. Acetonitrile is also rapidly and efficiently absorbed through skin and the gastrointestinal tract. Therefore, given the multi

- route exposure pathways (inhalation + dermalExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

18 + incidental ingestion), the internal acetonitrile concentrations are likely to have been much higher than target inhalation concentrations in animal studies

.
Other adverse effects:

Subchronic (NTP 1996 ) rodent studies : In rats, clinical signs of neurotoxicity during week 1 were hypoactivity and ruffle d fur at 800 and 1600 ppm; ataxia and abnormal posture and clonic convulsions in animals who died during the first week. Changes in the following organ weight were observed: (1) significantly decreased absolute and relative thymus weights in males and fem ales at 800 and 1600 ppm; (2) significantly increased absolute testis weight at 1600 ppm; and (3) significant increases in the absolute or relative weights of the heart, liver and kidney at 800 and 1600 ppm; an increase in absolute heart weight also occurr ed at lower doses (≥ 200 ppm). Other effects included: (1) a nonresponsive, normocytic and normochromic anemia in both sexes at 1600 ppm and in females at 800 ppm; and 20 decreased serum triiodothyronine (T3) concentrations in 1600 ppm females in the abs ence of alterations in serum throxine (T4) and thyroid

- stimulating hormone (TSH). No histopathology unrelated to early death was observed in any animals.

In mice, clinical signs of neurotoxicity occurred only during week 1 and consisted of hypoactivity a nd a hunched, rigid posture at 800 and 1600 ppm. As all animals in the 1600 ppm group died early, 800 ppm was the highest subchronic dose tested for systemic effects in mice. Relative to concurrent controls, the following statistically significant organ w eight changes were observed: (1) increased absolute liver weights in males at ≥ 200 ppm and in females at 800 ppm; (2) increased relative liver weights in all exposed male groups and in females at ≥ 400 ppm. Histopathology demonstrated the presence of mic roscopic lesions in the liver (i.e., hepatocellular cyptoplasmic vacuolation in both sexes at 400 and 800 ppm) and in the anterior forestomach (i.e., focal and multifocal hyperplasia/lesions/ulceration at exposure concentrations ≥ 400 ppm in males and ≥ 20 0 ppm in females).

Based on these effects, the critical effects for subchronic (systemic) toxicity in mice are hepatic and stomach. Forestomach histopathology was observed in all but the lowest dose group in females. Using this end point, the LOAEL and NOAEL would be 200 and 100 ppm, respectively. However, humans do not have a forestomach and thus the human relevance of these lesions is questionable. A more relevant endpoint is hepatic effects. Acetonitrile and its active metabolite cyanide have been shown to induce hepatic effects in other studies (concordance of information across studies). An increase in liver weight was observed at exposure concentrations which were less than or equal to the exposures at which liver histopathology was observed. This pattern of hepatic events is consistent with a cumulative toxic effect resulting from repeated

- dosing, and indicative of a concentration x time (C x T) effect. In contrast, there is no indication of a cumulative mortality effect.

Chronic studies : No mortality was observed in the chronic study in either rats exposed to vaporized acetonitrile at concentrations of 0, 100, 200 or 400 ppm (equivalent to 0, 168, 335 or 670 mg/m3) or in mice exposed to 0, 50, 100 or 200 ppm (equivalent to 0, 84, 168 or 335 mg/m3). Hepatoxicity was observed in male, but not female, rats. There was also a marginally significant increase in hepatic tumors (combined adenomas and carcinomas) in male, but not female, rats. In mice, aExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

19 dose

- related increase in the incidence o f hyperplasia of the forestomach epithelium was observed in both males and females (NTP 1996).

Developmental studies: Developmental effects were only observed at maternally toxic doses. This fact is not highlighted in the hazard assessment, as it is in WHO’s Environmental Health Criteria document (WHO 1993). According to current hazard and risk assessment guidelines, developmental effects observed at maternally toxic doses are assumed to be indirect (due to maternal toxicity). Therefore, acetonitrile would not be considered a developmental or reproductive toxicant according to current hazard and risk assessment guidelines. If mortality is statistically elevated at the highest dose tested, then it would be considered treatment

- related. However, develo pmental toxicity at that dose level would be attributed to maternal toxicity. Many compounds induce some degree of mortality at the highest dose tested.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

20 Commenter One

The answer to this question is: Yes. Based on the available human and animal data, I would anticipate that ACN could cause mortality as a result of chronic exposure, given the right species and the right period of exposure. My interpretation of the data that are available and presented in this document is outlined below.

The data indicate that ACN causes death in rabbits, mice and rats. It causes death in rabbits at much lower doses than that which was administered in the two year studies to mice and rats. Due to the similarities in metabolic enzymes between rabbits and humans and due to the fact that humans are less able t o than rats to detoxify cyanide, I would expect that health effects and mortality in humans, following human exposure to ACN, would be closer to that observed in rabbits than to that observed in mice and rats. Therefore, I would anticipate that human would be more sensitive to ACN actions than mice and rats. Thus, the data derived from the 2 year chronic exposure of rats and mice to low concentration of ACN should not be used to un

- list ACN from the current EPA regulations.

Furthermore, most, if not all, o f the conclusions are based on the assumption that the lack of apparent lethality from ACN in rats and mice in the two year studies using low concentrations is due to the ability of rats and mice to detoxify ACN metabolites, particularly cyanide moieties.

T he lack of such an apparent end point (mortality) of adverse effect does not mean that there were not any adverse effects that had occurred on the molecular levels within the experimental animal species utilized in these studies.

To explain this statement, I would like first to describe ACN metabolism. ACN is known to be oxidized by Cytochrome P450 2E enzyme system or by other oxidizing enzymes to yield a reactive intermediate, methyl cyanohydrine. The latter intermediate is in equ ilibrium with equal molar concentrations of cyanide ions and formaldehyde.

CH 3 CN

------------ CH 2 OHCN

--------- CH 2 O + HCN

Cyanide ions had been implicated in most aliphatic nitriles’ toxicity including that of ACN. The other metabolite, forma ldehyde (CH 2 O) has not been discussed in the interpretation of ACN toxicity and lethality. Formaldehyde on the other hand has its own set of toxic actions. Aliphatc aldehydes, in general, are known to block the activities and functions of several enzymes via the Chapter Three, Human Health Hazard Assessment 2. The U.S. EPA has determined that mortality is the critical endpoint for acetonitrile as described in the IRIS “Toxicological Review of Acetonitrile” (1999). Mortality has been observed in a 13

- week subchronic study as well as in developmental and reproductive toxi city studies. Mortality was not observed in a 2

- year chronic study conducted at doses below those that caused mortality in the 13

- week subchronic study. Based on the totality of available evidence, particularly the 13

- week and 2

- year studies, is it reasona ble to anticipate that acetonitrile can cause mortality as a result of chronic exposure? Please explain.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

21 formation of Schiff’s base with the amino groups in the enzymatic reactive sites. Also, CH 2 O is known to deplete anti

- oxidants in the cellular environment leading to cellular oxidative stress and oxidative damage. Both cyanide and aldehyde are k nown to alter mitochondrial chain reactions leading to mitochondrial membrane damage and the release of Cytochrome C, which activates apoptosomal enzymes, leading to the break down of nuclear DNA and the induction of cellular apoptosis. Because cellular ap optosis is a slow process, it is expected that mortality caused by cellular apoptosis to be a slow process during the life span of the cell. During these processes, tissues and organ degeneration may constitute a signal for tissue or organ injury that lead s to mortality.

Acute exposure to ACN will yield large number of formaldehyde molecules and cyanide ions. Both will cause severe oxidative stress and cause massive blockage of respiratory chain reactions involved in oxygen metabolism leading to the sudde n accumulation of reactive oxygen species, cellular, tissue, and organ damage, and subsequent death. Even in acute processes, delayed death usually occurs as a result of the sudden accumulation of cell, tissue and organ damage that could be caused by sudd en increase in oxidative damage and interference with oxygen metabolism.

Sub

- acutely, the processes of damage accumulation may take a while longer and when enough damage accumulates, organ malfunction may occur leading to death as indicated in the 13

- we ek ACN sub

- acute studies.

If exposure occurs at lower doses (such as that in the chronic studies), eventually enough molecular damage will accumulate at a longer time period (may be more than the two year period used in the studies conducted in the lea st sensitive species; rat and mice but a shorter period in the more sensitive species such as the rabbits and humans).

Therefore, I don’t believe that the lack of mortality indicated in the available chronic studies in rats and mice (2 year) justify the un

- listing of ACN from the criterion described because:

e. In the chronic studies described, highly insensitive species (rats and mice) were used. Thus

, the doses that would produce mortality following chronic exposure would be high.

f. The duration of two ye ars used for the studies described may not be enough to observe mortality in the species used.

g. Due to the known toxicity of ACN metabolites, CN and CH 2 O, molecular damage may have occurred in these species that may lead to altered physiological functions and other chronic disorders without the induction of death.

h. Due to the known teratogenic activities and developmental effects of ACN, and because of the sensitivity of developing fetuses to in

- utero exposure to very low levels of environmental chemicals, it is anticipated that molecular damage may occur in the fetus
atExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

22 low levels of exposur e. Such molecular damage during fetal development may lead to developmental abnormalities and impaired offspring that are susceptible to various diseases.

Commenter Two

The descriptive data do suggest that a few adverse effects occur at inhalation doses of acetonitrile below those that induce mortality. However, the document does not adequately explore the occurrence of effects that were anticipated in the subchronic and chronic experiments conducted by the National Toxicology Program. As indicated in t he general comments above, the metabolic conversion of acetonitrile, first to cyanide and then to thiocyanate, was expected to produce effects on thyroid function. Thyroid hormone measurements were made in the 13

- week study and there was some suggestion o f the expected effects on thyroid hormones in male and female rats at the 1600 ppm dose with a trend in the dose

- response data in lower doses. Moreover, there was evidence of increased C

- cell hyperplasia in the thyroid of female rats at all doses (statist ical analysis not explicitly presented). As these were expected toxicities of acetonitrile they should have been discussed in the document and a formal assessment made of whether these data were relevant to humans (there are reasons for not considering ra t data in where effects on thyroid function. Rats are much more sensitive to such effects than humans). In considering these effects on the thyroid, the Agency should consider whether the available reproductive and developmental toxicity studies were con ducted in a fashion that would allow effects of modified thyroid function on development to be evaluated. My review of these papers indicates that no treatments beyond the 20 th gestational day were performed. The critical period for thyroid

- dependent dev elopment events within the brain of the rat occurs in the postnatal period. Therefore, I suspect the Agency would ordinarily conclude that this is a critical data need and would modify their assessment to account for the deficiencies in the database.

The only reference to the possibility of thyroid effects is a reference to Hartung et al. (1982) in referring to the effects of thiocyanate. Thiocyanate is known to be a potent inhibitor of iodide transport in humans as well as experimental animals. Therefo re, it is critical that the document consider whether there has been an adequate experimental evaluation of risks that would arise from the generation of this important metabolite that arises in the metabolism of acetonitrile.

The evaluation of toxicities that could be potentially attributed to anti

- thyroid effects of the metabolite, thiocyanate, should be discussed and taken to a transparent conclusion in the “Conclusions” section, that is found at the beginning of the document.

Commenter Three

This re viewer believes that the available science supports the conclusion that acetonitrile can be reasonably anticipated to cause mortality as a result of chronic exposure. This opinion is based upon interpretation that mortalities in rodents observed after rep eat exposures during the subchronic 1996 NTP study occurred as a consequence of cumulative adverse effect of the chemical, rather than as an isolated, immediate response to one of the latter doses. Hence, it is reasonable to project that the same qualitat ive adverse effects would continue to accrue with additional dose applications upon extension of the exposure period. This would manifest as additional, later deaths in a chronic protocol at the lower effective dose level of the subchronicExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

23 study. Lack of direct determination of this anticipated result is a consequence of failure to include the subchronic LOAEL (400 ppm for mice; 800 ppm for rats) in the chronic study; absence of mortality with a 2

- fold lower dose does not preclude mortality occurring at t he subchronic LOAEL. Definitive evidence that mode of action for acetonitrile lethality upon acute exposure is equivalent for test animals and humans suggests relevance to irreversible chronic health effects to humans; however, confidence in this opinion i s lessened by a lack of direct evidence that the same mode of action mediates lethality from repeat dose, short term or

s ubchronic exposure to acetonitr ile in rodents as from high dose acute exposure to acetonitrile.

In isolation, one data set consistent with the petitioner’s interpretation is the high dose mouse mortality results of the 1996 NTP study. Death at 3 wk (Table 3

- 2) from an immediate lethal response to an isolated latter dose is feasible since the test dose yielding >50% deaths (6 hr, 1600
p pm) approximates the lowest reported, historical inhalation LC 50 (1

- 2 hr, 2300 ppm; p. 6, line 6). However, this reviewer is unaware of any examples of independent factors (eg., age, physiological hormonal status) of such rapid onset that would sensitize both male and female mice to a coincident, single interim toxicant dose of a repeat dose protocol. The occurrence of deaths in the next lower dose at a later times (>5 wk) would require that this spurious sensitization occurred randomly and repeatedly ove r a period of weeks during the trial, which seems unreasonable. The rat subchronic inhalation data do not support independent, immediate lethality to one of the interim doses since test doses with ~50% lethality a re 10

- fold less than the historical LC 50
. Rather, the rat results suggest attainment of the same qualitative adverse effect (lethality) upon multiple administrations of fractionated doses of a total, similar to what is done in radiation therapy to kill cancer cells

.
Another important issue for C ERCLA listing is relevance of rodent mortality from chronic acetonitrile exposure to humans. Extrapolation of rodent toxicities to humans is best supported by demonstrating equivalent mechanisms. It is clear from observations taken from emergency medicin e that acute acetonitrile poisoning in humans occurs via the same mechanism as acute animal lethality, i.e., through cytochrome P450

- dependent production of cyanide and consequential inhibition of mitochondrial cytochrome c oxidase, especially that of cent ral nervous system (CNS) respiratory control centers. The Technical Review suggests (p 5, line 2), that repeat exposure to acetonitrile will result in “continued generation of cyanide from the existing body burden of acetonitrile, coupled with the cyanide generated from new exposures” such that detoxication capacity for cyanide will be exceeded. The implication is that toxic effects from repeat dose mediated through the same mode of action, i.e., through cyanide, as acute effects. If so, relevance to hum ans is highly feasible. However, there is no direct experimental evidence presented to support equivalent mode of action for lethality from both exposure protocols. Information on blood and tissue cyanide levels after repeated acetonitrile doses associate d with lethality would strengthen the conclusion that biologically equivalent doses of reactive metabolite occurs with both acetonitrile exposure protocols. Comparable inhibition of target tissue (brain) cytoch r ome oxidase of rodents from high

- dose acute and lower, repeat

- dose acetonitrile exposures would indicate an equivalent molecular mechanism. In contrast, the 1996 NTP report (p. 18) describes a decrease in blood cyanide concentration with time in a 1994 NTP inhalation developmental study (cited in t he Technical Review as Mast et al

., 1994) from GD8 to GD18 and suggests induction of rhodanese may be responsible. If so, thiocyanate, which has known thyroid effects, would be expected to increase. A reduction in serum T 3 was reported in high dose rats of the 13

- wk study. Also, one lesion characteristic ofExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

24 low

- dose, chronic cyanide exposure, demyelination, was not reported in the 13

- wk histopathology of the 1996 NTP report (p. 32) although brain was included in the tissues examined. Collectively, these data gaps and additional observations cause this reviewer to question whether lethality from low repeat

- dose exposures occurs through the same mode of action as high

- dose acute acetonitrile poisoning and to be less confident about extrapolation of mortali ty from chronic acetonitrile exposure to humans.

The technical reviewers support similar mode of action of acetonitrile lethality in test animals vs. humans by comparing clinical symptoms (ataxia, convulsions and abnormal posture; p, 2, line 11). This sa me sentence, however, is supportive of similar modes of action of lethality from acute and repeat dose exposures, but the information is not apparent because it is unspecified that the comparison is between repeat dose animals (NTP 1996; p. 32, line 2) and acute poisonings of humans. Although these symptoms are nonspecific, a similar approach could be taken to provide some support for similarity of mode of action of lethality from acute vs. repeat dose acetonitrile exposure in animals. For example, Wilhit e ( 1981 ) describe s clinical symptoms of acute acetonitrile toxicity that include neurotoxic effects of tremors and convulsions. Such clinical symptoms of acute toxicity could be added to section 3.4.1. Similarly, clinical symptoms of the affected dams in the repeat dose, short term rabbit study (A r gus 1984, p IV

- 2) include ataxia, decreased motor activity and loss of righting reflex could be mentioned as qualified with Study Director Hoberman’s observation, expressed Apr 1, 2002, to Mr. D.E. Strother, tha t gastrointestinal irritation and decreased food consumption were coincident adverse effects. Although not as strong as a demonstration of equivalent biological dose at the target, explicit comparison of similar clinical symptoms indicating neurotoxicity observed in the acute, short term and subchronic studies should be presented to provide some support for same mode of action ( i.e., cyanide inhibition of CNS cytochrome oxidase) of lethality in the three protocols.

The technical re view has very convincingly presented evidence indicating that the effect of any one interim daily dose of a repeat dose protocol is likely augmented by residual effects from remaining fractions of uncleared acetonitrile from the preceding one or two days’ admi nistrations. Summarized information also makes a strong argument that a nearly continuous production of cyanide ensues. However, it has not been demonstrated that the accumulated tissue dose of cyanide can approach levels of disposition associated with h igh dose acute exposure causing lethality. Alternately, lethality from adverse effects of repeat dose can also result from cumulative, irreversible damage to a critical molecular/cellular target such that the summation of damage eventually exceeds a thres hold for non

- viability of the organism. Lack of lethality in the 2

- yr bioassay at 50% of the effective subchronic dose suggests that a threshold for the accrual of these lesions exists. Continued cyanide production at lower levels after termination of da ily acetonitrile exposure may contribute to the irreversibility of lesion s by inhibiting tissue repair. Cellular anoxia from cyanide inhibition of cytochrome oxidase could have lasting effects subsequent to toxicant clearance analogous to the oxidative da mage that occurs upon reoxygenation of a previously hypoxic tissue. These collective effects may or may not be equivalent mechanistically to that contributing to lethality upon high dose acute exposure. However, if accru al of functional molecular/cellular defects is relevant, it is unclear why some evidence of their presence was not detected histopathologically. Perhaps detection would require sample collection from nonmorbid animals, since target tissue (brain) is prone to artifacts upon death and delaye d immersion fixation. Histopathology of the 13

- wk studies of 1996 NTP study was restricted to animals that died on trial; hence, information from nonmorbid animals was notExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

25 available.

Commenter Four

I agree completely with the conclusion that mortalit y is the critical effect that must be addressed in any risk assessment of acetonitrile. However, in my opinion, this is an effect that arises as a consequence of cyanide

- induced toxicity, which is itself an acute or subacute (a misnomer) effect, i.e., an effect that arises (or not) from just a few days worth of exposure to a sufficiently high acetonitrile concentration. Death may be delayed some, perhaps by at most a few weeks, but the damage which results in death will have been done (or not) in just a few days worth of exposure. Thus, I disagree strongly with the assertion that it is reasonable to anticipate that acetonitrile would cause mortality as a result of chronic exposure.

First and foremost is the fact that survival was not affected adversel y by chronic inhalation exposure of mice (rats) to airborne acetonitrile concentrations up to 200 (400) ppm in the NTP study (1996). This maximum exposure level for mice of 200 ppm acetonitrile, which lasted 6 hours per day, 5 days per week, for up to 2 y ears, corresponds to a cumulative acetonitrile exposure of approximately 26,071 ppm

- days.

In the related subchronic study (NTP 1996) that was conducted in part to establish the doses for the chronic study, significant mortality was observed in mice expo sed to 800 ppm acetonitrile for as little as 3 weeks, and all mice exposed to 1600 ppm died within 3 weeks of exposure onset. These exposures correspond to cumulative acetonitrile exposure of only 3,000 and 6000 ppm

days, respectively.

Thus, if there w ere in fact a toxicity mechanism operating that caused mortality as a consequence of cumulative acetonitrile exposure, then increased mortality should have been readily apparent in the NTP chronic oncogenicity study, not only in the mice exposed to 200 ppm (26,071 ppm

days), but also in the mice exposed to 100 ppm (13,036 ppm

- days) and 50 ppm (6,518 ppm

days). Yet, there were no effects on clinical signs, body weight, survival, complete histopathology, hematology, or targeted organ weights in any of the ex posed groups of either mice (or rats) in the NTP (1996) study. In my opinion, the assertion that acetonitrile should be reasonably anticipated to cause mortality as a result of chronic exposure has no scientific support whatsoever.

Commenter Five

U.S. E PA is asking peer reviewers whether the existing science supports a conclusion that acetonitrile can be reasonably anticipated to cause mortality as a result of chronic exposure. Based on a critical weight

- of

- evidence evaluation of available data on cyani de and acetonitrile, my answer to this question is No.

The reasoning that U.S. EPA presents in support of its position that chronic or repeated

- dose exposure to acetonitrile causes mortality is not scientifically defensible. First, the data do not
supp ort this interpretation. Among repeated

- dose studies, only one subchronic study showed mortality; the deaths were clustered at the beginning of the 13

- week experiment (discussed inExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

26 more detail above). Second, based on what is known about the toxicokineti cs of acetonitrile and cyanide, and their modes of toxic action, mortality induced by chronic acetonitrile or cyanide exposure is biologically implausible.

Why? The mode of toxic action for cyanide

- associated mortality is acute. The dose

- response is

s teep but mortality occurs at high dose rates. A total cyanide dose load at lower daily concentrations over a longer period of time does not produce mortality. The mode of toxic action for cyanide

- associated mortality is also well characterized. Inhibitio n of cellular respiration by oxidative phosphorylation of cytochrome c oxidase, the terminal electron transporter in the mitochondrial respiratory chain, results in lack of energy production. If the inhibition is complete or near

- complete, asphyxiation is rapid and irreversible, and is followed by death. The delay in acetonitrile conversion to cyanide may result in a delay in onset of symptoms but the response is still acute.

Chronic exposure to acetonitrile would result in cyanide

- induced toxicity asso ciated with increased tissue burdens of thiocyanate, which is the metabolic detoxification product of cyanide. The conversion of acetonitrile

- derived cyanide to thiocyanate is catalyzed by the sulfurtransferase enzyme, rhodanase. Elevated concentrations o f thiocyanate are thyrotoxic. No evidence of thiocyanate

- induced thyrotoxicity was observed in any of the NTP or other subchronic studies.

Cyanide and acetonitrile are not known to accumulate in body tissues. If adverse effects were to be associated wit h the bioaccumulation of cyanide, then a concentration x time (C x T) effect would be anticipated. The clustering of deaths in the early weeks of the NTP (1996) subchronic study does not suggest bioaccumulation. Rather, the data indicate that the acute M TD was approximated or reached at the acetonitrile exposure concentrations at which the deaths occurred. Had this been a chronic effect, then mortality would have continued to occur at later times during the study, as “continued generation of cyanide from the existing body burden of acetonitrile, coupled with cyanide generated from new exposures, results in cyanide levels that exceed the capacity of enzymes to metabolize cyanide to thiocyanate” (Chapter 3, p. 23). This mode of toxic action is biologically i mplausible, given the lack of mortality at later times in the study or at lower doses (cumulative toxicity) and the absence of neurotoxicity symptoms in surviving animals (after the first week). Chronic tissue effects of repeated cyanide exposure, in addit ion to those of thiocyanate, include liver, kidney and reproductive organ toxicity and are associated with oxygen deprivation but are not lethal (ATSDR 1999). The absence of mortality in the NTP (1996) chronic bioassays and the Hazelton (1983) subchronic s tudies also demonstrates a lack of consistency and concordance of acetonitrile findings across similar studies. It also shows the absence of a C x T effect.

Thus, using a weight

- of

- evidence approach, it is scientifically indefensible and biologically implausible to conclude that acetonitrile can reasonably be anticipated to cause mortality as a result of chronic exposure.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

27 C. Specific Observations

Commenter One

The overall conclusion that ACN did not cause mortality at the administered doses i n rats and mice following inhalation or oral exposures may be accurate, however, deceiving. It is clearly presented in chapters one and three that species differences in ACN toxicity exist. Rabbits are much more sensitive to ACN than rats and mice. Death and maternal and fetal toxicities occurred in rabbits exposed to lower concentrations of ACN as compared to rats and mice. Furthermore, it is clear that mice are more sensitive to ACN than rats.

Page 2, para 2, line 2: Change to 201 mg/kg

- day.

Page 12, l ine 15: The equation does not show CH 3 CN production.

Page 34, line 8: On GD 29 surviving does were sacrificed?

Commenter Two

Whole body inhalation

– not sure how this is accomplished? This terminology is used throughout the document and needs to be ch anged to make the meaning clear . NTP used the term “Respiratory exposure, whole body”, which is a more accurate description of what was done. The main reason for using this designation is to distinguish these experiments from nose

only exposures.

Page 2 3, lines 11ff. This statement needs to be supported within the document, not simply stated as “it is possible...”

Page 28, line 14. Thiocyanate does interfere with thyroid function. There is no maybe about it.

Page 29, line10

- 11. This states that the conversion of acetonitrile to cyanide “is delayed”. That is not what the literature says. Conversion of acetonitrile to cyanide is slow such that it takes some time for toxic amounts to build up. That terminology is used elsewhere in the document, it is inaccurate, and is very confusing. There are data to indicate that coadministration of acetone with acetonitrile delays the formation of cyanide from acetonitrile (Freeman and Hayes, 1985) which is a different matter.

Page 33, line 12, line 23. Whole b ody inhalation. Hard to get the body through the nasal
passage.

Page 35, line 19

- 20. This sentence lacks clarity. Essentially, the lethal effects of cyanide arise from its binding to cytochrome aa 3 . That is a reversible bond, but it is fairly slow rate of dissociation. Therefore, if one reaches a systemic concentration of cyanide that binds this cytochrome on a continuous basis for a critical period it will be fatal. Therefore, the threshold is not the body burden of acetonitrile. The threshold i s the systemic concentration of cyanide that will produce death. Page 35, lines 20

- 23. This is nonsense. What is meant that the body can no longer successfullyExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

28 counter the toxic effects of cyanide? What happens is that cytochrome oxidase is inhibited to the point that cellular respiration can no longer accommodate the metabolic needs of critical tissues

– most specifically the brain.

There is also an issue of whether cyanide does not lead to apoptosis in brain cells. If that is massive enough, it could preclude recovery. I recommend that the authors' explore this question in the cyanide literature. It has been used to induce apoptosis in some in vitro systems. I would try to provide references except that would require a far ranging literature search that I am not being paid to explore.

Page 37, line 21 and 22. Avoid using jingoisms like “adaptive changes.” What is meant by this? If the adaptations are not identified, the statement is simply speculation.

Page 39, line 24. This compound sentence d oes not make sense. I assume it means to say that although there is no two

- generation study, there are data on some reproductive parameters from some developmental studies.

Page 41, lines 10

- 24. This discussion is a critical in to the attribution of ter atogenic effects of acetonitrile to cyanide. Could this be done in a more quantitative fashion? For example, is it possible to estimate the systemic cyanide concentrations that are produced by acetonitrile with those that are produced by direct treatment with cyanide? This requires some exploration of the cyanide literature.

Page 42, lines 14ff. Again, this discussion would be more convincing if a quantitative approach could be taken among different nitriles. Preferably it would be related to systemic concentrations of cyanide that would be achieved by different treatments. Barring that, one could compare doses in terms of cyanide equivalents (since inhalation doses are essentially in molar terms, it is easier to do with ppm in air than with mg/kg per day).

Page 44, lines 7

- 10. This needs to be stated more clearly or at least discussed more fully. The implication is that a microsomal system degrades cyanide that is administered directly, but not the cyanide produced from metabolism of acetonitrile. That is not a very satisfying dataset considering the emphasis placed in this document on the conversion of acetonitrile to cyanide as accounting for the toxicity of acetonitrile.

Commenter Three

a .) p. 28, lines 1

- 2: Inco rrect citation to Ahmed ( 1982 ) in reference to human case report. The Ahmed and Farooqi, 1982 paper describes rat oral acetonitrile exposures and immediate tissue distribution of cyanide.

b .) p. 28, lines 3

- 6: Disposition study with 2

- 14 C

- acetonitrile (Ahmed et al

. , 1992). The

summ ary statement of t his study could be misleading. The technical review authors are building the argument that cumulative tissue acetonitrile will be metabolized to cyanide, the mediator of toxicity. One question pertinent to cumulative effects of repeat do se is whether a significant fraction of previous dose(s) of parent acetonitrile is/are retained in target tissue at time of subsequent daily dose. The Technical Review sentence describing Ahmed et al

. ( 1992 ) could beExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

29 interpreted in this context to support widespread distribution of the parent molecule and tissue retention, since time of measurement is not specified. However, techniques used in the Ahmed et al 1992 study detected radioactivity due to non

- volatile metabolites from the methyl 14 C of the radi olabeled acetonitrile. Further, methods used by Ahmed et al. (1992) distinguis h ed radioactivity that was covalently bound. Their report includes immediate times after pulse dosing with tracer acetonitrile, but also 24 and 48 h points, which would appear to support 48 h retention of acetonitrile. However, this is more likely due to formaldehyde adducts. The wording in the Technical Review is the same as in the EPA (1999) report, where it is constructed to emphasize that disposition in stomach can arise f rom a systemic source after inhalation exposure. That wording may give incorrect emphasis to the present context.

Commenter Four

In reviewing the public comments provided, I found those submitted by Gradient Corporation (Rhomberg and Goodman) and Dr. Eu gene McConnell to be cogent and compelling. I believe that U

. S

. EPA’s responses to these comments, particularly to the sections of the Gradient comments that dealt with mortality are weak and unconvincing at best.

There is simply no way that chronic mor tality can be demonstrated in subchronic studies or those of shorter duration. There is no evidence to support convincingly the assertion that mortality should be a reasonably anticipated consequence of chronic acetonitrile exposure. All of the mortality that has been observed to date in subchronic and shorter duration studies can be explained readily in terms of the acute toxicity that is associated with the metabolism of acetonitrile to cyanide.

Section 3.1, p 25, lines 9

- 12: T he phrase “uncertainty as to the significance of certain results” sounds very mysterious. The specific concerns ought to be identified explicitly.

Section 3.1, p 29, lines 5

- 6: L ower affinity is the same as higher Km, but lower rate is the same as lower Vmax. This should be made clear. Also, the assertion that humans are thus les s able than rats to detoxify cyanide is speculative. Under what specific conditions, such as high dose, or low dose, might this be true? Can one be certain that rhodanese is not an inducible enzyme

, perhaps more inducible in human than rodents?

Section 3.1, p 29, lines 21

- 24: I do not believe that the mentioned points “strongly suggest” anything about accumulation of acetonitrile or cyanide from day to day during repeat exposures. This is a possi bility, but so is enzyme induction, which could lead to more rapid metabolism and clearance.

Section 3.1, p 30, lines 4

- 7: “I t is likely” is again too strong a phrase. This is speculation. See previous comment about the possibility of enzyme induction.

Section 3.3.1.2, p 32, lines 15

- 18: Comments of two of the three principal reviewers of the NTP study to the effect that higher doses should have been employed may support EPA’s position, but they make little sense to me. Had NTP tested at the next high er dose from the subchronic study (400 ppm) , they would have seen the same thing that was seen in the subchronic study at that dose, namely, early mortality, which might ultimately have compromised the chronic study’sExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

30 validity, since a treatment related de ath was observed in the 400 ppm group on day 13 of the subchronic study.

Section 3.3.1.4, p 35, lines 8

- 16: The argument that the modest delay in the times of death of some animals in the subchronic study to somewhat beyond two weeks relative to time of first exposure does not imply that the mortality dose

- response relationships for mortality for short

term and subchronic exposure are distinct from those for acute exposure duration. I think all it

m eans is that the animals took longer to die than they wo uld have at a higher dose. The proportion dying at a given dose may not have changed at all!

Commenter Five

p. 30

- 31. Section 3.3.1. The discussion about why U.S. EPA (1999) did not apply an uncertainty factor for subchronic to chronic exposure duratio n is not clear or rational.

p. 33, Section 3.3.1.3. Mast et al. (1994) is only available as a summary report and thus interpretation of this study should be qualified wherever the findings are discussed or referenced (e.g. Mast et al., 1994, as cited in OECD

- SIDS draft 1997

– this reference is outdated; the final SIDS has been completed ]. Same comments apply to Mast et al. (1994) discussed on p. 42, lines 3

- 13.

p. 33, Line 18. Acetonitrile and cyanide concentrations were determined for maternal blood. Where are the data? This would be useful information. If these data are not reported, then the sentence should be omitted.

p. 34, lines 1

- 4. This is an example where lack of rigor is demonstrated. Mortality in the Mast et al. (1994) study was 1/33 at 181 mg/kg/day in pregnant females, 1/10 and 2/10 in pregnant and nonpregnant females at 543 mg/kg/day. Assuming the control rates are 0 (and control rates are not given in most study summaries including tables and need to be added), these mortality rates have neither statistical nor toxicological significance! In contrast, in Saillenfait et al. (1993), a lethality incidence of 8/20 dams in the highest dose group (what were the incidences in the other dose groups) would be both statistically and toxicolog ically significant. So there is no divergence or discrepancy in the findings between the studies. Mast et al. did not observe significant treatment

- associated mortality whereas at higher doses, Saillenfait et al. did.

This discussion is also repeated on p. 42

- 43 comparing the two studies.

Repeated misinterpretation of data in a similar manner occurs throughout the repor t.

p.36, lines 3

- 5. As noted previously, this default statement is insufficient documentation of human relevance according to current scientific standards. Both qualitative and quantitative interspecies comparisons are discussed.

p. 36, lines 7

- 10. This sentence does not make sense. Why would this process “also occur in humans albeit at concentrations considerably higher with aceton itrile than with the majority of these other cyanogenic compounds”?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

31 Which cyanogenic compounds (which “saturated aliphatic nitriles”)? Why at higher concentrations? If this process occurs in humans but only at very high acetonitrile concentrations, what is the human relevance ?

Please note that nitriles have a wide range of toxic properties and according to Hartung (1982) should not be grouped together as “organic nitriles”.

Beginning page 39, under Developmental and Reproductive Toxicity. Please no te that any effects at maternally toxic doses are not considered by current scientific standards and regulatory guidance to be toxic to the embryo/fetus. The discussion in some studies suggesting otherwise is misleading. This is an example of the lack of objectivity.

e.g., p. 41, lines 22

- 24; p. 42, 23

- 24; p. 43, 18

- 19.

p. 43, lines 11

- 12. Emaciation is not a frank effect unless the animals are morib und and death is imminent. Please clarify or modify.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

32 IV. RESPONSE TO ECOTOXICOLOGY CHARGE

B. General Impressions

Commenter Six

The overall contents of the Technical Review of Acetonitrile are acceptable. However, after reviewing the document, it was not clear what the overall conclusions were with regards to the status of acetonitrile and reporting requirements of the EPCRA. In addition, several areas either need clarification or additional documentation. Specifically, the review indicates t hat most of the acetonitrile produced in the United States is released into the envir onment by deep well injection. The section discussing the potential for migration from on

- site injection wells to groundwater and the persistence of acetonitrile in ground water should be expanded. The review also indicates that in mammalian systems acetonitrile is metabolized to cyanide (the toxic metabolite). It is not clear if this pathway does (or does not) exist in ecological receptors. This issue should be clarified . The review also indicates that acetonitrile is “not expected to present a significant concern” for ecological receptors because the acute toxicity is greater than or equal to 100 mg/L. The justification for using this value as a determinant for minimal toxicity should be included in the text. While it is understood that 100 mg/L is a large dose, this should be explained in relevance to other toxins or regulatory standards.

Commenter Seven

The overall impression is of an adequate summary of available information on the physicochemical and toxicological behavior of methyl cyanide. There are, however, some unanswered questions that make it difficult to use this document to fully judge this chemical’s potential to impact humans or the environme nt and where any such impacts may occur. The description of biotic and abiotic degradation processes is not clear whether these processes differ in different media or what degradation byproducts might be expected in different media (if any). Use of the E PIWIN model to assess the distribution of methyl cyanide following release was good , but more discussion of its transport and fate in groundwater (an important disposal media) is needed. The review of what is known about the ecological hazards posed by th is chemical suffers from the lack of available information on chronic effects or on population

- level
responses.

Commenter Eight

The January 15, 2008 Draft Technical Review of Acetonitrile is a very clear and balanced document. It is well

- written with no area lacking clarity and coverage. The only slight confusion that I had was associated with the large amounts of acetonitrile injected underground. Given this major input to the environment and the mention of possible groundwater contamination on page 11, I was left wondering why there was not more discussion of exposure via groundwater. Also the efficiency of the other major disposal method, on

- site incineration, might be described more.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

33 C. Response to Charge Questions

Commenter Six

Overall

, the chemical and physical properties are clearly described. As stated above, additional details regarding the fate of acetonitrile from underground injection wells would be valuable to the review. Section 2.6.5

, Toxic Transformation Products

, is brief a nd could be improved by expanding the discussion. Additional editorial comments are included in the section on specific comments (below).

Commenter Seven

This chapter appears accurate, relatively clear, and unbiased with respect to the physicochemical properties of methyl cyanide. Given their importance in estimating transport and fate, corroborating references (if available) for the values of KOW, Henry’s Law, and hydroxyl reaction would be helpful (some are given in the text but not in the table).

Page 53, Table 4

- 1. This table reproduces many of the values cited in Table 2

- 1 but with more complete source citations. It appears possible to combine these tables into Table 2

- 1 and simply cite this table in Chapter 4.

Commenter Eight

Yes. The descri ption is clear and concise

. Chapter Two, Chemistry and Environ mental Fate 1. Has an objective (accurate, clear, complete, and unbiased) and technically accurate description of acetonitrile been provided in terms of chemical and physical properties?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

34 Commenter Six

The report objectively addresses the fate of acetonitrile in soil and groundwater. To a more limited extent, fate and transport in the atmosphere is also included. Since approximately 90% of acetonitrile is disposed of in on

- site injection wells, this section should be expanded. The discussion should include information regarding the potential for release of contami nants from injection wells into groundwater and the fate and transport of contaminants if they ar e released into groundwater. Also, the text indicates that 30 million tons of acetonitrile are burned on

- site each year. Are data available to determine the e fficiency of these methods? What levels of acetonitrile may be released into the atmosphere as a result of incomplete combustion (or are their other toxic by

- products of combustion)?

Commenter Seven

• Page 10, line 27. The volatilization pathway would on ly be accessible to methyl cyanide disposed of on the soil or water surface. Yet it appears (from Lines 22

- 25) that the bulk of releases are to groundwater or to air following combustion. Is there any information on methyl cyanide’s transport and fate in groundwater? It is there any information on whether the combustion of methyl cyanide produces toxic by

products? (I would think not but some information to this effect, if available, would be helpful.)

• Page 11, Line 2 et seq . What are the degradation b yproducts of methyl cyanide? Do these byproducts differ with respect to media and degradation pathway? If such information is available, it would helpful in assessing the nature and extent of any problems caused by methyl cyanide’s breakdown in the envir onment. It would be helpful to summarize what is known about degradation by pathway and byproduct into a simple table.

• Page 14, Section 2.6.1. Given that large amounts of methyl cyanide are disposed of via underground injection, the seeming lack of info rmation on its transport and fate in groundwater is troublesome. There are scattered mentions (e.g., Page 11, Line 8 and Page 15, Line 13) of its potential to contaminate groundwater but no detailed exposition of this process or its consequences. Even if such information is not available, some mention should be made of groundwater as a separate media and of methyl cyanide’s possible transport and fate in this media.

• Page 14, Line 15. Please specify these “various” waste streams.

• Page 15, Section 2.6.2. Except for its cryptic last sentence (Page 16, Line 1), this Chapter Two, Chemistry and Environmental Fate 2. Does the report objectively address the fate of the chemical in the environment including both transport between environmental compartments (atmosphere, aquatic and terrestrial) and transformation (e.g., degradation), including both abiotic and biotic processes where relevant?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

35 section makes it appear that abiotic degradation only occurs in air. Is this true? And what are the presumed byproducts of such degradation?

• Page 16, Line 11. The “...most significant degrad ation process...” Please clarify whether this refers to all su ch processes or just the biotic.

• Page 16, Line 22. The compound is degraded into what?

• Page 17, Line 15. Please define anaerobic “consortium.” There is also no mention between Lines 14 and 23 of the environmental media in which these experiments were conducted.

• Page 17, Line 17. When you say anaerobic degradation results can be “applied” to groundwater, do you mean by extrapolation of tests in other media or because test were actually con ducted in groundwater? Given (per Page 18, Lines 3

- 4) that groundwater is a source of methyl cyanide contamination, it would seem important to provide as much information as is available about its behavior in this media.

• Page 18, Section 2.6.4. Using EP IWIN v 3.20 and the physicochemical parameters listed in Table 2

- 1, I was able to reproduce the half

- life, but not the mass amount, estimates given in Table 2

- 2. For example, my estimate for air was 11.8% rather than the 51.7% given in Table 2

- 2. Either an error was made or not all of the assumptions used to produce 51.7% and the other mass amount values were clearly stated. Given the importance of the EPIWIN results to understanding the fate of this chemical, all of the inputs/assumptions used for this model should be disclosed and listed (possibly in a separate appendix), that replication of results is more straightforward.

• Page 18, Line 9. The EPIWIN model can release 1000 kg/hr to all three compartments simultaneously or one at a time. Which of the se release scenarios was followed? Was an investigation made of what affect releasing to only one compartment at a time would have on the environmental distribution of methyl cyanide? In other words, are all release points equal or do they produce differ ing outcomes or is Table 2

- 2 always the same regardless of release point?

• Page 18, Lines 18

- 21. These are redundant with Page 10, Lines 22

- 25.

• Page 18, Line 10

- 11. This sentence is unclear. Please clarify whether these estimated half

- lives those estim ated internally by EPIWIN or user

- entered?

• Page 18, Lines 16

- 24. The vast majority of releases occur in injection wells, which have shown the potential to contaminate groundwater. Yet the EPIWIN model does not include groundwater as one of its environme ntal compartments. As has been said previously, this leaves open the question of the transport and eventual fate of methyl cyanide in groundwater.

• Page 18, Line 22. Is there a reference for the statement “...can be anticipated to be significantly slower. ..”?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

36 • Page 19, Line 8 lists the toxic byproducts as acetic acid and ammonia

- is this always the case? What about cyanide?

Commenter Eight

Yes. A minor issue has already been mentioned above. Except for brief comments and a few sentences on page 18, t here was not much detail about the transport and transformation of acetonitrile in groundwater, a major location for its disposal.

Section 2.6.4. is a sound description of the modeling. A Monte Carlo

- based exercise that included uncertainties would provid e more insight about the environmental distribution under a range of conditions or realistic scenarios.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

37 Commenter Six

The information on ecological toxicity has been objectively described. I am not aware of any additional studies that should be included. The text could be more clea rly presented, specific comments are provided below.

Commenter Seven

There are two concerns with this chapter: (a) an exclusive focus on lower trophic level aquatic and terrestrial species with no discussion of terrestrial vertebrates and (b) the lack of robust available information on the chronic toxicity of methyl cyanide, particularly in vertebrate species.

Ecological receptors include more than plants, invertebrates, and fish. Was there no information available on methyl cyanide effects in terrestri al vertebrates? If this is the case, it would be helpful to make a clear statement to this effect in Section 4.1.

Although somewhat problematic, would it not have been possible to draw some tentative conclusions about terrestrial vertebrate responses in the wild from the rat and mouse laboratory data presented in Chapter 3?

• Page 51, Line 15. Please add “or biomagnify” after “bioaccumulate” to distinguish these two different processes. Also on Page 57, Line 11.

• Page 51, Section 4.1. There is no mention of whether there is any information available on the mechanism of toxicity of methyl cyanide in various ecological receptors. If no information is available, then it would be helpful to make a clear statement to this effect in Section 4.1. Couldn ’t what is known in this regard about cyanide be used to make some informed judgments about methyl cyanide?

• Page 53. Separate tables for acute and chronic data would be helpful.

• Page 53, Table 4

- 2. It is not clear whether footnotes “b” and “c” apply to all or just selected studies listed [It would also help if the font size of the footnotes was increased to, say, 10 pt]. Also, is the “d” in the “Value” column supposed to be a footnote?

• Page 55, Section 4.2.2. The chronic toxicity testing was performe d on embryos of a

non

- standard, fairly robust, test species. It has been shown (for cyanide) that juveniles and adults, and not embryos, are the more sensitive life stages. The text also notes several other weaknesses in the very limited studies on chron ic toxicity in fish. The ECOSAR estimated chronic values are lower by a factor of 2 to 10 than Chapter Four, Ecological Hazard Assessment 1. Has the available information on the ecological toxicity of acetonitrile been objectively described? Are there any additional studies that should be included?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

38 those from these deficient studies.

Commenter Eight

It is my opinion that the ecological toxicity was objectively described. In addition, the earlier chapter on health effects clearly presented useful insight germane to the ecological effects discussion. A few additional references might be useful. They ar e given below in Point 13 of my Specific Observations.

It must be noted (Specific Observations 14

- 17 below) that the toxicity data that the EPA had available was not high quality information. Given the widespread and high use of this compound, I found thi s very surprising. However, I did not find much more information during a brief journal search.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

39 Commenter Six

The concl usion appears to be supported with respect to ecological receptors. Supporting information is needed for the statement that “acute toxicity values . . . greater than or equal to 100 mg/L indicating relatively low acute aquatic toxicity .” While I agree with this conclusion, documentation (either by regulatory guidelines or comparison with values for aquatic toxicity for other chemicals) is needed. In addition, minimal information exists with regards to terrestrial toxicity, population effects, or general effects on the environment. Thus, the conclusion (in Section 4.1) should be qualified that it primarily addresses ecological receptors in aquatic environments.

Commenter Seven

No, for two primary reasons. First, it does not appear possible to determine if there is a concern without som e information on typical environmental concentrations for comparison with these toxicity values. Second, the already noted weaknesses in chronic toxicity information make it particularly difficult to assess population

- level effects from, for example, impa irment of reproductive ability due to chronic exposures to low levels (e.g., from a groundwater seep to surface water).

All of this suggests that available information is insufficient to make unequivocal statements about the chronic toxicity of methyl cya nide. This also limits this technical document’s ability to support broad, supportable statements about a lack of concern. Because what is of “concern” is typically largely a policy determination, it is not clear why a technical review has to make this d etermination?

Commenter Eight

Yes. This seems a reasonable conclusion from the facts presented. Acetonitrile does not appear to be very toxic but the toxicity data are not high quality information. Acetonitrile does not have any properties suggesting tha t it would bioaccumulate to any level of concern. My only minor reservation is de scribed below in Point 2 of my Specific Observations. Although the acetonitrile has low toxicity as expressed as effect concentrations, it is released from diverse sources in large amounts. The amounts and widespread nature of its release suggests the need for extra diligence to possible ecological effects under various exposure scenarios despite its low toxicity. Chapter Four, Ecological Hazard Assessment 2. Is the conclusion presented in section 4.1, “Acetonitrile does not present a concern for adverse effects on t he environment” supported by the data/information presented?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

40 C. Specific Observations

Commenter Six

Section 1.2 Conclusions :

Pg. 2; Lines 3

- 6: The introductory statement to this section is very long and somewhat confusing. Suggest the following: “The EPA has concluded that it is reasonable to anticipate mortality in animals chronically exposed to acetonitrile. This conclusion is based on observations of mortality in acute, short

- term and subchronic repeated exposure studies in animals. In addition, acetonitrile has been shown to have a steep dose

- response curve; and, the mode of action for acetonitrile toxicity (i.e., conversion to cyanide) also supports this conclusion.”

In addition, the conclusions should be clearly stated and perhaps bulleted at the introduction of this section. For example:

• Acetonitrile has been found to produce mortality in acute, shor t

- term and subchronic animal studies;

• Due to it’s mode of action and steep dose

- response curve, it is reasonable to assume that mortality would also occur under chronic conditions;

• In ecological studies, acetonitrile has been found to have minimal toxici ty (i.e., concentrations of greater than 100 mg/L are required to produce mortality in aquatic studies);

• Acclimated microbes have been found to be capable of rapidly degrading acetonitrile under aerobic conditions; however, under anaerobic conditions or wi th unacclimated microbes, acetonitrile may persist in the environment.

Pg. 2; Line 9: R evise text to read “. . . oxygen needed for cellular respiration. The lack of oxygen in cells leads to cytotoxic ano xia (Albaum et al., 1946).”

Pg. 2; Line 12: T he text states that “similar clinical signs have been observed in humans”. Are these case reports involving humans exposed to cyanide or acetonitrile? This should be clarified in the text.

Pg. 2; Line 15: D elete “animal” (before repeat

- dose toxicity).

Pg . 2; Line 18: D elete “the” (before 13

- week inhalation studies).

Pg. 2; Line 20: D elete “a delayed period of time (weeks)” and replace with “several weeks .”

Section 1.3 Chemistry and Environmental Fate :

Pg. 3; Line 17: The text describing volatilizatio n of acetonitrile from a river should include the parameters (temperature and wind speed) that were used in developing this estimate. Pg. 3; Line 19: D elete “land” and replace with “soil

. ”External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

41 Pg. 3; Lines 20

- 21: R evise text to read

– “sorption coefficient and high water solubility it is predicted that acetonitrile will migrate rapidly through permeable soils.”

Pg. 4; Line 5: I nsert “acetonitrile in the presence of” after “Half

- lives for

. ”

Pg. 4; Line 7: R evise text to read “for non

- acclimated or anaerob ic bacteria” (assuming this is correct?).

Section 1.4 Human Health Hazard Assessment Summary :

Pg. 4; Line 19: Revise text to read “oxygen needed for cellular respiration. The reduction of oxygen levels in cells may result in cytotoxic anoxia

. ”

Pg. 4; Line 20: Insert “in mammalian systems” after “converted to cyanide

. ”

Pg. 4; Line 23: D elete “then” (before “it may be partially”)

.
Pg. 4; Line 24: Place a period (.) after Hartung, 1982 citation; delete “resulting” and revise text to read “This proces s results in a greater retention time for cyanide

. ”

Pg. 5; Line 1: D elete “occurs” and replace with “does not occur until several

. ”

Pg. 5; Lines 2

- 5: D elete text “Following repeated . . .metaboliz e cyanide to thiocyanate.” R eplace with “Following repea ted exposure to acetonitrile, it is likely that cyanide will accumulate in the body due to the generation of cyanide from the existing body burden as well as new exposures to acetonitrile. This accumulation may result in cyanide levels in tissues that

exc eed the capacity of enzymes to metabolize cyanide to isocyanate.”

Pg. 5; Line 6

- 7: R evise text to read “The accumulation of toxic concentrations of cyanide and the metabolic rate at which lethal cyanide concentrations are reached will large ly depend on t he balance . . .”

Pg. 6; Line 8: T here is almost a 10

- fold difference between mice and rats for LD50. Is there any metabolic difference between these organisms that can be attributed to this difference? And, if so, what is the relevance o f this difference between rats and mice for acetonitrile toxicity in humans? [Note, it is also interesting that acetonitrile is 10

- fold more toxic in rats; and, male rats appear to be the sensitive species with regards to acetonitrile carcinogenicity].

Pg. 6; L ines 13

- 14: T he text indicates that there is an “overall lack of genotoxic potential” for acetonitrile in cell culture bioassays. This may be an incorrect conclusion. Typically, negative results in cell culture bioassays reflect an insensitivity of the assay more than a potential lack of response in mammalian systems. Acetonitrile did produce a positive response in several cell culture bioassays (SCE in CHO cells, gene conversions in S. cerevisiae , and chromosome aberrations in CHO cells). This should be more clearly stated in the summary. Pg. 7; Lines 20

- 21: R evise text to read “This conclusion is based on the observation that the minimum acute toxicity values for aquatic organisms is greater than or equal to 100 mg/L. InExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

42 comparison with other chemic als, acetonitrile has a relatively low aquatic toxicity.”

Pg. 8; Line 4: D elete “experimental

. ”

2. CHEMISTRY AND ENVIRONMENTAL FATE

2.2 SUMMARY

Pg. 10; Lines 13

- 21: Information included in the technical summary should be included in this section; and, the text on production values should be revised to match the technical s ummary (which is more clear). I s uggest the following revisions:

“Acetoni trile is produced as a by

- product of acrylonitrile production. The principal uses of acetonitrile include: as a solvent in hydrocarbon extraction processes, as a specialty solvent, as a reaction solvent in the production of synthetic pharmaceuticals, as a solvent for the extraction of fatty acids, and as a solvent in an alytical instrumentation (Lewis 2 001; Budavari et al., 1996; WHO 1993). The estimated US production of acetonitrile has varied over the past decade between 10 million and 100 million pounds per year (US EPA 2002). Of the millions of pounds produced each year, a commercial market exists for only half of the inventory. The US production capacity for acetonitrile in 1994 was estimated to be 46 million pounds (McKinney and DeVito, 1998).”

Pg. 10; Lines 24

- 25: The text indicates that 30 million pounds of acetonitrile were burned on

site for energy production. Are there any estimates of the efficiency of the combustion procedures? Or, how much acetonitrile (or other combustion by

- products) ma y be released by this process?

Pg. 10; Line 26: I nsert “most of” before “the chemical will volatilize”

Pg. 11; Line 1: I nsert “predicted to be” before “the environmental compartment”

Pg. 11; Line 2: D elete “distribute” and replace with “partition”

P g. 11; Line 12: I nsert “from” before “weeks to months”

Pg. 12; Line 12: I nsert “an” before “oxidation catalyst”

Pg. 12; Line 15

- 16: T his reaction should be shown in it’s complete form (or as stated in the text : the reaction produces acrylonitrile with acetonitrile and hydrogen cyanide as principal by

products).

Pg. 14; Line 14: R evise text to read “The production and wide use of acetonitrile as a solvent

. ”

Pg. 15; Line 2: W hat temperature was used in estimating the half

- life in a model river? Pg. 1 6; Lines 1

- 2: R ather than indicate hydrolysis is “extremely slow ,” can a half

- life be provided and products of abiotic hydrolysis identified?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

43 Pg. 16; Line 13: I nsert “enzyme” before “transforms the nitriles

. ”

Pg. 16; Lines 19

- 20: R evise text to read “Activated sludge and sewage have been found to contain populations of mixed microorganisms that are capable of transforming organic and inorganic substances into less toxic forms.”

Pg. 16; Lines 22

- 23: D elete “degraded with” and replace with “exhibited” , and delete “More dated studies” and replace with “Several older studies have

. ”

Pgs. 16

- 17; Lines 24

- 1: R evise text to read “proceeded slowly with unacclimated microorganisms

. ”

Pg. 17; Lines4

- 5: D elete “develop a strain” and replace with “enrich popul ations”, and on following line, revise text to read “cultures achieved a rate of degradation of 58% for acetonitrile in water in 5 days (BOD test)

. ”

Pg. 17; Line 8: I nsert “concentrations ranging from” before “0.1

- 25 mg/L .”

Pg. 17; Lines 23

- 24: R evise text to read “anaerobic degradation is roughly four times slower than aerobic degradation”, and on following line, delete “beyond aerated” and replace with “under anoxic conditions.”

Pg. 18; Line 1: R evise text to read “Due to large differences in tempera ture, pressure, and other conditions these estimated degradation rates for acetonitrile cannot be applied to underground injection.”

Pg. 18; Lines 4

- 5: Because such a large volume of the waste product is disposed in these wells, this section should be expanded to discuss more specifically the solubility of acetonitrile and the potential for migration from the injection zone to aquifers. It may be adequate to in dicate that since acetonitrile is highly soluble in water any material discharged into an aquifer from deep

well injection is likely to be transported with groundwater migration. It should also be indicated that the environmental conditions (i.e., temperature, nutrient concentration an d microbial populations) in groundwater are not conducive to the degradation of any material that does reach the aquifer.

Pg. 18; Lines 19

- 20: I t is preferable to use the same units in discussing TRI and injection, suggest that the TRI release for 2003 be rounded off to 20.8 million pounds.

Pg. 18; Lines 21

- 23: R evise text to read “It is anticipated that the rate of acetonitrile degradation following release from an underground injection well will be very slow. Chemical concentrations from an undergr ound release are likely to be relatively high; and, anaerobic or oxygen limiting conditions are likely to exist. Optimal conditions for acetonitrile degradation are generally assumed to require aerobic conditions and relatively low chemical concentrations .” Pg. 19; Section 2.6.5 Toxic Transformation Products. Additional explanation is needed for this statement. Are these transformation products from aerobic degradation? Are there no pathways which would result in the release of cyanide to the environmen t?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

44 3. HUMAN HEALTH HAZARD ASSESSMENT (not reviewed)

4. ECOLOGICAL HAZARD ASSESSMENT

Pg. 51; Line 1: R evise text to read “Acetonitrile is not expected to present a significant risk for adverse effects in ecological receptors.”

Pg. 51; Line 11: D elete “ranged” and replace with “are also relatively low

, ranging from 160 to 7300 mg/L.”

Pg. 51; Line 24: R evise text to read “found for acetonitrile. Limited data . . .”

Table 4.2

– You would have to assume if you have footnotes for a table from “a

- v” that something is not quite right! This table is very difficult to follow or interpret. I believe it would be easier to follow if broken into Table 4.2, Acute Toxicity and Table 4.3 Chronic Toxicity.

Also, since the conclusions rely heavily on predicted v alues, it would help the reader if some example of predictability were provided; i.e., compare toxicity values derived under experimental conditions with those predicted using the ECOSAR model. While it is assumed that the model provides a conservative es timate of toxicity values, this should be demonstrated in the text.

Finally, the conclusions for ecological receptors would be easier to follow if a summary table were included that provided ranges of values for acute, chronic, aquatic

, and terrestrial to xicity.

Commenter Seven

Chapter One, Integrated Technical Summary:

• Page 2, Section 1.2. This section appears to focus exclusively on potential adverse health outcomes in humans, while there is a separate section (Section 1.5) for potential ecological i mpacts. It would be better to have one comprehensive conclusion section, encompassing both human and ecological effects, for the whole report, rather than leave the impression that its conclusions are based exclusively on human impacts.

• Page 7, Lines 8

- 9 . It is not clear how you can assert that this chemical does not pose a concern for the environment without some knowledge o f its levels in the

environment. If there is information on known environmental levels, it does not appear to have been presented in this report.

• Page 7, Lines 19

- 20. The information presented on chronic ecological outcomes is not robust enough to support the statement that methyl cy anide “...does not pose a concern for ... adverse effects on the environment...” The statement “This conclusion is based on ...” with reference to acute effects does not follow from theExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

45 previous sentence. It is also questionable whether acute outcomes ar e a reasonable basis for conclusions about chronic outcomes. Chronic ecological outcomes could be of importance in instances where, for example, groundwater leakage to surface water provides a continual, low

- level source of methyl cyanide to aquatic recep tors (c.f., Page 15, Line 13 and Page 18, Lines 4

- 5).

Specific comments and observations for Chapters 2 and 4 appear above under the relevant chapter.

Commenter Eight

Minor technical suggestions:

1. Page 1, Line 10. Please spell out INEOS at its first use

.

2. Page 1, Lines 18

- 25. The goal of an assessment is to sufficiently understand the risk of specified effects to gauge what level of regulatory attention is warranted. Although the other qualities of acetonitrile suggest relatively low risk, the sheer magn itude of its annual release and its introduction underground via injection wells suggests that an elevated level of diligence might be warranted in this decision process. The amounts released from diverse sources increases the probability of an adverse ef fect relative to an equivalent chemical that is released in small amounts by only a limited number of users.

3. Page 13, Lines 13

- 14. One sentence paragraph. Perhaps this sentence could be moved to the second paragraph of Section 2.1?

4. Page 16, Line 24. Perhaps the word “sluggishly” is too vague for this technical description?

5. Page 17, Line 4. Change “strain” to “culture”?

6. Page 17, Line 23. Change to read “... anaerobic degradation rate is roughly...”?

7. Page 18, Lines 7

- 15. A Monte Carlo

- based model that in corporated uncertainty in estimates would enhance this presentation.

8. Page 18, Line 20. Change to “... were disposed on

- site in ...”?

9. Page 24, 1 st full paragraph (and elsewhere). A figure might help the reader understand these details better.

10. Page 24, Lines 16

- 18. Consider shifting the clause at the end to the beginning of the
sentence.

11. Page 39, Section 3.3.3. The (undefined) power associated with analysis of these data is likely very low because the number of observed exposed females was so low. I wonder ab out the value of any discussion of these data which might be used to weakly imply low risk.

12. Page 51, Line 6. Here and elsewhere the phrase “the fact that” could easily be omitted.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

46 13. The following r eferences might also be helpful: Chen, C

- Y, S

- L Chen and ER Christensen. 2005. Individual and combined toxicity of nitriles and aldehydes to Raphidocelis subcapitata. Environ. Toxicol. Chem. 24(5): 1067

- 1073. Chen, C

- Y and C

- F Huang. 1996. Toxicity of organic mixtures containing cyanogenic toxicants. Environ. Toxicol. Chem. 15(9): 1464

- 1469. Grogan, J, SC DeVito, RS Pearlman and KR Korzekwa. 1992 Modeling cyanide release from nitriles: prediction of cytochrome P450 mediated acute nitrile toxicity. Chem. Res. Toxicol. 5: 548

- 552.

14. Issues r elated to the Tong publications: I found the citation for Tong and Hongiun (2001) to be Zhang, T. and H. Jin. 2001. Aquatic toxicity of acetonitrile and its water quality criteria for the protection of aquatic life in China. Inter. J. Environ. Pollut. 15(5): 568

- 575 . Perhaps, the first names might incorrectly be inverted in the draft EPA report or by the journal editor? The bottom of the first page of the journal article indicates Zhang and Jin is the correct way to cite the article. However, this individual’s other publications suggest that Tong is his proper name.

Relative to Tong and Hongiun (2001), note that, in addition to the shortcomings mentioned in the draft report about this paper, it appears that nominal concentrations were used. Measured concentrations are much better. Also the applied trimmed Spearman

- Karber LC50 estimates are not as good as parametric methods. There are other issues with this publication relative to methods and statistical analyses. So, I agree with the concern stated on page 56, line 9 about these studies.

Relative to Tong et al. (1996), the least powerful option, the Steel’s many

- one test was used because the data failed tests for normality. Yet, there is no discussion of conventional transformations that might have resulted in norma l data. The associated NOEC/LOEC/ACR values are likely not of sufficient quality to give them much weight in this EPA draft report.

15. There are also statistical compromises in Barahona

- Gomariz et al . (1994) that produce considerable uncertainty in my mi nd about the estimated effect metrics. The applied methods were not very powerful

, and power influences the effect metric estimation very much.

16. The Henderson et al. (1961, or 1960?) paper uses a “static with no renewal” design that was standard in th e 1960s but would be considered unacceptable today. I suspect that the acetonitrile concentrations dropped from the nominal concentrations during the test and, as a result, the effect metrics are expressed in concentrations higher than what they should be.

17. Generally, the Ewell et al. (1986) paper used a rudimentary extrapolation to estimate LC50 values for the tested compounds because they did not have enough partial kills. The lack of high concentration treatments resulted in a <100 mg/L estimate fo r what seemed to be a static test. Nominal concentrations were used instead of measured concentrations.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 1 APPENDIX A

PEER REVIEW COMMENTS FROM COMMENTER ONE.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 2 Commenter One

Review Report on Technical Review of Acetonitrile (Methyl Cyanide) CAS Registry Number 75

- 05

- 8

Office of Environmental Information Environmental Analysis Division Analytical Support Branch

J une 15, 2008External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 3 I. GENERAL COMMENTS:

The document “Technical review of Aceto nitrile,” case registry #75

- 05

- 8, is well written. The information in chapter three on “Human Health Hazard Assessment” of acetonitrile (ACN) is well described and clearly presented. Chapter one, “The integrated technical summary”, clearly summarizes the i nterpretation of the technical data available to the authors on the chemistry, environmental occurrences, and health hazards of ACN.

Chapter three describes in some details the studies available on ACN

- induced human and animal acute, sub

- acute, and chron ic toxicities. However, the data available are relatively old information that is derived from accidental human ingestion of ACN and relatively few idealized animal studies in mice rats and rabbits. Also, the interpretations of the results are based on

som ewhat obsolete mechanistic information.

Therefore, the overall conclusion that “ACN

- induced acute, sub

- acute and chronic toxicities and subsequent mortality is due to the amount of un

- detoxified cyanide moieties alone” is not valid because of the current understanding of the molecular mechanisms of actions of cyanide ions and other reactive metabolites of ACN.

II. RESPONSE TO CHARGE QUESTIONS:

1. Has the available information .................?

The available information that resulted from some limited, older studies has been adequately described in the document. The interpretations presented in the document are based on the interpretations of the results that are presented by the authors of original studies and or from previous USEPA documents.

To my knowledg e, there are no new studies on ACN adverse activities. There are, however, several recent studies that describe the molecular mechanisms of toxicities of the cyanide moieties and how cyanide actually interferes with mitochondrial respiratory chain mechanis ms. Also, there are modern studies that describe the molecular mechanisms of other ACN metabolites such as formaldehyde (see below).

2. The USEPA has determined that ........................ Based on the totality of available evidence, is it reasonable to anticipate that A CN can cause mortality as a result of chronic exposure?

The answer to this question is: Yes. Based on the available human and animal data, I would anticipate that ACN could cause mortality as a result of chronic exposure, given the right species and the right period of exposure. My interpretation of the data that are available and presented in this document is outlined below.

The data indicate that ACN causes death in rabbits, mice and rats. It causes death in rabbits at much lower doses than that w hich was administered in the two year studies to mice and rats. Due to the similarities in metabolic enzymes betweenExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 4 rabbits and humans and due to the fact that humans are less able to than rats to detoxify cyanide, I would expect that health effects and m ortality in humans, following human exposure to ACN, would be closer to that observed in rabbits than to that observed in mice and rats. Therefore, I would anticipate that human would be more sensitive to ACN actions than mice and rats. Thus, the data deri ved from the 2 year chronic exposure of rats and mice to low concentration of ACN should not be used to un

- list ACN from the current EPA regulations.

Furthermore, most, if not all, of the conclusions are based on the assumption that the lack of apparent l ethality from ACN in rats and mice in the two year studies using low concentrations is due to the ability of rats and mice to detoxify ACN metabolites, particularly cyanide moieties.

The lack of such an apparent end point (mortality) of adverse effect doe s not mean that there were not any adverse effects that had occurred on the molecular levels within the experimental animal species utilized in these studies.

To explain this statement, I would like first to describe ACN metabolism. ACN is known to be oxidized by Cytochrome P450 2E enzyme system or by other oxidizing enzymes to yield a reactive intermediate, methyl cyanohydrine. The latter intermediate is in equilibrium with equal molar concentrations of cyanide ions and formaldehyde.

CH 3 CN

-- ---------- CH 2 OHCN

--------- CH 2 O + HCN

Cyanide ions had been implicated in most aliphatic nitriles’ toxicity including that of ACN. The other metabolite, formaldehyde (CH 2 O) has not been discussed in the interpretation of ACN toxicity and lethality. Formaldehyde on the other hand has its own set of toxic actions. Aliphatc aldehydes, in general, are known to block the activities and functions of several enzymes via the for mation of Schiff’s base with the amino groups in the enzymatic reactive sites. Also, CH 2 O is known to deplete anti

- oxidants in the cellular environment leading to cellular oxidative stress and oxidative damage. Both cyanide and aldehyde are known to alte r mitochondrial chain reactions leading to mitochondrial membrane damage and the release of Cytochrome C, which activates apoptosomal enzymes, leading to the break down of nuclear DNA and the induction of cellular apoptosis. Because cellular apoptosis is a slow process, it is expected that mortality caused by cellular apoptosis to be a slow process during the life span of the cell. During these processes, tissues and organ degeneration may constitute a signal for tissue or organ injury that leads to mortali ty.

Acute exposure to ACN will yield large number of formaldehyde molecules and cyanide ions. Both will cause severe oxidative stress and cause massive blockage of respiratory chain reactions involved in oxygen metabolism leading to the sudden accumulati on of reactive oxygen species, cellular, tissue, and organ damage, and subsequent death. Even in acute processes, delayed death usually occurs as a result of the sudden accumulation of cell, tissue and organ damage that could be caused by sudden increase in oxidative damage and interference with oxygen metabolism.

Sub

- acutely, the processes of damage accumulation may take a while longer and when enough damage accumulates, organ malfunction may occur leading to death as indicatedExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 5 in the 13

- week ACN sub

- a cute studies.

If exposure occurs at lower doses (such as that in the chronic studies), eventually enough molecular damage will accumulate at a longer time period (may be more than the two year period used in the studies conducted in the least sensitive species; rat and mice but a shorter period in the more sensitive species such as the rabbits and humans).

Therefore, I don’t believe that the lack of mortality indicated in the available chronic studies in rats and mice (2 year) justify the un

- listing of ACN from the criterion described because:

a. In the chronic studies described, highly insensitive species (rats and mice) were used. Thus the doses that would produce mortality following chronic exposure would be high. b. The duration of two years used for t he studies described may not be enough to observe mortality in the species used.

c. Due to the known toxicity of ACN metabolites, CN and CH 2 O, molecular damage may have occurred in these species that may lead to altered physiological functions and other chron ic disorders without the induction of death. d. Due to the known teratogenic activities and developmental effects of ACN, and because of the sensitivity of developing fetuses to in

- utero exposure to very low levels of environmental chemicals, it is anticipate d that molecular damage may occur in the fetus at low levels of exposure. Such molecular damage during fetal development may lead to developmental abnormalities and impaired offspring that are susceptible to various diseases.

III. SPECIFIC OBSERVATIONS:

T he overall conclusion that ACN did not cause mortality at the administered doses in rats and mice following inhalation or oral exposures may be accurate, however, deceiving. It is clearly presented in chapters one and three that species differences in ACN toxicity exist. Rabbits are much more sensitive to ACN than rats and mice. Death and maternal and fetal toxicities occurred in rabbits exposed to lower concentrations of ACN as compared to rats and mice. Furthermore, it is clear that mice are more sensiti ve to ACN than rats.

Page 2, para 2, line 2: Change to 201 mg/kg

- day.

Page 12, line 15: The equation does not show CH 3 CN production.

Page 34, line 8: On GD 29 surviving does were sacrificed?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 1 APPENDIX B

PEER REVIEW COMMENTS FROM COMMENTER TWOExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 2 

### Peer Review Comments on Technical Review of Acetonitrile (Methyl Cyanide)

Commenter Two 5

- 7

- 08

I. GENERAL IMPRESSIONS

The intent of this technical report is not clear. It is stated that it is related to the review of a petition to remove acetonitrile from the list of chemicals subject to reporting requirements. A major concern is how consistent this document is with the rationales that underlie other regulatory activities of the Agency. This document does make reference to IRIS reviews of acetonitrile and the analysis appears consistent with what is provided in the IRIS document. The present document makes the conclusi on that the effects of acetonitrile are related to its conversion to cyanide. The conclusions of this document are not consistent with the Office of Ground Water and Drinking Water's (OGWDW) Consumer Factsheet on Cyanide, where effects of cyanide on thyro id function and neurological effects which are related to its conversion to thiocyanate. Unfortunately, OGWDW has no transparent link to the criteria document that supported the cyanide regulation. It is my recollection that the concern over thyroid effe cts of cyanide largely relied on human data related to exposure to cyanogenic compounds (i.e. similar to acetonitrile) in the diet that lead to inhibited thyroid hormone synthesis. This literature needs to be revisited and an explicit determination made a s to its applicability to the problem the present document is supposed to address. I recognize that many of the papers that address this issue are old and may well be confounded with other socioeconomic factors. Nevertheless, they need to be explicitly c onsidered in this document because of the huge emphasis that is placed upon the interpretation that the toxicity of acetonitrile is attributable to its conversion to cyanide.

The document was generally well written and the review of data that has been dir ectly associated with studies of acetonitrile were largely accurate. Below, there are further comments on whether the document adequately explored potential thyroid effects of this chemical.

II. RESPONSE TO CHARGE QUESTIONS

Provide narrative response s to each of the charge questions below.

1. Has the available information supporting the hazard assessment of acetonitrile been objectively described? Are there any additional studies that should be included?

Comments:

I did not identify any studies of acetonitrile, itself, in the literature that were of importance that were not included in this review. The information that is discussed appears to be objectively described. The toxicological data on acetonitrile is pretty sparse. The major studies have been performed via inhalation exposure, although there are some reproductive and developmental data that have been obtained using gavage.

While the data are sparse, this review does not make adequate use of the available data to form the conclusions of the report. One example is the handling of the genotoxicity data. A secondExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 3 was the failure to utilize the limited metabolism data to support their hypothesis that the lethal effects (and other effects) of acetonitrile were attributable to the generation of cyanide as a metabolite. A third example, addressed further in the response to the next question, is that potential effects of acetonitrile on thyroid function was not adequately discussed and evaluated in this review.

The data appear to clearly estab lish that acetonitrile produced chromosome aberrations and aneuploidy in several test systems. The document treats these as "only marginal effects", which is a prejudicial editorial comment made without discussing why the data were marginal. The
sentence talks about "potential" of acetonitrile being demonstrated. If the results were statistically significant, one should conclude that it produced the effect. It is entirely possible that these data can be dismissed by a more thorough discussion of the dat a, including questions of consistency among tests measuring the same endpoint, that is, comparing the doses used in positive vs. negative effects in tests measuring the same endpoint. That discussion should come to a judgment based on the data as a whole. As this section now stands, it comes to no conclusions and largely leaves the issue open.

The metabolism and pharmacokinetic data on acetonitrile and its metabolites are limited, but there are sufficient details available to allow more defensible conclusions to be made related whether mortality is the key event. Some of elements of this discus sion are provided on page 29 of the document, but the discussion of the data is interspersed with a series of speculative comments that detract from the proper use of these data (e.g. "overwhelming enzymatic detoxifying mechanisms" and "reconversion of thi ocyanate to cyanide"). These statements should not be made without specifically discussing how those two things might occur or have been shown to occur. I would strongly suggest the inclusion of a diagram that lays out the metabolism of acetonitrile to c yanide and thiocyanate. In that diagram, all the important routes of elimination of acetonitrile and these two metabolites should be estimated. That will mean some exploration of the cyanide and thiocyanate literature to obtain the important quantitative data (hopefully in humans as well as rats and/or mice).

As pointed out, the half

- life of acetonitrile in humans poisoned with acetonitrile was 32 hours while that of cyanide is about 15 hours. Therefore, the slow conversion of acetonitrile will not all ow substantive amounts of cyanide to accumulate at low doses. Thus, it would appear that the "detoxification mechanism" alluded to is rhodanese conversion of cyanide to thiocyanate. The authors need to explore the kinetics of this latter conversion to de termine what systemic concentrations of cyanide will begin to saturate this enzyme. Until that occurs, there will not be a sharp accumulation of cyanide. The idea that the reconversion of thiocyanate back to cyanide can contribute to this build

- up also n eeds to be supported. As written, the implication is that the elimination of thiocyanate is slower than that of cyanide, but limited quantitative data are provided to support this contention (i.e. simply stated that thiocyanate is not entirely eliminated between doses). This needs to be confirmed, not simply be a suggestion by an author (line 1, page 30). To get accumulation cyanide, it needs to be established from the literature that this is the main route of cyanide elimination, and that the relative r ate of cyanide generation is more rapid than its elimination by rhodanese. One way to slow this conversion is to build up levels of thiocyanate. However, that is simply a question approaching equilibrium concentrations of cyanide and thiocyanate in the s ystemic circulation. It does not mean that there is a net conversion of thiocyanate conversion back to cyanide, but that they are at equilibrium (or more properly in a steady state that is controlled by the rate of thiocyanate elimination).External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 4 The question of whether acetonitrile would be likely to produce inhibition of iodide uptake and thus decrease thyroid hormone synthesis will depend upon the systemic concentrations at which this steady

- state establishes itself with continued treatment.

2. The U.S. EPA has determined that mortality is the critical endpoint for acetonitrile as described in the IRIS “Toxicological Review of Acetonitrile” (1999). Mortality has been observed in a 13

- week subchronic study as well as in developmental and reproductive toxicity studies. Mortality was not observed in a 2

- year chronic study conducted at doses below those that caused mortality in the 13

- week subchronic study. Based on the totality of available evidence, particularly the 13

- week and 2

- year studies, is it reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure? Please explain.

Comments:

The descriptive data do suggest that a few adverse effects occur at inhalation doses of acetonitrile below those that induce mortality. Howev er, the document does not adequately explore the occurrence of effects that were anticipated in the subchronic and chronic experiments conducted by the National Toxicology Program. As indicated in the general comments above, the metabolic conversion of ac etonitrile, first to cyanide and then to thiocyanate, was expected to produce effects on thyroid function. Thyroid hormone measurements were made in the 13

- week study and there was some suggestion of the expected effects on thyroid hormones in male and fe male rats at the 1600 ppm dose with a trend in the dose

- response data in lower doses. Moreover, there was evidence of increased C

- cell hyperplasia in the thyroid of female rats at all doses (statistical analysis not explicitly presented). As these were e xpected toxicities of acetonitrile they should have been discussed in the document and a formal assessment made of whether these data were relevant to humans (there are reasons for not considering rat data in where effects on thyroid function. Rats are mu ch more sensitive to such effects than humans). In considering these effects on the thyroid, the Agency should consider whether the available reproductive and developmental toxicity studies were conducted in a fashion that would allow effects of modified thyroid function on development to be evaluated. My review of these papers indicates that no treatments beyond the 20 th gestational day were performed. The critical period for thyroid

- dependent development events within the brain of the rat occurs in the postnatal period. Therefore, I suspect the Agency would ordinarily conclude that this is a critical data need and would modify their assessment to account for the deficiencies in the database.

The only reference to the possibility of thyroid effects is a reference to Hartung et al. (1982) in referring to the effects of thiocyanate. Thiocyanate is known to be a potent inhibitor of iodide transport in humans as well as experimental animals. Therefore, it is critical that the document consider whether the re has been an adequate experimental evaluation of risks that would arise from the generation of this important metabolite that arises in the metabolism of acetonitrile.

The evaluation of toxicities that could be potentially attributed to anti

- thyroid eff ects of the metabolite, thiocyanate, should be discussed and taken to a transparent conclusion in the "Conclusions" section, that is found at the beginning of the document.

III. SPECIFIC OBSERVATIONS

Whole body inhalation

– not sure how this is accomplished? This terminology is usedExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 5 throughout the document and needs to be changed to make the meaning clear . NTP used the term "Respiratory exposure, whole body", which is a more accurate description of what was done. The ma in reason for using this designation is to distinguish these experiments from nose

only exposures.

Page 23, lines 11ff. This statement needs to be supported within the document, not simply stated as "it is possible..."

Page 28, line 14. Thiocyanate doe s interfere with thyroid function. There is no maybe about it.

Page 29, line10

- 11. This states that the conversion of acetonitrile to cyanide "is delayed". That is not what the literature says. Conversion of acetonitrile to cyanide is slow such that it takes some time for toxic amounts to build up. That terminology is used elsewhere in the document, it is inaccurate, and is very confusing. There are data to indicate that coadministration of acetone with acetonitrile delays the formation of cyanide from acetonitrile (Freeman and Hayes, 1985) which is a different matter.

Page 33, lin e 12, line 23. Whole body inhalation. Hard to get the body through the nasal
passage.

Page 35, line 19

- 20. This sentence lacks clarity. Essentially, the lethal effects of cyanide arise from its binding to cytochrome aa 3 . That is a reversible bond, bu t it is fairly slow rate of dissociation. Therefore, if one reaches a systemic concentration of cyanide that binds this cytochrome on a continuous basis for a critical period it will be fatal. Therefore, the threshold is not the body burden of acetonitri le. The threshold is the systemic concentration of cyanide that will produce death.

Page 35, lines 20

- 23. This is nonsense. What is meant that the body can no longer successfully counter the toxic effects of cyanide? What happens is that cytochrome ox idase is inhibited to the point that cellular respiration can no longer accommodate the metabolic needs of critical tissues

– most specifically the brain.

There is also an issue of whether cyanide does not lead to apoptosis in brain cells. If that is

mas sive enough, it could preclude recovery. I recommend that the authors' explore this question in the cyanide literature. It has been used to induce apoptosis in some in vitro systems. I would try to provide references except that would require a far rang ing literature search that I am not being paid to explore.

Page 37, line 21 and 22. Avoid using jingoisms like "adaptive changes." What is meant by this? If the adaptations are not identified, the statement is simply speculation.

Page 39, line 24. Thi s compound sentence does not make sense. I assume it means to say that although there is no two

- generation study, there are data on some reproductive parameters from some developmental studies.

Page 41, lines 10

- 24. This discussion is a critical in to t he attribution of teratogenic effects of acetonitrile to cyanide. Could this be done in a more quantitative fashion? For example, is it possible to estimate the systemic cyanide concentrations that are produced by acetonitrile with those that are produce d by direct treatment with cyanide? This requires some exploration of theExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 6 cyanide literature.

Page 42, lines 14ff. Again, this discussion would be more convincing if a quantitative approach could be taken among different nitriles. Preferably it would b e related to systemic concentrations of cyanide that would be achieved by different treatments. Barring that, one could compare doses in terms of cyanide equivalents (since inhalation doses are essentially in molar terms, it is easier to do with ppm in ai r than with mg/kg per day).

Page 44, lines 7

- 10. This needs to be stated more clearly or at least discussed more fully. The implication is that a microsomal system degrades cyanide that is administered directly, but not the cyanide produced from metabol ism of acetonitrile. That is not a very satisfying dataset considering the emphasis placed in this document on the conversion of acetonitrile to cyanide as accounting for the toxicity of acetonitrile.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 1 APPENDIX C

PEER REVIEW COMM ENTS FROM COMMENTER SIXExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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### Peer Review Comments on Technical Review of Acetonitrile (Methyl Cyanide)

Commenter Six

June 9, 2008

I. GENERAL IMPRESSIONS The overall contents of the Technical Review of Acetonitrile are acceptable. However, after reviewing the document, it was not clear what the overall conclusions were with regards to the status of acetonitrile and reporting requirements of the EPCRA. In addition, several areas either need clarification or additional documentation. Specifically, the review indicates that most of the acetonitrile produced in the United States is released into the environment by deep well injection. The section discussing the potential for migration from on

- site injection wells to groundwater and the persistence of acetonitrile in groundwater should be expanded. The review also indicates that in mammalian systems acetonitrile is metabolized to cyanide (the toxic metabolit e). It is not clear if this pathway does (or does not) exist in ecological receptors. This issue should be clarified. The review also indicates that acetonitrile is “not expected to present a significant concern” for ecological receptors because the acu te toxicity is greater than or equal to 100 mg/L. The justification for using this value as a determinant for minimal toxicity should be included in the text. While it is understood that 100 mg/L is a large dose, this should be explained in relevance to o ther toxins or regulatory standards.

II. RESPONSE TO CHARGE QUESTIONS Chapter Two, Chemistry and Environmental Fate 1. Has an objective (accurate, clear, complete and unbiased) and technically accurate description of acetonitrile been provided in terms of chem ical and physical properties?

Comments: Overall, the chemical and physical properties are clearly described. As stated above, additional details regarding the fate of acetonitrile from underground injection wells would be valuable to the review. Sectio n 2.6.5 Toxic Transformation Products is brief and could be improved by expanding the discussion. Additional editorial comments are included in the section on specific comments (below).

2. Does the report objectively address the fate of the chemical in t he environment including both transport between environmental compartments (atmosphere, aquatic and terrestrial) and transformation (e.g., degradation), including both abiotic and biotic processes where relevant?

Comments: The report objectively addresse s the fate of acetonitrile in soil and groundwater. To a more limited extent, fate and transport in the atmosphere is also included. Since approximately 90% of acetonitrile is disposed of in on

- site injection wells, this section should be expanded. The di scussion should include information regarding the potential for release of contaminants from injection wells into groundwater and the fate and transport of contaminants if they are released into groundwater. Also, the text indicates that 30 million to ns of acetonitrile are burned on

- siteExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 3 each year. Are data available to determine the efficiency of these methods? What levels of acetonitrile may be released into the atmosphere as a result of incomplete combustion (or are their other toxic by

- products of combustion)?

Chapter Four, Ecological Hazard Assessment 1. Has the available information on the ecological toxicity of acetonitrile been objectively described? Are there any additional studies that should be included?

Comments: The information on ecological toxicity has been objectively described. I am not aware of any additional studies that should be included. The text could be more clearly presented, specific comments are provided below.

2. Is the conclusion presented in Section 4.1, “Acetonitri le does not present a concern for adverse effects on the environment” supported by the data/information presented?

Comments: The conclusion appears to be supported with respect to ecological receptors. Supporting information is needed for the statement that “acute toxicity values . . . greater than or equal to 100 mg/L indicating relatively low acute aquatic toxicity.” While I agree with this conclusion, documentation (either by regulatory guidelines or comparison with values for aquatic toxicity for ot her chemicals) is needed. In addition, minimal information exists with regards to terrestrial toxicity, population effects, or general effects on the environment. Thus, the conclusion (in Section 4.1) should be qualified that it primarily addresses ecolo gical receptors in aquatic environments.

III. SPECIFIC OBSERVATIONS

Section 1.2 Conclusions

Pg. 2; Lines 3

- 6: The introductory statement to this section is very long and somewhat confusing. Suggest the following: “The EPA has concluded that it is reasona ble to anticipate mortality in animals chronically exposed to acetonitrile. This conclusion is based on observations of mortality in acute, short

- term and subchronic repeated exposure studies in animals. In addition, acetonitrile has been shown to have a steep dose

- response curve; and, the mode of action for acetonitrile toxicity (i.e., conversion to cyanide) also supports this conclusion.”

In addition, the conclusions should be clearly stated and perhaps bulleted at the introduction of this section. For example:

• Acetonitrile has been found to produce mortality in acute, short

- term and subchronic animal studies;

• Due to it’s mode of action and steep dose

- response curve, it is reasonable to assume that mortality would also occur under chronic conditions ;

• In ecological studies, acetonitrile has been found to have minimal toxicity (i.e., concentrations of greater than 100 mg/L are required to produce mortality in aquatic studies);External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 4 • Acclimated microbes have been found to be capable of rapidly degrading acet onitrile under aerobic conditions; however, under anaerobic conditions or with unacclimated microbes, acetonitrile may persist in the environment.

Pg. 2; Line 9: Revise text to read “. . . oxygen needed for cellular respiration. The lack of oxygen in ce lls leads to cytotoxic anoxia (Albaum et al., 1946).

Pg. 2; Line 12: The text states that “similar clinical signs have been observed in humans”. Are these case reports involving humans exposed to cyanide or acetonitrile? This should be clarified in the text.

Pg. 2; Line 15: Delete “animal” (before repeat

- dose toxicity).

Pg. 2; Line 18: Delete “the” (before 13

- week inhalation studies).

Pg. 2; Line 20: Delete “a delayed period of time (weeks)” and replace with “several weeks.”

Section 1.3 Chemistry and Environmental Fate

Pg. 3; Line 17: The text describing volatilization of acetonitrile from a river should include the parameters (temperature and wind speed) that were used in developing this estimate.

Pg. 3; Line 19: Delete “land” and replace with “soil.”

Pg. 3; Lines 20

- 21: Revise text to read

– “sorption coefficient and high water solubility it is predicted that acetonitrile will migrate rapidly through permeable soils.”

Pg. 4; Line 5: Insert “acetonitrile in the presence of” after “Half

- lives for.”

Pg. 4; Line 7: Revise text to read “for non

- acclimated or anaerobic bacteria” (assuming this is correct?).

Section 1.4 Human Health Hazard Assessment Summary

Pg. 4; Line 19: Revise text to read “oxygen needed for cellular respiration. The redu ction of oxygen levels in cells may result in cytotoxic anoxia”

Pg. 4; Line 20: Insert “in mammalian systems” after “converted to cyanide.”

Pg. 4; Line 23: Delete “then” (before “it may be partially”).

Pg. 4; Line 24: Place a period (.) after Hartung , 1982 citation; delete “resulting” and revise text to read “This process results in a greater retention time for cyanide.”

Pg. 5; Line 1: Delete “occurs” and replace with “does not occur until several.”

Pg. 5; Lines 2

- 5: Delete text “Following repeated . . .metabolize cyanide to thiocyanate.” Replace with “Following repeated exposure to acetonitrile, it is likely that cyanide willExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 5 accumulate in the body due to the generation of cyanide from the existing body burden as well as new exposures to acetonitrile. This accumulation may result in cyanide levels in tissues that exceed the capacity of enzymes to metabolize cyanide to isocyanate.”

Pg. 5; Line 6

- 7: Revise text to read “The accumulation of toxic con centrations of cyanide and the metabolic rate at which lethal cyanide concentrations are reached will largely depend on the balance . . . “

Pg. 6; Line 8: There is almost a 10

- fold difference between mice and rats for LD50. Is there any metabolic differ ence between these organisms that can be attributed to this difference? And, if so, what is the relevance of this difference between rats and mice for acetonitrile toxicity in humans? [Note, it is also interesting that acetonitrile is 10

- fold more toxic in rats; and, male rats appear to be the sensitive species with regards to acetonitrile carcinogenicity].

Pg. 6; Lines 13

- 14: The text indicates that there is an “overall lack of genotoxic potential” for acetonitrile in cell culture bioassays. This may be an incorrect conclusion. Typically, negative results in cell culture bioassays reflect an insensitivity of the assay more than a potential lack of response in mammalian systems. Acetonitrile did produce a positive response in several cell culture bioa ssays (SCE in CHO cells, gene conversions in S. cerevisiae , chromosome aberrations in CHO cells). This should be more clearly stated in the summary.

Pg. 7; Lines 20

- 21: Revise text to read “This conclusion is based on the observation that the minimum ac ute toxicity values for aquatic organisms is greater than or equal to 100 mg/L. In comparison with other chemicals, acetonitrile has a relatively low aquatic toxicity.”

Pg. 8; Line 4: Delete “experimental.”

2. CHEMISTRY AND ENVIRONMENTAL FATE

2.1 SUMMAR Y

Pg. 10; Lines 13

- 21: Information included in the technical summary should be included in this section; and, the text on production values should be revised to match the technical summary (which is more clear). I suggest the following revisions:

“Acet onitrile is produced as a by

- product of acrylonitrile production. The principal uses of acetonitrile include: as a solvent in hydrocarbon extraction processes, as a specialty solvent, as a reaction solvent in the production of synthetic pharmaceuticals, a s a solvent for the extraction of fatty acids, and as a solvent in analytical instrumentation (Lewis 2001; Budavari et al., 1996; WHO 1993). The estimated US production of acetonitrile has varied over the past decade between 10 million and 100 million pou nds per year (US EPA, 2002). Of the millions of pounds produced each year, a commercial market exists for only half of the inventory. The US production capacity for acetonitrile in 1994 was estimated to be 46 million pounds (McKinney and DeVito 1998).”

Pg. 10; Lines 24

- 25: The text indicates that 30 million pounds of acetonitrile were burned on

site for energy production. Are there any estimates of the efficiency of the combustion procedures? Or, how much acetonitrile (or other combustion by

- products) may be released by this process?External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 6 Pg. 10; Line 26: Insert “most of” before “the chemical will volatilize.”

Pg. 11; Line 1: Insert “predicted to be” before “the environmental compartment.”

Pg. 11; Line 2: Delete “distribute” and replace with “partition.”

Pg. 11; Line 12: Insert “from” before “weeks to months.”

Pg. 12; Line 12: Insert “an” before “oxidation catalyst.”

Pg. 12; Line 15

- 16: This reaction should be shown in it’s complete form (or as stated in the text the reaction produces ac rylonitrile with acetonitrile and hydrogen cyanide as principal by

products).

Pg. 14; Line 14: Revise text to read “The production and wide use of acetonitrile as a solvent.”

Pg. 15; Line 2: What temperature was used in estimating the half

- life in a mo del river?

Pg. 16; Lines 1

- 2: Rather than indicate hydrolysis is “extremely slow”, can a half

- life be provided and products of abiotic hydrolysis identified?

Pg. 16; Line 13: Insert “enzyme” before “transforms the nitriles.”

Pg. 16; Lines 19

- 20: Rev ise text to read “Activated sludge and sewage have been found to contain populations of mixed microorganisms that are capable of transforming organic and inorganic substances into less toxic forms.”

Pg. 16; Lines 22

- 23: Delete “degraded with” and replace with “exhibited”, and delete “More dated studies” and replace with “Several older studies have.”

Pgs. 16

- 17; Lines 24

- 1: Revise text to read “proceeded slowly with unacclimated microorganisms.”

Pg. 17; Lines4

- 5: Delete “develop a strain” and replace w ith “enrich populations”, and on following line, revise text to read “cultures achieved a rate of degradation of 58% for acetonitrile in water in 5 days (BOD test).”

Pg. 17; Line 8: Insert “concentrations ranging from” before “0.1

- 25 mg/L.”

Pg. 17; Lines 23

- 24: Revise text to read “anaerobic degradation is roughly four times slower than aerobic degradation,” and on following line, delete “beyond aerated” and replace with “under anoxic conditions.”

Pg. 18; Line 1: Revise text to read “Due to large differences in temperature, pressure, and other conditions these estimated degradation rates for acetonitrile cannot be applied to underground injection.”

Pg. 18; Lines 4

- 5: Because such a large volume of the waste product is disposed in these wells,External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 7 t his section should be expanded to discuss more specifically the solubility of acetonitrile and the potential for migration from the injection zone to aquifers. It may be adequate to indicate that since acetonitrile is highly soluble in water any material discharged into an aquifer from deep

well injection is likely to be transported with groundwater migration. It should also be indicated that the environmental conditions (i.e., temperature, nutrient concentration and microbial populations) in groundwater are not conducive to the degradation of any material that does reach the aquifer.

Pg. 18; Lines 19

- 20: It is preferable to use the same units in discussing TRI and injection, suggest that the TRI release for 2003 be rounded off to 20.8 million pounds.

Pg. 18; Lines 21

- 23: Revise text to read “It is anticipated that the rate of acetonitrile degradation following release from an underground injection well will be very slow. Chemical concentrations from an underground release are likely to be relatively high; and, anaerobic or oxygen limiting conditions are likely to exist. Optimal conditions for acetonitrile degradation are generally assumed to require aerobic conditions and relatively low chemical concentrations.”

Pg. 19; Section 2.6.5 Toxic Transfor mation Products: Additional explanation is needed for this statement. Are these transformation products from aerobic degradation? Are there no pathways which would result in the release of cyanide to the environment?

3. HUMAN HEALTH HAZARD ASSESSMENT (no t reviewed)

4. ECOLOGICAL HAZARD ASSESSMENT

Pg. 51; Line 1: Revise text to read “Acetonitrile is not expected to present a significant risk for adverse effects in ecological receptors.”

Pg. 51; Line 11: Delete “ranged” and replace with “are also relati vely low, ranging from 160 to 7300 mg/L.”

Pg. 51; Line 24: Revise text to read “found for acetonitrile. Limited data . . .”

Table 4.2

– You would have to assume if you have footnotes for a table from “a

- v” that something is not quite right! This table is very difficult to follow or interpret. I believe it would be easier to follow if broken into Table 4.2, Acute Toxicity and Table 4.3 Chronic Toxicity.

Also, since the conclusions rely heavily on predicted values, it would help the reader if some

e xample of predictability were provided; i.e., compare toxicity values derived under experimental conditions with those predicted using the ECOSAR model. While it is assumed that the model provides a conservative estimate of toxicity values, this should be demonstrated in the text.

Finally, the conclusions for ecological receptors would be easier to follow if a summary table were included that provided ranges of values for acute, chronic, aquatic, and terrestrial toxicity.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 1 APPENDIX D

PEER REVIEW COMMENTS FROM COMMENTER SEVENExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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### Peer Review Comments on Technical Review of Acetonitrile (Methyl Cyanide)

Commenter Seven , 22 May 2008

I. GENERAL IMPRESSIONS

Provide overall impressions (approximately 1/2 page in length) addressing the accuracy of information presented, clarity of presentation, and soundness of conclusions.

• The overall impression is of an adequate summary of available information on the physicochemical and toxicological behavior of methyl cyanide. There are, however, some unanswered questions that make it difficult to use this document to fully judge this chemical’s potential to impact humans or the environment and where any such impacts may occur. The description of biotic and abiotic degradatio n processes is not clear whether these processes differ in different media or what degradation byproducts might be expected in different media (if any). Use of the EPIWIN model to assess the distribution of methyl cyanide following release was good but, m ore discussion of its transport and fate in groundwater (an important disposal media) is needed. The review of what is known about the ecological hazards posed by this chemical suffers from the lack of available information on chronic effects or on popula tion

- level responses.

II. RESPONSE TO CHARGE QUESTIONS

Provide narrative responses to each of the four charge questions below.

• I found it awkward to separate narrative responses from specific observations for each chapter, so both are presented together in the preceding sections.

Chapter One, Integrated Technical Summary

Comments:

• The charge cover letter also requested comments and observations on the relevant sections of Chapter One, the Integrated Technical Summary.

• Page 2, Section 1.2. Th is section appears to focus exclusively on potential adverse health outcomes in humans, while there is a separate section (Section 1.5) for potential ecological impacts. It would be better to have one comprehensive conclusion section, encompassing both hu man and ecological effects, for the whole report, rather than leave the impression that its conclusions are based exclusively on human impacts.

• Page 7, Lines 8

- 9. It is not clear how you can assert that this chemical does not pose a concern for the envir onment without some knowledge of its levels in the environment? If there is information on known environmental levels, it does not appear to have been presented in this report.

• Page 7, Lines 19

- 20. The information presented on chronic ecological outcome s is not robust enough to support the statement that methyl cyanide “...does not pose a concern for ... adverse effects on the environment...” The statement “This conclusion is based on ...” withExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 3 reference to acute effects does not follow from the previou s sentence. It is also questionable whether acute outcomes are a reasonable basis for conclusions about chronic outcomes. Chronic ecological outcomes could be of importance in instances where, for example, groundwater leakage to surface water provides a continual, low

- level source of methyl cyanide to aquatic receptors (c.f., Page 15, Line 13 and Page 18, Lines 4

- 5).

Chapter Two, Chemistry and Environmental Fate

1. Has an objective (accurate, clear, complete, and unbiased) and technically accurate description of acetonitrile been provided in terms of chemical and physical properties?

Comments:

• This chapter appears accurate, relatively clear, and unbiased with respect to the physicochemical properties of methyl cyanide. Given their importance in e stimating transport and fate, corroborating references (if available) for the values of KOW, Henry’s Law, and hydroxyl reaction would be helpful (some are given in the text but not in the table).

• Page 53, Table 4

- 1. This table reproduces many of the values cited in Table 2

- 1 but with more complete source citations. It appears possible to combine these tables into Table 2

- 1 and simply cite this table in Chapter 4.

2. Does the report objectively address the fate of the chemical in the environment incl uding both transport between environmental compartments (atmosphere, aquatic and terrestrial) and transformation (e.g., degradation), including both abiotic and biotic processes where relevant?

Comments:

• Page 10, line 27. The volatilization pathway woul d only be accessible to methyl cyanide disposed of on the soil or water surface. Yet it appears (from Lines 22

- 25) that the bulk of releases are to groundwater or to air following combustion. Is there any information on methyl cyanide’s transport and fat e in groundwater? It is there any information on whether the combustion of methyl cyanide produces toxic by

- products? (I would think not but some information to this effect, if available, would be helpful.)

• Page 11, Line 2 et seq . What are the degradati on byproducts of methyl cyanide? Do these byproducts differ with respect to media and degradation pathway? If such information is available, it would helpful in assessing the nature and extent of any problems caused by methyl cyanide’s breakdown in the e nvironment. It would be helpful to summarize what is known about degradation by pathway and byproduct into a simple table.

• Page 14, Section 2.6.1. Given that large amounts of methyl cyanide are disposed of via underground injection, the seeming lack of information on its transport and fate in groundwater is troublesome. There are scattered mentions (e.g., Page 11, Line 8 and Page 15, Line 13) of its potential to contaminate groundwater but no detailed exposition of this process or its consequences. Eve n if such information is not available, some mention should be made of groundwater as a separate media and of methyl cyanide’s possible transport and fate in this media.

• Page 14, Line 15. Please specify these “various” waste streams.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 4 • Page 15, Section 2. 6.2. Except for its cryptic last sentence (Page 16, Line 1), this section makes it appear that abiotic degradation only occurs in air. Is this true? And what are the presumed byproducts of such degradation?

• Page 16, Line 11. The “...most significant d egradation process...” Please clarify whether this refers to all such processes or just the biotic?

• Page 16, Line 22. The compound is degraded into what?

• Page 17, Line 15. Please define anaerobic “consortium.” There is also no mention between Lines 1 4 and 23 of the environmental media in which these experiments were conducted.

• Page 17, Line 17. When you say anaerobic degradation results can be “applied” to groundwater, do you mean by extrapolation of tests in other media or because test were actually conducted in groundwater? Given (per Page 18, Lines 3

- 4) that groundwater is a source of methyl cyanide contamination, it would seem important to provide as much information as is available about its behavior in this media.

• Page 18, Section 2.6. 4. Using EPIWIN v 3.20 and the physicochemical parameters listed in Table 2

- 1, I was able to reproduce the half

- life, but not the mass amount, estimates given in Table 2

- 2. For example, my estimate for air was 11.8% rather than the 51.7% given in Table 2

- 2. Either an error was made or not all of the assumptions used to produce 51.7% and the other mass amount values were clearly stated. Given the importance of the EPIWIN results to understanding the fate of this chemical, all of the inputs/assumptions us ed for this model should be disclosed and listed (possibly in a separate appendix), that replication of results is more straightforward.

• Page 18, Line 9. The EPIWIN model can release 1000 kg/hr to all three compartments simultaneously or one at a time. Which of these release scenarios was followed? Was an investigation made of what affect releasing to only one compartment at a time would have on the environmental distribution of methyl cyanide? In other words, are all release points equal or do they pr oduce differing outcomes or is Table 2

- 2 always the same regardless of release point?

• Page 18, Lines 18

- 21. These are redundant with Page 10, Lines 22

- 25.

• Page 18, Line 10

- 11. This sentence is unclear. Please clarify whether these estimated half

lives those estimated internally by EPIWIN or user

- entered?

• Page 18, Lines 16

- 24. The vast majority of releases occur in injection wells, which have shown the potential to contaminate groundwater. Yet the EPIWIN model does not include groundwater as one of i ts environmental compartments. As has been said previously, this leaves open the question of the transport and eventual fate of methyl cyanide in groundwater.

• Page 18, Line 22. Is there a reference for the statement “...can be anticipated to be signific antly slower...”?

• Page 19, Line 8 lists the toxic byproducts as acetic acid and ammonia

- is this always the case? What about cyanide? Chapter Four, Ecological Hazard AssessmentExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 5 1. Has the available information on the ecological toxicity of acetonitri le been objectively described? Are there any additional studies that should be included?

Comments:

• There are two concerns with this chapter: (a) an exclusive focus on lower trophic level aquatic and terrestrial species with no discussion of terrestrial vertebrates and (b) the lack of robust available information on the chronic toxicity of methyl cyanide, particularly in vertebrate species.

• Ecological receptors include more than plants, invertebrates, and fish. Was there no information available on methyl cyanide effects in terrestrial vertebrates? If this is the case, it would be helpful to make a clear statement to this effect in Section 4.1.

• Although somewhat problematic, would it not have been possible to draw some tentative conclusions about t errestrial vertebrate responses in the wild from the rat and mouse laboratory data presented in Chapter 3?

• Page 51, Line 15. Please add “or biomagnify” after “bioaccumulate” to distinguish these two different processes. Also on Page 57, Line 11.

• Page 5 1, Section 4.1. There is no mention of whether there is any information available on the mechanism of toxicity of methyl cyanide in various ecological receptors. If no information is available, then it would be helpful to make a clear statement to this e ffect in Section 4.1. Couldn’t what is known in this regard about cyanide be used to make some informed judgments about methyl cyanide?

• Page 53. Separate tables for acute and chronic data would be helpful.

• Page 53, Table 4

- 2. It is not clear whether f ootnotes “b” and “c” apply to all or just selected studies listed [It would also help if the font size of the footnotes was increased to, say, 10 pt]. Also, is the “d” in the “Value” column supposed to be a footnote?

• Page 55, Section 4.2.2. The chronic toxicity testing was performed on embryos of a non

standard, fairly robust, test species. It has been shown (for cyanide) that juveniles and adults, and not embryos, are the more sensitive life stages. The text also notes several other weaknesses in the very limited studies on chronic toxicity in fish. The ECOSAR estimated chronic values are lower by a factor of 2 to 10 than those from these deficient studies.

2. Is the conclusion presented in section 4.1, “Acetonitrile does not present a concern for ad verse effects on the environment” supported by the data/information presented?

Comments:

• No, for two primary reasons. First, it does not appear possible to determine if there is a concern without some information on typical environmental concentrations f or comparison with these toxicity values. Second, the already noted weaknesses in chronic toxicity information make it particularly difficult to assess population

- level effects from, for example, impairment of reproductive ability due to chronic exposure s to low levels (e.g., from a groundwater seep to surface water).External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 6 • All of this suggests that available information is insufficient to make unequivocal statements about the chronic toxicity of methyl cyanide. This also limits this technical document’s abil ity to support broad, supportable statements about a lack of concern. Because what is of “concern” is typically largely a policy determination, it is not clear why a technical review has to make this determination?



### III. SPECIFIC OBSERVATIONS

Provide specific observations, corrections, or comments on the document, mentioning page, paragraph, and/or line number.

• Specific comments and observations are combined above under the relevant chapter.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 1 APPENDIX E

PEER REVIEW COMMENTS FROM COMMENTER THREEExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 2 

### Peer Review Comments on Technical Review of Acetonitrile (Methyl Cyanide)

Commenter Three May 25, 2008

I. GENERAL IMPRESSIONS

This Technical Review of Acetonitrile is a well written document and organized such that information necessary to address this reviewer’s charge question was readily accessed. Statements summarizing cogent information from primary resources are clearly written and accurately represent releva nt study results with minimal extraneous detail. The conclusion reached by the reviewers that mortality can be reasonable anticipated as an effect of chronic acetonitrile exposure is based upon application of generally accepted principles of toxicology. This conclusion is, in this reviewer’s opinion, unquestionably sound for rodents in standard test protocols. Extrapolation of this conclusion to humans is not as definitively supported since similarity of mode of action of lethality from acute and chronic exposures is not evidenced with review of direct experimental data. Instead, the review presents a persuasive argument for human relevance based upon similar acetonitrile toxicokinetics, slow rate of metabolism to reactive metabolite cyanide and recyclin g of detoxication product thiocyanat e in both animals and humans.

The reviewers have made a compelling case for acceptance of mortality as a toxic endpoint of chronic exposure for this chemical. Since CNS regulation of respiration is so critical for viab ility of the organism, it is feasible that its dysfunction could cause rapid onset of mortality in the apparent absence of preceding clinical symptoms and histopathological lesions detectable with techniques typical of standard toxicology test protocols. The argument that mortality is not an endpoint of chronic toxicity is based upon an inherent contradiction imposed operationally by the design guidelines for chronic studies specifying avoidance of lethality as a criterion for dose selection. An exception seems appropriate in this case.

One citation error and one study summary statement that could be misleading were found in the review; these are detailed in section III, items a and b, of these comments. Also, I would recommend o n first occurrence of des cription of the cyanide molecular defect (p. 2, line 5) , use of terminology “ chemical reduction of oxygen” to distinguish from “reduction” meaning loss of quantity. Also, semicolons and commas are used inconsistently to separate items of a series in the l ast column of Table 3.1.

II. RESPONSE TO CHARGE QUESTIONS

1. Has the available information supporting the hazard assessment of acetonitrile been objectively described? Are there any additional studies that should be included? A comprehensive review of studies pertinent to the hazard assessment of acetonitrile has been presented. The Technical Review includes all relevant studies on health effects of acetonitrile that this reviewer identified through independent searching of NTP, EPA and Medline databas es. Results are summarized objectively except with a possible misleading context for one mention of the Ahmed et al 1992 study that I have detailed in Section III, item b.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 3 No additional studies were identified that would impact on my overall opinion. Ho wever, I believe addition information on clinical symptoms from the reviewed studies should be presented and explicitly used to provide some evidence for equivalence of mode of action of lethality from acute, short term and subchronic exposure to acetonitr ile. The rationale for calling for this additional material is detailed below in paragraph 4 of Section II.2. Summary of clinical symptoms from the Argus 1984 study, however, should be qualified by disclosure of the Study Director’s (Dr. A.M Hoberman) o bservation, expressed in the Apr 1, 2002 to Mr.D.E. Strother, that gastrointestinal irritation and decreased food consumption were coincident adverse effects.

The Technical Review is weighted in presenting support for the concept that a biological dose of reactive metabolite cyanide at target tissue can be achieved with low dose repeat exposure of acetonitrile that is equivalent to that associated with high dose acute exposure causal to lethality. This argument is made based upon acetonitrile metabolis m, rates of conversion and clearance/recycling of toxic metabolite cyanide and cumulative functional effects. Brief mention should be given to alternative or complementary mechanisms, such as expansion of the comment

on thyroid toxicity from thiocyan a te ( p. 31, lines 12

– 15). If the authors believe these mechanisms are not relevant, their reasoning should be given.

2. The U.S. EPA has determined that mortality is the critical endpoint for acetonitrile as described in the IRIS “Toxicological Review of Ac etonitrile” (1999). Mortality has been observed in a 13

- week subchronic study as well as in developmental and reproductive toxicity studies. Mortality was not observed in a 2

- year chronic study conducted at doses below those that caused mortality in the 13

- week subchronic study. Based on the totality of available evidence, particularly the 13

- week and 2

- year studies, is it reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure? Please explain.

This reviewer believes that the available science supports the conclusion that acetonitrile can be reasonably anticipated to cause mortality as a result of chronic exposure. This opinion is based upon interpretation that mortalities in rodents observed after repeat exposures du ring the subchronic 1996 NTP study occurred as a consequence of cumulative adverse effect of the chemical, rather than as an isolated, immediate response to one of the latter doses. Hence, it is reasonable to project that the same qualitative adverse effe cts would continue to accrue with additional dose applications upon extension of the exposure period. This would manifest as additional, later deaths in a chronic protocol at the lower effective dose level of the subchronic study. Lack of direct determin ation of this anticipated result is a consequence of failure to include the subchronic LOAEL (400 ppm for mice; 800 ppm for rats) in the chronic study; absence of mortality with a 2

- fold lower dose does not preclude mortality occurring at the subchronic LO AEL. Definitive evidence that mode of action for acetonitrile lethality upon acute exposure is equivalent for test animals and humans suggests relevance to irreversible chronic health effects to humans; however, confidence in this opinion is lessened by la ck of direct evidence that the same mode of action mediates lethality from repeat dose, short term or

s ubchronic exposure to acetonitr ile in rodents as from high dose acute exposure to acetonitrile.

In isolation, one data set consistent with the petition er’s interpretation is the high dose mouse mortality results of the 1996 NTP study. Death at 3 wk (Table 3

- 2) from an immediate lethal response to an isolated latter dose is feasible since the test dose yielding >50% deaths (6 hr, 1600 ppm) approximates t he lowest reported, historical inhalation LC 50 (1

- 2 hr, 2300 ppm; p. 6, line 6). However, this reviewer is unaware of any examples of independent factors (eg., age,External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 4 physiological hormonal status) of such rapid onset that would sensitize both male and fema le mice to a coincident, single interim toxicant dose of a repeat dose protocol. The occurrence of deaths in the next lower dose at a later times (>5 wk) would require that this spurious sensitization occurred randomly and repeatedly over a period of week s during the trial, which seems unreasonable. The rat subchronic inhalation data do not support independent, immediate lethality to one of the interim doses since test doses with ~50% lethality a re 10

- fold less than the historical LC 50 . Rather, the rat r esults suggest attainment of the same qualitative adverse effect (lethality) upon multiple administrations of fractionated doses of a total, similar to what is done in radiation therapy to kill cancer cells

.
Another important issue for CERCLA listing is r elevance of rodent mortality from chronic acetonitrile exposure to humans. Extrapolation of rodent toxicities to humans is best supported by demonstrating equivalent mechanisms. It is clear from observations taken from emergency medicine that acute aceto nitrile poisoning in humans occurs via the same mechanism as acute animal lethality, i.e., through cytochrome P450

- dependent production of cyanide and consequential inhibition of mitochondrial cytochrome c oxidase, especially that of central nervous system (CNS) respiratory control centers. The Technical Review suggests (p 5, line 2), that repeat exposure to acetonitrile will result in “continued generation of cyanide from the existing body burden of acetonitrile, coupled with the cyanide generated from ne w exposures” such that detoxication capacity for cyanide will be exceeded. The implication is that toxic effects from repeat dose mediated through the same mode of action, i.e., through cyanide, as acute effects. If so, relevance to humans is highly feas ible. However, there is no direct experimental evidence presented to support equivalent mode of action for lethality from both exposure protocols. Information on blood and tissue cyanide levels after repeated acetonitrile doses associated with lethality w ould strengthen the conclusion that biologically equivalent doses of reactive metabolite occurs with both acetonitrile exposure protocols. Comparable inhibition of target tissue (brain) cytoch r ome oxidase of rodents from high

- dose acute and lower, repeat

- dose acetonitrile exposures would indicate an equivalent molecular mechanism. In contrast, the 1996 NTP report (p. 18) describes a decrease in blood cyanide concentration with time in a 1994 NTP inhalation developmental study (cited in the Technical Revie w as Mast et al

., 1994) from GD8 to GD18 and suggests induction of rhodanese may be responsible. If so, thiocyanate, which has known thyroid effects, would be expected to increase. A reduction in serum T 3 was reported in high dose rats of the 13

- wk study . Also, one lesion characteristic of low

- dose, chronic cyanide exposure, demyelination, was not reported in the 13

- wk histopathology of the 1996 NTP report (p. 32) although brain was included in the tissues examined. Collectively, these data gaps and add itional observations cause this reviewer to question whether lethality from low repeat

- dose exposures occurs through the same mode of action as high

- dose acute acetonitrile poisoning and to be less confident about extrapolation of mortality from chronic ac etonitrile exposure to humans.

The technical reviewers support similar mode of action of acetonitrile lethality in test animals vs. humans by comparing clinical symptoms (ataxia, convulsions and abnormal posture; p, 2, line 11). This same sentence, however, is supportive of similar mod es of action of lethality from acute and repeat dose exposures, but the information is not apparent because it is unspecified that the comparison is between repeat dose animals (NTP 1996; p. 32, line 2) and acute poisonings of humans. Although these sympt oms are nonspecific, a similar approach could be taken to provide some support for similarity of mode of action of lethality from acute vs. repeat dose acetonitrile exposure in animals. For example, Wilhite ( 1981 ) describe s clinical symptoms of acuteExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 5 acet onitrile toxicity that include neurotoxic effects of tremors and convulsions. Such clinical symptoms of acute toxicity could be added to section 3.4.1. Similarly, clinical symptoms of the affected dams in the repeat dose, short term rabbit study (A r gus 1 984, p IV

- 2) include ataxia, decreased motor activity and loss of righting reflex could be mentioned as qualified with Study Director Hoberman’s observation, expressed in the Apr 1, 2002 to Mr. D.E. Strother, that gastrointestinal irritation and decreased food consumption were coincident adverse effects. Although not as strong as a demonstration of equivalent biological dose at the target, explicit comparison of similar clinical symptoms indicating neurotoxicity observed in the acute, short term and subchr onic studies should be presented to provide some support for same mode of action ( i.e., cyanide inhibition of CNS cytochrome oxidase) of lethality in the three protocols.

The technical re view has very convincingly presented evidence indicating that the ef fect of any one interim daily dose of a repeat dose protocol is likely augmented by residual effects from remaining fractions of uncleared acetonitrile from the preceding one or two days’ administrations. Summarized information also makes a strong argumen t that a nearly continuous production of cyanide ensues. However, it has not been demonstrated that the accumulated tissue dose of cyanide can approach levels of disposition associated with high dose acute exposure causing lethality. Alternately, lethali ty from adverse effects of repeat dose can also result from cumulative, irreversible damage to a critical molecular/cellular target such that the summation of damage eventually exceeds a threshold for non

- viability of the organism. Lack of lethality in th e 2

- yr bioassay at 50% of the effective subchronic dose suggests that a threshold for the accrual of these lesions exists. Continued cyanide production at lower levels after termination of daily acetonitrile exposure may contribute to the irreversibility of lesion s by inhibiting tissue repair. Cellular anoxia from cyanide inhibition of cytochrome oxidase could have lasting effects subsequent to toxicant clearance analogous to the oxidative damage that occurs upon reoxygenation of a previously hypoxic tiss ue. These collective effects may or may not be equivalent mechanistically to that contributing to lethality upon high dose acute exposure. However, if accru al of functional molecular/cellular defects is relevant, it is unclear why some evidence of thei r presence was not detected histopathologically. Perhaps detection would require sample collection from nonmorbid animals, since target tissue (brain) is prone to artifacts upon death and delayed immersion fixation. Histopathology of the 13

- wk studies of 1996 NTP study was restricted to animals that died on trial; hence, information from nonmorbid animals was not available.

III. SPECIFIC OBSERVATIONS

a .) p. 28, lines 1

- 2: Incorrect citation to Ahmed ( 1982 ) in reference to human case report. The Ahmed and Farooqi, 1982 paper describes rat oral acetonitrile exposures and immediate tissue distribution of cyanide.

b .) p. 28, lines 3

- 6: Disposition study with 2

- 14 C

- acetonitrile (Ahmed et al

. , 1992). The

summ ary statement of t his study could be misleading. The technical review authors are building the argument that cumulative tissue acetonitrile will be metabolized to cyanide, the mediator of toxicity. One question pertinent to cumulative effects of repeat do se is whether a significant fraction of previous dose(s) of parent acetonitrile is/are retained in target tissue at time of subsequent daily dose. The Technical Review sentence describing Ahmed et al

. ( 1992 ) could be interpreted in this context to support widespread distribution of the parent molecule and tissueExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 6 retention, since time of measurement is not specified. However, techniques used in the Ahmed et al . ( 1992 ) study detected radioactivity due to non

- volatile metabolites from the methyl 14 C of the r adiolabeled acetonitrile. Further, methods used by Ahmed et al. distinguis h ed radioactivity that was covalently bound. Their report includes immediate times after pulse dosing with tracer acetonitrile, but also 24 and 48 h points, which would appear to s upport 48 h retention of acetonitrile. However, this is more likely due to formaldehyde adducts. The wording in the Technical Review is the same as in the EPA (1999) report, where it is constructed to emphasize that disposition in stomach can arise from a systemic source after inhalation exposure. That wording may give incorrect emphasis to the present context.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 1 APPENDIX F

PEER REVIEW COMMENTS FROM COMMENTER EIGHTExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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## Peer Review Comments on Technical Review of Acetonitrile (Methyl Cyanide)

Commenter Eight May 16, 2008

I. GENERAL IMPRESSIONS

The January 15, 2008, Draft Technical Review of Acetonitrile is a very clear and balanced document. It is well

- written with no area lacking clarity and coverage. The only slight confusion that I had was associated with the large amounts of acetonitrile injected underground. Given this major input to the environment and the mention of possible groundwater contamination on page 11, I was left wondering why there was not more discussion of exposure via groundwater. Also the efficiency of the other major disposal method, on

- site incineration, might be described more.

II. RESPONSE TO CHARGE QUESTIONS

Chapter Two. Chemistry and Environmental Fate

1. Has an objective (accurate, cl ear, complete, and unbiased) and technically accurate description of acetonitrile been provided in terms of chemical and physical properties?

Yes. The description is clear and concise.

2. Does the report objectively address the fate of the chemical in the e nvironment including both transport between environmental compartments (atmosphere, aquatic and terrestrial) and transformation (e.g., degradation), including both abiotic and biotic processes where relevant?

Yes. A minor issue has already been mentioned above. Except for brief comments and a few sentences on page 18, there was not much detail about the transport and transformation of acetonitrile in groundwater, a major location for its disposal.

Section 2.6.4. is a sound description of the modeling. A Monte Carlo

- based exercise that included uncertainties would provide more insight about the environmental distribution under a range of conditions or realistic scenarios.

Chapter Four. Ecological Hazard Assessment

1. Has the available information on the ecological toxicity of acetonitrile been objectively described? Are there any additional studies that should be included?

It is my opinion that the ecological toxicity was objectively described. In addition, the earlie r chapter on health effects clearly presented useful insight germane to the ecological effects discussion. A few additional references might be useful. They are given below in Point 13 of III. Specific Observations. It must be noted (Specific Observations 14

- 17 below) that the toxicity data that the EPAExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 3 had available was not high quality information. Given the widespread and high use of this compound, I found this very surprising. However, I did not find much more information during a brief journal search.

2 . Is the conclusion presented in Section 4.1, “Acetonitrile does not present a concern for adverse effects on the environment “ supported by the data/information presented?

Yes. This seems a reasonable conclusion from the facts presented. Acetonitrile does not appear to be very toxic but the toxicity data are not high quality information. Acetonitrile does not have any properties suggesting that it would bioaccumulate to any level of concern. My only minor reservation is described below in Point 2 of I II. Specific Observations. Although the acetonitrile has low toxicity as expressed as effect concentrations, it is released from diverse sources in large amounts. The amounts and widespread nature of its release suggests the need for extra diligence to po ssible ecological effects under various exposure scenarios despite its low toxicity.

III. SPECIFIC OBSERVATIONS

Minor technical suggestions:

1. Page 1, Line 10. Please spell out INEOS at its first use.

2. Page 1, Lines 18

- 25. The goal of an assessment is to sufficiently understand the risk of specified effects to gauge what level of regulatory attention is warranted. Although the other qualities of acetonitrile suggest relatively low risk, the sheer magnitude of its annual release and its introduction undergr ound via injection wells suggests that an elevated level of diligence might be warranted in this decision process. The amounts released from diverse sources increases the probability of an adverse effect relative to an equivalent chemical that is released in small amounts by only a limited number of users. 3. Page 13, Lines 13

- 14. One sentence paragraph. Perhaps this sentence could be moved to the second paragraph of Section 2.1?

4. Page 16, Line 24. Perhaps the word “sluggishly” is too vague for this technical description?

5. Page 17, Line 4. Change “strain” to “culture”?

6. Page 17, Line 23. Change to read “... anaerobic degradation rate is roughly...”?

7. Page 18, Lines 7

- 15. A Monte Carlo

- based model that incorporated uncertainty in estimates would enhance this presen tation.

8. Page 18, Line 20. Change to “... were disposed on

- site in ...”?

9. Page 24, 1 st full paragraph (and elsewhere). A figure might help the reader understand these details better.

10. Page 24, Lines 16

- 18. Consider shifting the clause at the end to the beginni ng of the
sentence.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 4 11. Page 39, Section 3.3.3. The (undefined) power associated with analysis of these data is likely very low because the number of observed exposed females was so low. I wonder about the value of any discussion of these data which might be used to weakly imply low risk.

12. Page 51, Line 6. Here and elsewhere the phrase “the fact that” could easily be omitted.

13. The following references might also be helpful. Chen, C

- Y, S

- L Chen and ER Christensen. 2005. Individual and combined toxicity of

n itriles and aldehydes to Raphidocelis subcapitata. Environ. Toxicol. Chem. 24(5): 1067

- 1073. Chen, C

- Y and C

- F Huang. 1996. Toxicity of organic mixtures containing cyanogenic toxicants. Environ. Toxicol. Chem. 15(9): 1464

- 1469. Grogan, J, SC DeVito , RS Pearlman and KR Korzekwa. 1992 Modeling cyanide release from nitriles: prediction of cytochrome P450 mediated acute nitrile toxicity. Chem. Res. Toxicol. 5: 548

- 552.

14. Issues related to the Tong publications. I found the citation for Tong and Hongi un (2001) to be Zhang, T. and H. Jin. 2001. Aquatic toxicity of acetonitrile and its water quality criteria for the protection of aquatic life in China. Inter. J. Environ. Pollut. 15(5): 568

- 575. Perhaps, the first names might incorrectly be inverted in th e draft EPA report or by the journal editor? The bottom of the first page of the journal article indicates Zhang and Jin is the correct way to cite the article. However, this individual’s other publications suggest that Tong is his proper name.

Relative to Tong and Hongiun (2001), note that, in addition to the shortcomings mentioned in the draft report about this paper, it appears that nominal concentrations were used. Measured concentrations are much better. Also the applied trimmed Spearman

- Karber LC50 estimates are not as good as parametric methods. There are other issues with this publication relative to methods and statistical analyses. So, I agree with the concern stated on page 56, line 9 about these studies.

Relative to Tong et al. (1996), the le ast powerful option, the Steel’s many

- one test was used because the data failed tests for normality. Yet, there is no discussion of conventional transformations that might have resulted in normal data. The associated NOEC/LOEC/ACR values are likely not of sufficient quality to give them much weight in this EPA draft report.

15. There are also statistical compromises in Barahona

- Gomariz et al. (1994) that produce considerable uncertainty in my mind about the estimated effect metrics. The applied methods wer e not very powerful, and power influences the effect metric estimation very much.

16. The Henderson et al. (1961, or 1960?) paper uses a “static with no renewal” design that was standard in the 1960s but would be considered unacceptable today. I suspect that the acetonitrile concentrations dropped from the nominal concentrations during the test and, as a result, the effect metrics are expressed in concentrations higher than what they should be.

17. Generally, the Ewell et al. (1986) paper used a rudimen tary extrapolation to estimate LC50External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 5 values for the tested compounds because they did not have enough partial kills. The lack of high concentration treatments resulted in a <100 mg/L estimate for what seemed to be a static test. Nominal concentrations were used instead of measured concentrations.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 1 APPENDIX G

PEER REVIEW COMMENTS FROM COMMENTER FOURExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 2 Peer Review Comments on Chapter 3 (Human Health Hazard Assessment) and Relevant Sections of Chapter 1 (Integrated Technical Summary) of USEPA’s 15 January 2008 Draft “Technical Review of Acetonitrile (Methyl Cyanide)”

Commenter Four, 10 June 2008

I. Genera l Impressions

Chapter 3 of the draft document is comprised of four main sections: 3.1 Summary, 3.2 Kinetics and Metabolism, 3.3 Effects of Repeated Exposure, and 3.4 Effects of Acute Exposure. These sections are generally well

- written, logically organize d, and easy to follow. They also appear to be reasonably comprehensive in their coverage of the relevant scientific information. In addition to the chapter’s primary focus on mortality as the critical health endpoint in both single and repeat dose studie s, studies of the potential for acetonitrile to cause carcinogenic, genotoxic, reproductive and developmental, as well as neurotoxic effects were identified, reviewed, and summarized.

This reader was left, however, with the distinct impression that certai n data pertaining to acetonitrile pharmacokinetics and mortality were not simply being reported in an objective, unbiased manner, but rather, were being marshaled to support the Agency’s principal assertion regarding acetonitrile that “ it is reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure ”, without appropriate and balanced consideration of the alternatives. While this lack of balance is discussed in greater detail below, I believe this Agency position is altog ether unjustified by the available information. In my opinion, the data presented and summarized in this draft chapter point convincingly toward mortality arising solely from intense (high concentration) acetonitrile exposures of quite short durations (fr om hours to at most a few weeks). Thus, if excessive mortality were to be seen in a chronic study, and it has not, it would be reasonably anticipated to occur during and as a consequence of the first few weeks of acetonitrile exposure. This view is consi stent with what is known of the kinetics of acetonitrile (and cyanide) uptake, distribution, metabolism, and elimination in laboratory animals and humans.

II. Response to Charge Questions

1. “Has the available information supporting the hazard assessment of ace tonitrile been objectively described? Are there any additional studies that should be included?

Most of the information presented in the draft chapter has been objectively described. However, there are specific areas where there seems to be a lack of balance. In Section 3.2, Kinetics and Metabolism, for example, the lower measured affinity for cyanide (higher K m ) of rhodanese from human nasal tissues and lower measured maximal rate (smaller V max ) of cyanide metabolism, relative to that in rat nasal ti ssues, were highlighted as key results from the study by Lewis et al. (1991) (see p 29 lines 1

- 8). A comment follows immediately that humans are thus “less able [than rats] to detoxify cyanide to thiocyanate,” with the implication that humans would thusExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 3 b e at greater risk of cyanide

- induced toxicity.

However, the draft chapter neglects to point out that Lewis et al. were unable to saturate the human rhodanese enzyme despite the use of potassium cyanide concentrations of 500 mM and greater. In fact, Lew is et al. reported that the rhodanese enzyme exhibited activation , not saturation, in one of their samples, consistent with previous work by Jarabak and Westley (1974) (cited in Lewis et al., 1991). This suggests that a simple Michaelis

- Menten model of cy anide metabolism fails to adequately describe the actual dose

- response relationship obtained by Lewis et al., particularly at high concentrations (see their Figure 2)! Such enzyme induction in human nasal tissues and, possibly, liver would also, presumabl y, make humans less sensitive to cyanide exposure than rats at least at sufficiently high concentrations where the rat metabolic response is saturated but the human response is not.

Lewis et al. (1991) also provide highly relevant discussion regarding the high “maximum detoxication” capacity for cyanide metabolism observed in the rat nasal cavity: “This capacity is sufficient to completely metabolize a maximum of 2800 ppm inhaled hydrogen cyanide ...” This high detoxication capacity may explain in part the reduced sensitivity of rats, compared to mice, to cyanide

- induced toxicity that was observed in animals exposed to high acetonitrile concentrations in the NTP subchronic inhalation study (NTP 1996).

Section 3.2 also fails to mention that CYP2E1, the P450 enzyme responsible for the metabolism of acetonitrile to cyanide, is an enzyme that is inducible by a variety of substrates, including ethanol, c.f., Lu and Cederbaum, Free Radic Riol Med 2008, 44(5)723

- 38). This property could lead to significantly higher rates of cyanide production from inhaled acetonitrile, as well as shortening of the half

- life of acetonitrile in the body.

Section 3.2 of the draft ends with two highly speculative par agraphs. They assert that the slow metabolism of acetonitrile to cyanide, species differences in the tissue distribution of rhodanese, and the slow elimination rate of the thiocyanate, at least in some species, when taken together, strongly suggest that ac etonitrile from one day’s dose is not totally eliminated before the next day’s dose in repeat dose studies (in what species?, at what doses?, after how many doses?), leading to an accumulation of both acetonitrile and cyanide “to the point of overwhelming detoxifying mechanisms.” However, I am aware of no direct evidence that this cascade of events occurs during chronic exposure to acetonitrile, thus producing excess mortality as a chronic endpoint, as is stated in Section 3.3 of the draft document.

I w as also surprised by the primarily qualitative nature of the discussion of acetonitrile pharmacokinetics. This is a topic that cries out for quantitative treatment with a PBPK model, yet a brief search of the literature using PubMed failed to identify any recent work in this area. Perhaps this is a significant data gap that needs to be filled with additional research.

2. The USEPA has determined that mortality is the critical endpoint for acetonitrile as described in the IRIS “Toxicological Review of Aceton itrile” (1999). MortalityExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

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- 4 has been observed in a 13

- week subchronic study as well as in developmental and reproductive toxicity studies. Mortality was not observed in a 2

- year chronic study conducted at doses below those that caused mortality in the 13

- w eek subchronic study. Based on the totality of available evidence, particularly the 13

- week and 2

- year studies, is it reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposure? Please explain.

I agree completely with the conclusion that mortality is the critical effect that must be addressed in any risk assessment of acetonitrile. However, in my opinion, this is an effect that arises as a consequence of cyanide

- induced toxicity, which is itself an acute or subacute (a misnomer) effect, i.e., an effect that arises (or not) from just a few days worth of exposure to a sufficiently high acetonitrile concentration. Death may be delayed some, perhaps by at most a few weeks, but the damage which results in death will have been done (or not) in just a few days worth of exposure. Thus, I disagree strongly with the assertion that it is reasonable to anticipate that acetonitrile would cause mortality as a result of chronic exposure.

First and foremost is the fact that survi val was not affected adversely by chronic inhalation exposure of mice (rats) to airborne acetonitrile concentrations up to 200 (400) ppm in the NTP study (1996). This maximum exposure level for mice of 200 ppm acetonitrile, which lasted 6 hours per day, 5 days per week, for up to 2 years, corresponds to a cumulative acetonitrile exposure of approximately 26,071 ppm

- days.

In the related subchronic study (NTP 1996) that was conducted in part to establish the doses for the chronic study, significant mortalit y was observed in mice exposed to 800 ppm acetonitrile for as little as 3 weeks, and all mice exposed to 1600 ppm died within 3 weeks of exposure onset. These exposures correspond to cumulative acetonitrile exposure of only 3,000 and 6000 ppm

- days, respec tively.

Thus, if there were in fact a toxicity mechanism operating that caused mortality as a consequence of cumulative acetonitrile exposure, then increased mortality should have been readily apparent in the NTP chronic oncogenicity study, not only in the mice exposed to 200 ppm (26,071 ppm

- days), but also in the mice exposed to 100 ppm (13,036 ppm

- days) and 50 ppm (6,518 ppm

- days). Yet, there were no effects on clinical signs, body weight, survival, complete histopathology, hematology, or targeted org an weights in any of the exposed groups of either mice (or rats) in the NTP (1996) study. In my opinion, the assertion that acetonitrile should be reasonably anticipated to cause mortality as a result of chronic exposure has no scientific support whatsoev er.

III. Other Comments

In reviewing the public comments provided, I found those submitted by Gradient Corporation (Rhomberg and Goodman) and Dr. Eugene McConnell to be cogent and compelling. I believe that USEPA’s responses to these comments, particularly to the sections of the Gradient comments that dealt with mortality are weak and unconvincing at best.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

G

- 5 There is simply no way that chronic mortality can be demonstrated in subchronic studies or those of shorter duration. There is no evidenc e to support convincingly the assertion that mortality should be a reasonably anticipated consequence of chronic acetonitrile exposure. All of the mortality that has been observed to date in subchronic and shorter duration studies can be explained readily in terms of the acute toxicity that is associated with the metabolism of acetonitrile to cyanide.

Specific Comments

Section 3.1, p 25, lines 9

- 12: The phrase “uncertainty as to the significance of certain results” sounds very mysterious. The specific c oncerns ought to identified explicitly.

Section 3.1, p 29, lines 5

- 6: Lower affinity is the same as higher K m , but lower rate is the same as lower V max . This should be made clear. Also, the assertion that humans are thus les able than rats to detoxify c yanide is speculative. Under what specific conditions, such as high dose, or low dose, might this be true? Can one be certain that rhodanese is not an inducible enzyme, perhaps more inducible in human than rodents?

Section 3.1, p 29, lines 21

- 24: I do not believe that the mentioned points “strongly suggest” anything about accumulation of acetonitrile or cyanide from day to day during repeat exposures. This is a possibility, but so is enzyme induction, which could lead to more rapid metabolism and clear ance.

Section 3.1, p 30, lines 4

- 7: “It is likely” is again too strong a phrase. This is speculation. See previous comment about the possibility of enzyme induction.

Section 3.3.1.2, p 32, lines 15

- 18: Comments of two of the three principal reviewers of the NTP study to the effect that higher doses should have been employed may support EPA’s position, but they make little sense to me. Had NTP tested at the next higher dose from the subchronic study (400 ppm) , they would have seen the same thing that was seen in the subchronic study at that dose, namely, early mortality, which might ultimately have compromised the chronic study’s validity, since a treatment related death was observed in the 400 ppm group on day 13 of the subchronic study.

Section 3.3. 1.4, p 35, lines 8

- 16: The argument that the modest delay in the times of death of some animals in the subchronic study to somewhat beyond two weeks relative to time of first exposure does not imply that the mortality dose

- response relationships for morta lity for short

- term and subchronic exposure are distinct from those for acute exposure duration. I think all it means is that the animals took longer to die than they would have at a higher dose. The proportion dying at a given dose may not have changed at all!External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

H

- 1 APPENDIX H

PEER REVIEW COMMENTS FROM COMMENTER FIVEExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

40 

### External Peer Review of the “Technical Review of Acetonitrile (Methyl

Cyanide)” produced by EPA’s Office of Environmental Information

Prepared for:

Versar, Inc., Springfield, VA.

Prepared by:

Commenter Five

May, 2008External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

40 I. GENERAL IMPRESSIONS

Provide overall impressions (approximately 1/2 page in length) addressing the accuracy of information presented, clarity of presentation, and soundness of conclusions

.
The health assessment chapter is repetitive and does not provide a thorough, integrated hazard characterization with a weight

- of

- evidence analysis. It also is not sufficiently rigorous, co mplete and transparent to meet current scientific standards and regulatory guidance.

Table 3

- 1 is misleading. First, no statistical significance is reported. A single death or even two deaths in a dose group would not be considered an effects level ev en if higher rates of mortality occur at higher doses because the incidence is too low to differ significantly from control rates and could have occurred by chance. Second, in developmental studies, all fetotoxic or reproductively toxic effects occurred a t maternally toxic doses and thus acetonitrile is not a fetal/emybro toxicant. Third, some studies showing no treatment effect reported in the text are not summarized in this table (e.g., Hazelton 1983). I discuss these issues under Charge Questions

.
The conclusions are not supported by the data.

Human and animal data should be presented separately.

Human relevance is inadequately characterized.

II. CHARGE QUESTIONS

1. Has the available information supporting the hazard assessment of acetonitrile been objectively described? Are there any additional studies that should be included?

There is a lack of objectivity in the health hazard assessment. The hypothesis or postulated mode of lethality is presented in the first paragraph of the s ummary. Data are subsequently presented in a manner which supports this position, without providing a thoughtful discussion and integration of all available data and consideration of alternatives.

Although new data are not available, existing data are not framed in accordance with the currently

- accepted standards of hazard characterization. In particular, insufficient information is presented on mode of toxic action and the human relevance of animal studies, and a weight

- of

evidence narrative is incom plete. There is also a reasonable lack of objectivity, as indicated by the almost

- complete focus on mortality with little discussion of other health outcomes (e.g., liver, lung, forestomach, blood), incomplete presentation of toxicokinetics and mode

- of

- ac tion data, and lack of rigor in assessing and interpreting findings from individual studies in accordance with current standards and regulatory guidance.

There is a developmental toxicity study discussed on p. 18 of the NTP (1996) report on the toxicology and carcinogenesis of acetonitrile, in which cyanide and acetonitrile were measured in Sprague

- Dawley rats exposed to acetonitrile at concentrations of 100, 400 or 1200 ppm for 6External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

41 hours/day on gestation days 6

- 19. The only reported effects were “a few” ma ternal deaths at 1200 ppm and one at 400 ppm. Analysis indicated “significant exposure

- related concentrations of acetonitrile in the blood of all exposed groups and the presence of cyanide in the blood of the 1200 ppm group (cyanide was detected at a very low level in one 400 ppm rat at each time point but could not be quantified”

– presumably because it was too low. And: “The concentration of cyanide in the blood of 1200 ppm rats declined from approximately 2 mg/mL on GD 8 to approximately 0.8 mg/mL on G D 18, while acetonitrile concentration remained essentially constant during the same period...........due to induction of rhodanese.” These data do not support the hypothesis that cyanide bioaccumulates and are consistent with other data that also do not indica te bioaccumulation. This study should be added, as should others referenced in WHO/EHC (1993), which address blood levels of acetonitrile, cyanide, and/or thiocyanate.

References which might be useful for a more integrated assessment of acetonitrile toxicity include the following documents.

WHO. 2004. World Health Organization. Concise International Chemical Assessment Document (CICAD): 61. Hydrogen Cyanide and Cy anides: Human Health Aspects. Geneva, Switzerland.

Way J.L. 1984. Cyanide intoxication and its mechanism of antagonism. Ann Rev Pharmacol and Toxicol. 24:451

- 481.

Sylvester, M. and C. Sander. 1990. Immunohistochemical localization of rhodanese. His tochem. J. 22(4):197

- 200.

Melnick, R.L., Roycroft, J.H., Chou, B.J., Regan HA and Miller. RA 1990. Inhalation toxicology of isoprene in F344 rats and B6C3F2 mice. Environ. Health Perspect. 86:93

- 98.

U.S. EPA. 2005. Guidelines for Carcinogen Risk Asse ssment. [ See section on writing a weight

of

- evidence hazard narrative, using a modified set of Bradford Hill (1965) criteria]

Boobis AR

, Doe JE

, Heinrich

- Hirsch B

, Meek ME

, Munn S

, Ruchirawat M

, Schlatter J

, Seed J

, Vickers C . 2008. IPCS framework for analyzing the relevance of a noncancer mode of action for humans. Crit Rev Toxicol. 38(2):87

- 96.

Hill AB. The environment and disease: association or causation? Proc Royal S oc Med 1965; 58:295

- 300.

NAS 2005. Health Effects of Perchlorate Ingestion. National Research Council. Washington, D.C. [ Thyrotoxicity induced by ions which competitively inhibit iodide uptake in the thyroid gland

– as thiocyanate does

– is thoroughly discussed in this report .]

1. Toxicokinetic and mode of toxic action data are incompletely summarized

.
Data which are omitted or inadequately summarized include the following:External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

42 (a). Acetonitrile is more slowly metabolized to cyanide than cyanide salts or some other nitriles (WHO/EHC 1993; Hartung 1982). Therefore, during acetonitrile exposure, the body/tissue burden of acetonitrile is also increased. Both human case reports of poisoned individuals and animal studies show that acetonitrile is distribut ed fairly uniformly throughout the body (EHC 1993).

(b) Acetonitrile is also excreted unchanged in urine and in expired air. Therefore, the clinical and subclinical effects is likely to be a result of the combined effects of the parent molecule and it s cyanide metabolite (Hartung 1982; EHC 1993). Further, animal studies do not indicate that either acetonitrile or cyanide has the potential for bioaccumulation (EHC 1993; ATSDR 1999).

(c) Released cyanide is metabolized to thiocyanate by rhodanese, a sulfur transferase enzyme, which is readily excreted in the urine. Rhodanese is an inducible enzyme and levels of rhodanese increase in response to increasing blood and tissue cyanide concentrations. Sites with the greatest abundance of rhodanese are lo cated to maximize conversion of cyanide to thiocyanate following both oral and inhalation exposure (Sylvester and Sander, 1990 ). Detoxification of cyanide by rhodanese exhibits zero

- order kinetics relative to cyanid e; the concentration of sulfur

- containing donor molecules is the rate

- limiting factor.

(d) The role of metabolism is crucial in determining the acute toxicity of ingested cyanide. Both the total amount administered and the rate of absorption are important (U.S. EPA 1992) because acute toxicity results from rapid ingestion of high doses of cyanide at a rate that exceeds the body’s capacity for metabolism (detoxification). In contrast, s low absorption of the same or greater total oral load of cyanide may allow for complete metabolism. Unmetabolized cyanide can be both acutely and chronically toxic, although cyanide is not known to accumulate in the body.

The principal features of the acute toxicity profile for cyanide are its severe toxicity, including lethality, by all routes of administration, with a steep rate

- dependent dose

– effect curve. The toxic effects of cyanide ion in humans and animals are generally similar and are believed to result from inactivation of cytochrome oxidase and inhibition of cellular respiration during the terminal reaction of the electron transport chain. This prevents the formation of adenosine triphosphate (ATP) via oxidative phosphorylation. Although ac ute oral doses of cyanide cause cardiovascular, respiratory, and neurophysiological changes, the brain appears to be the organ most sensitive to acute cyanide toxicity (WHO 2004). Death from cyanide poisoning is believed to result from central nervous sys tem depression, subsequent to inhibition of brain cytochrome oxidase activity (WHO 2004).

Thiocyanate is not acutely toxic; approximately 60

- 80% of an ingested dose of inorganic cyanide is converted to thiocyanate, the majority of which is rapidly excrete d in the urine. However, elevated long

- term ingested cyanide can result in sustained serum levels of thiocyanate leading to inhibition of iodide uptake at the level of the sodium

- iodide transporter in the thyroid gland, subsequent thyrotoxicity and result ing neurotoxicity (iodide deficiency

- associated neuropathy and mental retardation).External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

43 USEPA’s hazard assessment suggests that: “With repeated exposure to acetonitrile, it is possible that continued generation of cyanide from the existing body burden of acetonitrile, coupled with cyanide generated from new exposures, results in cyanide levels that exceed the capacity of enzymes to metabolize cyanide to thiocyanate.”

However, if this is the case, then thiocyanate levels in both urine and blood would be ex pected to be significantly elevated and abnormalities in the thyroid gland (e.g., goiter) and thyroid hormone homeostasis (i.e., increased TSH and/or T4) would be expected to occur. Chronic exposure to cyanide, resulting in excessive production of thiocy anate, can interfere with iodine uptake by the thyroid gland, leading to enlargement of the thyroid (e.g., goiter) and abnormal levels of thyroid hormones (increased TSH and/or T4). The most prominent health hazard of chronic exposure to elevated levels o f cyanide is thyrotoxicity resulting in neurotoxicity. There is no evidence of either thyroidal or neurotoxic effects that could be attributed to prolonged or repeated cyanide exposure in either the subchronic or chronic NTP acetonitrile studies.

The p otential effects of thiocyanate, which are associated with chronic cyanide toxicity, are not discussed in this assessment. The only thyroid

- related finding was noted in the subchronic rat study by NTP (1996) and was reported as a statistically significant increase in T3 in the absence of any changes in T4 or TSH. This is puzzling. T3 is the bioactive thyroid hormone and is converted from T4 in situ by specific iodinase enzymes (NAS 2005). A change in iodide uptake in the thyroid gland because of the com petitive inhibition of thiocyanate usually results in an increase in TSH and/or a change in T4. A change only in T3 might indicate an increased energy or metabolic tissue demand, as could occur with cyanide

- induced oxygen deprivation, which is met by incr eased conversion of stored T4 to T3.

(e ) The results from both the subchronic and chronic studies do not support the following assumptions :

“With repeated exposure to acetonitrile, it is possible that continued generation of cyanide from the existin g body burden of acetonitrile, coupled with cyanide generated from new exposures, results in cyanide levels that exceed the capacity of enzymes to metabolize cyanide to thiocyanate” and

“As some deaths from acetonitrile did not occur immediately, but inst ead occurred after longer exposure, the results suggest that certain key biologic processes may be gradually overwhelmed or gradually degraded in function such that the body can no longer successfully counter the toxic effects of cyanide generated from ace tonitrile (p. 35).” Based on the findings, the above statements are not biologically plausible scenarios. If there had been cumulative toxicity resulting from repeated

- dosing, then the results should indicate a temporal relationship between effect and exp osure

- duration. If Concentration x Time (C x T) effects are invoked as a possible explanation for the mortality findings, an increase in mortality with increasing exposure duration should have also been observed. This did not occur. The animal deaths i n the subchronic study were concentrated in weeks 1

- 3; a gradual degradation in function or an overwhelming of key biologic processes would predict that mortality wouldExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

44 increase in subsequent exposure weeks, especially in the higher dose groups. Similarly

, mortality would be expected in the 2

- year chronic study at lower concentrations.

It is understandable that USEPA is concerned about possible mortality associated with a cyanide

containing compound because mortality is a very frank, irreversible effect and cyanide has a steep acute concentration

- response curve. Lethality is well known as an acutely toxic response. However, it is the dose rate that determines the acute lethality of cyanide. The weight

- of

evidence shows that a lower dose rate for a lo nger period of time is detoxified and it is only when the detoxification system is acutely overwhelmed that lethality occurs. The data on mortality in the subchronic studies, in conjunction with the significant amount of knowledge on the mode of toxic act ion suggest that the observed findings were due to acute toxicity. In this context, the MTD was exceeded in the higher doses. Repeated dosing (6 hours/day, 5 days per week

– note that it was not continuous) is better construed as a series of acute toxic ity doses with regard to lethality as an end point.

The pattern of responses under very high atmospheric concentrations of acetonitrile for a 6

- hour period is strongly supportive of an acutely toxic response. The clustering of deaths at the beginning of the study, in conjunction with an absence of mortality in the chronic study and in the Hazelton (1983) subchronic studies, indicates that there is no CxT response. What is known about the acute and chronic modes of toxic action of cyanide provide furth er evidence in support of assigning mortality to acute, rather than chronic, toxicity.

With regard to USEPA’s statement that mortality was not observed in the chronic studies because exposure concentrations were significantly lower than those in the sub chronic studies, I note that the acetonitrile concentrations in the chronic study are still orders of magnitude greater than relevant environmental concentrations. This is also a tautological argument.

(f) The pattern of dosing would also predict mortality as an acutely to xic response, not as a chronic one.

The dosing regime was 6 hours/day, 5 days/week for 13 weeks. Thus, animals were not exposed for 18 hours a day. There was also a non

- exposure period of approximately 60 hours over the weekend (Friday evening to Monday morning). Given what is known about cyanide, its toxicity and modes of toxic action and detoxification, cyanide levels would have been significantly reduced during non

- dosing periods. Mortality is thus an acute response. Chronic effects, if they occurred, would be manifested in organ toxicity, such as liver toxicity, and would be associated with repeated oxygen deprivati on in tissues. 2 . Human relevance is not adequately addressed

.
Human relevance is noted in only one sentence and framed as a default statement (i.e., in the absence of data to the contrary, animal studies are assumed to be relevant to humans). The state

- of

- the

- science has progressed significantly since the times when this type of sentence would be considered sufficient for inter

- species extrapolation (see Boobis et al. 2008 cited above). Human relevance topic should be expanded to indicate that th ere are sufficient data to indicate whether there are sufficient data to conclude that the mode of toxic action of acetonitrile and cyanide are qualitatively similar among species, including humans. How well are theExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

45 quantitative differences among species (including absorption and metabolism) described? Further, as implied on p. 36, lines 7

- 10, if effects observed in animals are only likely to occur at very high acetonitrile atmospheric exposures in humans (which are orders of magnitude above environmenta lly relevant concentrations), is there human relevance? 3

. The weight

- of

- evidence narrative does not consider all data and is incomplete

.
This hazard assessment does not meet current quality standards for integration and synthesis of all available data . USEPA (2005) provides an updated framework for a weight

- of

- evidence narrative evaluation, based on the Bradford Hill criteria for causality in epidemiology (Hill 1965).

Briefly, in a weight

- of

- evidence narrative, all data is considered simultaneously a nd evaluated for (1) strength of association; (2) consistency of association; (3) specificity of association; (4) temporality of association; (5) dose

- response or “biological gradient”; (6) biological plausibility; (7) coherence; and (8) other factors.

4. There is a lack of rigor in assessing and interpreting individual study findings

a. The health hazard assessment focuses on mortality and does not give sufficient consideration to other potentially adverse health effects, or lack thereof (see below).

b. No statistical significance evaluation has been conducted on any of the reported studies. As a result, the LOAEL/NOAELs of various studies are overestimates

.
The statistical significance of cancer findings is presented and discussed. A major omissi on is lack of consideration of statistical significance of noncancer findings. Current guidance mandates an evaluation of the statistical significance of all findings.

Dose groups in which one or two animals die are not effect

- levels because as compare d to concurrent (or historical) controls, the increase in mortality would not significantly differ from chance. This increase would not be either statistically or toxicologically significant. Although mortality is a frank effect, the death of one or two a nimals in a dose group is usually considered a

random event which can also occur in control animals, even if there is a significant increase in mortality incidence at higher doses. Therefore, a dose level in which 1 or 2 deaths occur does not constitut e an effects level.

In the NTP (1996) 13

- week rat studies, the mortality incidence for males was 0/10 in each of the 0, 100, 200 and 400 ppm groups, 1/10 in the 800 group (one death occurring in week 1), and 6/10 in the 1600 group (four deaths occurrin g during week 1, one death occurring during week 2, one death occurring during week 4). For females, the mortality incidence was 0/10 in each of the 0, 100, 200, 400 and 800 ppm groups, and 3/10 in the 1600 ppm group (one death occurring during week 1, tw o deaths occurring during week 2). In the 13

- week mouse studies, the mortality incidence in males was 0/10 in each of the 0, 100, 200 and 400 ppm groups, 1/10 in the 800 ppm group (death occurring sometime during study weeks 6

- 13), and 10/10 in the 1600 p pmExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

46 group (two deaths occurring during week 1, eight deaths occurring during week 3). In females, the mortality incidence was 0/10 for the 0, 100 and 200 ppm groups, 1/10 in the 400 ppm group (occurring during week 2), 4/10 in the 800 ppm group (one occurring during week 2, two occurring during week 3, and one occurring during weeks 6

- 13), and 10/10 in the 1600 ppm group (four occurring during week 1, six occurring during week 3). Inspection of these data for statistical significance suggests that (1) the data would be statistically positive in a trend test; and (2) pairwise comparisons would indicate a significant effects level of 1600 ppm for rats and 800 ppm for mice, not 800 ppm for rats and 400 ppm for mice.

c. The pattern of results indicate that a t the higher exposures , the maximum tolerated dose (MTD) was exceeded.

The temporal pattern of deaths would suggest to objective reviewers that the MTD had been exceeded. This rationale also accounts for why NTP ( 1996 ) reduced the highest exposure concentration for the chronic bioassay to 400 ppm for rats and 200 ppm for mice.

The MTD may be defined as the maximum daily dose which induces signs of toxicity but not lethality or significant morbidity. The 100% mortality incidence in mice and the 30

- 60% mortality incidence in rats at an exposure concentration of 1600 ppm demonstrates that the MTD was exceeded. The 10

- 30% mortality incidence in mice and the 10% mortality incidence in rats at 800 ppm indicate that this exposure concentration is near t he MTD and the deaths are associated with individual variability in susceptibility.

Although concern for acetonitrile is based on its metabolism to cyanide which has a steep concentration

- response curve, cyanide

- induced lethality is an acute response and has a steep concentration

- response slope. For cyanide or cyanide

- containing compounds, high

- dose mortality is associated with acute toxicity so that the MTD is also acute. For an acutely toxic end point, a 6

- hour daily dosing regime for 5 days/week is t oxicologically equivalent to a series of repeated acute

- dosing periods.

Lower exposures to cyanide induce a range of effects associated with tissue oxygen deprivation or no effects. Although peak cyanide concentrations following acetonitrile exposure are delayed (due to low metabolic rate for conversion to cyanide), there is no evidence to suggest that cyanide bioaccumulates.

All but two of the deaths in both rats and mice at the two highest exposure concentrations occurred during the first three weeks of the study. This pattern is not unusual for a range of compounds in which the MTD has been exceeded. Typically, mortality occurring early in a repeated

- dose study results in a reduction of the doses/concentrations during the study to a level
at w hich lethality does not occur.

d. A fuller discussion of adverse findings, including mortality, is needed for completeness, comprehensiveness and transparency.

NTP (1996) reports that that (1) the majority of deaths occurred during the first two wee ks of exposure; and (2) the spectrum of lesions reported in animals dying early was similar to thoseExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

47 encountered in early

- death animals in other NTP studies (in addition to being lesions consistent with previous descriptions of acetonitrile toxicity). Thu s, it is reasonable to assume that these early deaths were associated with overdosing. Overdosing with a range of compounds typically results in clinical signs of neurotoxicity which may be followed by death.

NTP (1996) also notes that (1) the charact er of the forestomach lesions (i.e., hyperplasia with and without inflammation, focal ulceration) observed in mice surviving to the end of the study has also been seen in mice in other NTP inhalation studies (Melnick et al., 1990), and (2) this suggests th at the effect may not be specifically related to acetonitrile exposure but rather to “some other factor associated with the conditions of exposure” (p.55). The most likely explanation for this histopathology is incidental ingestion which would be increase d by grooming

- and

- swallowing behavior during whole body exposure.

It is useful to note that acetonitrile is water

- soluble and is also irritating to the skin and the respiratory tract. Acetonitrile is also rapidly and efficiently absorbed through skin an d the gastrointestinal tract. Therefore, given the multi

- route exposure pathways (inhalation + dermal + incidental ingestion), the internal acetonitrile concentrations are likely to have been much higher than target inhalation concentrations in animal stu dies

.
Other adverse effects:

Subchronic (NTP 1996 ) rodent studies : In rats, clinical signs of neurotoxicity during week 1 were hypoactivity and ruffled fur at 800 and 1600 ppm; ataxia and abnormal posture and clonic convulsions in animals who died during the first week. Changes in the following organ weight were observed: (1) significantly decreased absolute and relative thymus weights in males and females at 800 and 1600 ppm; (2) significantly increased absolute te stis weight at 1600 ppm; and (3) significant increases in the absolute or relative weights of the heart, liver and kidney at 800 and 1600 ppm; an increase in absolute heart weight also occurred at lower doses (≥ 200 ppm). Other effects included: (1) a

no nresponsive, normocytic and normochromic anemia in both sexes at 1600 ppm and in females at 800 ppm; and 20 decreased serum triiodothyronine (T3) concentrations in 1600 ppm females in the absence of alterations in serum throxine (T4) and thyroid

- stimulatin g hormone (TSH). No histopathology unrelated to early death was observed in any animals.

In mice, clinical signs of neurotoxicity occurred only during week 1 and consisted of hypoactivity and a hunched, rigid posture at 800 and 1600 ppm. As all animals in the 1600 ppm group died early, 800 ppm was the highest subchronic dose tested for systemic effects in mice. Relative to concurrent controls, the following statistically significant organ weight changes were observed: (1) increased absolute liver weights in males at ≥ 200 ppm and in females at 800 ppm; (2) increased relative liver weights in all exposed male groups and in females at ≥ 400 ppm. Histopathology demonstrated the presence of microscopic lesions in the liver (i.e., hepatocellular cyptoplasmic vacuolation in both sexes at 400 and 800 ppm) and in the anterior forestomach (i.e., focal and multifocal hyperplasia/lesions/ulceration at exposure concentrations ≥ 400 ppm in males and ≥ 200 ppm in females).External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

48 Based on these effects, the critical effec ts for subchronic (systemic) toxicity in mice are hepatic and stomach. Forestomach histopatholgy was observed in all but the lowest dose group in females. Using this end point, the LOAEL and NOAEL would be 200 and 100 ppm, respectively. However, humans do not have a forestomach and thus the human relevance of these lesions is questionable. A more relevant endpoint is hepatic effects. Acetonitrile and its active metabolite cyanide have been shown to induce hepatic effects in other studies (concordance of information across studies). An increase in liver weight was observed at exposure concentrations which were less than or equal to the exposures at which liver histopathology was observed. This pattern of hepatic events is consistent with a cumulative toxi c effect resulting from repeated

- dosing, and indicative of a concentration x time (C x T) effect. In contrast, there is no indication of a cumulative mortality effect.

Chronic studies : No mortality was observed in the chronic study in either rats exposed to vaporized acetonitrile at concentrations of 0, 100, 200 or 400 ppm (equivalent to 0, 168, 335 or 670 mg/m3) or in mice exposed to 0, 50, 100 or 200 ppm (equivalent to 0, 84, 168 or 335 mg/m3). Hepatoxicity was observed in male, but not female, rats. There was also a marginally significant increase in hepatic tumors (combined adenomas and carcinomas) in male, but not female, rats. In mice, a dose

- related increase in the incidence o f hyperplasia of the forestomach epithelium was observed in both males and females (NTP 1996).

Developmental studies: Developmental effects were only observed at maternally toxic doses. This fact is not highlighted in the hazard assessment, as it is in WHO’s Environmental Health Criteria document (WHO 1993). According to current hazard and risk assessment guidelines, developmental effects observed at maternally toxic doses are assumed to be indirect (due to maternal toxicity). Therefore, acetonitrile would not be considered a developmental or reproductive toxicant according to current hazard and risk assessment guidelines. If mortality is statistically elevated at the highest dose tested, then it would be considered treatment

- related. However, develo pmental toxicity at that dose level would be attributed to maternal toxicity. Many compounds induce some degree of mortality at the highest dose tested.

2. The U.S. EPA has determined that mortality is the critical endpoint for acetonitrile as descr ibed in the IRIS “Toxicological Review of Acetonitrile” (1999). Mortality has been observed in a 13

- week subchronic study as well as in developmental and reproductive toxicity studies. Mortality was not observed in a 2

- year chronic study conducted at doses below those that caused mortality in the 13

- week subchronic study. Based on the totality of available evidence, particularly the 13

- week and 2

- year studies, is it reasonable to anticipate that acetonitrile can cause mortality as a result of chronic exposu re? Please explain.

U.S. EPA is asking peer reviewers whether the existing science supports a conclusion that acetonitrile can be reasonably anticipated to cause mortality as a result of chronic exposure. Based on a critical weight

- of

- evidence evaluation of available data on cyanide and acetonitrile, my answer to this question is No.

The reasoning that U.S. EPA presents in support of its position that chronic or repeated

- doseExternal Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

49 exposure to acetonitrile causes mortality is not scientifically defensible. F irst, the data do not support this interpretation. Among repeated

- dose studies, only one subchronic study showed mortality; the deaths were clustered at the beginning of the 13

- week experiment (discussed in more detail above). Second, based on what is kn own about the toxicokinetics of acetonitrile and cyanide, and their modes of toxic action, mortality induced by chronic acetonitrile or cyanide exposure is biologically implausible.

Why? The mode of toxic action for cyanide

- associated mortality is acu te. The dose

- response is steep but mortality occurs at high dose rates. A total cyanide dose load at lower daily concentrations over a longer period of time does not produce mortality. The mode of toxic action for cyanide

- associated mortality is also wel l characterized. Inhibition of cellular respiration by oxidative phosphorylation of cytochrome c oxidase, the terminal electron transporter in the mitochondrial respiratory chain, results in lack of energy production. If the inhibition is complete or nea r

- complete, asphyxiation is rapid and irreversible, and is followed by death. The delay in acetonitrile conversion to cyanide may result in a delay in onset of symptoms but the response is still acute.

Chronic exposure to acetonitrile would result in cy anide

- induced toxicity associated with increased tissue burdens of thiocyanate, which is the metabolic detoxification product of cyanide. The conversion of acetonitrile

- derived cyanide to thiocyanate is catalyzed by the sulfurtransferase enzyme, rhodanase. Elevated concentrations of thiocyanate are thyrotoxic. No evidence of thiocyanate

- induced thyrotoxicity was observed in any of the NTP or other subchronic studies.

Cyanide and acetonitrile are not known to accumulate in body tissues. If adverse effect s were to be associated with the bioaccumulation of cyanide, then a concentration x time (C x T) effect would be anticipated. The clustering of deaths in the early weeks of the NTP (1996) subchronic study does not suggest bioaccumulation. Rather, the dat a indicate that the acute MTD was approximated or reached at the acetonitrile exposure concentrations at which the deaths occurred. Had this been a chronic effect, then mortality would have continued to occur at later times during the study, as “continued generation of cyanide from the existing body burden of acetonitrile, coupled with cyanide generated from new exposures, results in cyanide levels that exceed the capacity of enzymes to metabolize cyanide to thiocyanate” (Chapter 3, p. 23). This mode of tox ic action is biologically implausible, given the lack of mortality at later times in the study or at lower doses (cumulative toxicity) and the absence of neurotoxicity symptoms in surviving animals (after the first week). Chronic tissue effects of repeated cyanide exposure, in addition to those of thiocyanate, include liver, kidney and reproductive organ toxicity and are associated with oxygen deprivation but are not lethal (ATSDR 1999). The absence of mortality in the NTP (1996) chronic bioassays and the H azelton (1983) subchronic studies also demonstrates a lack of consistency and concordance of acetonitrile findings across similar studies. It also shows the absence of a C x T effect.

Thus, using a weight

- of

- evidence approach, it is scientifically indefe nsible and biologically implausible to conclude that acetonitrile can reasonably be anticipated to cause mortality as a result of chronic exposure.External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

50 III. SPECIFIC OBSERVATIONS

Provide specific observations, corrections, or comments on the document, mention ing page, paragraph, and/or line number.

p. 30

- 31. Section 3.3.1. The discussion about why U.S. EPA (1999) did not apply an uncertainty factor for subchronic to chronic exposure duration is not clear or rational.

p. 33, Section 3.3.1.3 Mast et al. (1994) is only available as a summary report and thus interpretation of this study should be qualified wherever the findings are discussed or referenced (e.g. Mast et al. 1994, as cited in OECD

- SIDS draft 1997

– this reference is outdated; the final SIDS has been completed ]. Same comments apply to Mast et al. (1994) discussed on p. 42, lines 3

- 13.

p. 33 Line 18. Acetonitrile and cyanide concentrations were determined for maternal blood. Where are the data? This would be useful information. If these d ata are not reported, then the sentence should be omitted.

p. 34, lines 1

- 4. This is an example where lack of rigor is demonstrated. Mortality in the Mast et al. (1994) study was 1/33 at 181 mg/kg/day in pregnant females, 1/10 and 2/10 in pregnant and

n onpregnant females at 543 mg/kg/day. Assuming the control rates are 0 (and control rates are not given in most study summaries including tables and need to be added), these mortality rates have neither statistical nor toxicological significance! In contr ast, in Saillenfait et al. 1993, a lethality incidence of 8/20 dams in the highest dose group (what were the incidences in the other dose groups) would be both statistically and toxicologically significant. So there is no divergence or discrepancy in the findings between the studies. Mast et al. did not observe significant treatment

- associated mortality whereas at higher doses, Saillenfait et al. did.

This discussion is also repeated on p. 42

- 43 comparing the two studies.

Repeated misinterpretation of data in a similar manner occurs throughout the repor t.

p.36, lines 3

- 5. As noted previously, this default statement is insufficient documentation of human relevance according to current scientific standards. Both qualitative and quantitative interspecie s comparisons are discussed.

p. 36, lines 7

- 10. This sentence does not make sense. Why would this process “also occur in humans albeit at concentrations considerably higher with acetonitrile than with the majority of these other cyanogenic compounds”? W hich cyanogenic compounds (which “saturated aliphatic nitriles”)? Why at higher concentrations? If this process occurs in humans but only at very high acetonitrile concentrations, what is the human relevance ?

Please note that nitriles have a wide rang e of toxic properties and according to Hartung (1982) should not be grouped together as “organic nitriles.”External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide) Produced by EPA’s Office of Environmental Information

51 Beginning page 39, under Developmental and Reproductive Toxicity. Please note that any effects at maternally toxic doses are not considered by cur rent scientific standards and regulatory guidance to be toxic to the embryo/fetus. The discussion in some studies suggesting otherwise is misleading. This is an example of the lack of objectivity.

e.g., p. 41, lines 22

- 24; p. 42, 23

- 24; p. 43, 18

- 19.

p. 43, lines 11

- 12. Emaciation is not a frank effect unless the animals are moribund and death is imminent. Please clarify or modify.