i

Response to the External Peer Review of

“Technical Review of Acetonitrile (Methyl Cyanide)”

January 24, 2013

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. RESPONSE TO HUMAN HEALTH PEER REVIEW COMMENTS ................................ 2

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

B. Response to Charge Questions............................................................................................... 6

C. Specific Observations .......................................................................................................... 35

IV. RESPONSE TO CHEM-FATE & ECOTOXICOLOGY PEER REVEW COMMENTS .. 40

A. General Impressions............................................................................................................. 40

B. Response to Charge Questions............................................................................................. 47

C. Specific Observations .......................................................................................................... 59

APPENDICE: External Peer Review of EPA’s Draft Technical Review of Acetonitrile (Methyl 

Cyanide)....................................................................................................................77
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I. BACKGROUND AND DOCUMENT FORMAT

In response to a petition to delist acetonitrile for the Toxics Release 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 the 

draft report. Within this document (Response to Peer Review), the reviewer’s names have been 

masked. Five of the scientists (Commenter One, Commenter Two, Commenter Three, 

Commenter Four and Commenter Five), had expertise in human health hazard assessments

(human health toxicologists). Commenter Six, Commenter Seven, and Commenter Eight were

ecotoxicologists, selected for their expertise in environmental fate and ecological hazard 

assessments.

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

Chapter 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.

The 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 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.

The three ecotoxicologists were asked to review Chapter Two (Chemistry and Environmental 

Fate) and Chapter Four (Ecological Hazard Assessment) and any relevant sections of Chapter 

One (Integrated Technical 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., 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?

Sections III and IV of this report capture the peer reviewer’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 review 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 

animal studies in mice rats and rabbits. Also, the interpretations of the results are based on 

somewhat obsolete mechanistic information.
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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: Commenter 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 “ACNinduced acute, sub-acute and chronic toxicities and subsequent mortality is 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 been 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 for 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 assessment 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).

 
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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 

petition 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 environmental 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 document 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, OGWDW 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 acetonitrile) 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 of 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 toxicity 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: Commenter 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 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 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 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.

Comment III-A-7: Commenter 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 standards and regulatory 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 as 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-week 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 

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 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.

EPA Response III-A-8:

The EPA agrees 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 mortality 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 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).

Table 3-1 has been extended to include the studies included in the text and have been 

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)
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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 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.” 

B. Response to Charge Questions

Chapter Three, Charge Question 1.

Has the 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 describe 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 in 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 of 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 adequate 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 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 were not adequately discussed and evaluated 

in this review.

EPA Response III-B-Ch3-Q1-2:
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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 data. 

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 acetonitrile 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 has 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 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 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 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 acetonitrile 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 point 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 studies. 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 with 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. EPA, 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 incidence 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). 

Positive 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 microtubules. 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 (Zimmermann 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 interfere 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 discussion 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 thiocyanate 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 cyanide 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 Health 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 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 

allow 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 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 cyanide 
9

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 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 conversion 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 of 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. Thyroid 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 metabolism 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 NTP 
10

(1996) studies. This suggests adequate thyroid hormone (T3 and T4) 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 thyroid 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 sensitivity.

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 Toxicological 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

However, 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 acetonitrile. 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) observation, 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 modified 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 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.”

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 gastrointestinal 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.
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EPA Response III-B-CH3-Q1-8:

The technical review has been revised to present a more balanced discussion of the human health 

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 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 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 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 cyanide 

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, 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 

detoxification” 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 detoxification 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).

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 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 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 species with lower 
12

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 nature 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 lower in smokers than in nonsmokers, and individual Km and Vmax 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-B-CH3-Q1-10: Commenter 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 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.

EPA Response III-B-CH3-Q1-10:

This information has been integrated 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-B-CH3-Q1-11: Commenter Four

Section 3.2 of the draft ends with two highly speculative paragraphs. They assert that the slow 

metabolism of acetonitrile to cyanide, 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 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-B-CH3-Q1-11:

The statements made in the two referenced paragraphs have been removed from the Technical 

Review of Acetonitrile (Methyl Cyanide).

Comment III-B-CH3-Q1-12: Commenter Four

I was also surprised by the primarily qualitative nature of the discussion of acetonitrile 
13

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-B-CH3-Q1-12:

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 analysis of the pharmacokinetics is not available in the 

peer-reviewed literature.

Comment III-B-CH3-Q1-13: 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-B-CH3-Q1-13:

As detailed 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 mortality 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-B-CH3-Q1-14: 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-ofevidence narrative is incomplete. 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 action 

data, and lack of rigor in assessing and interpreting findings from individual studies in 

accordance with current standards and regulatory guidance.

EPA Response III-B-CH3-Q1-14:

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 analysis 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 

Cyanide) 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 listing was mortality. The 

petitioner focused their second petition on this effect, thus EPA’s focus was on this effect. 
14

Comment III-B-CH3-Q1-15: Commenter Five

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” – 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 

indicate 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-B-CH3-Q1-15:

EPA has revised the discussion of mode of action related 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-B-CH3-Q1-16: 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 Perspect. 86:93-98.

U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. [See section on writing a weightof-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.
15

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.]

EPA Response III-B-CH3-Q1-16:

As noted in this and other reviewer’s comments “new data are not available” specific to 

acetonitrile. The studies listed by this reviewer, 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-B-CH3-Q1-17: Commenter Five

Toxicokinetic and mode of toxic action data are incompletely summarized. 

EPA Response III-B-CH3-Q1-17:

See specific responses to subsections (a) through (f) of III-B-CH3-Q1-17.

Comment III-B-CH3-Q1-17(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 distributed fairly uniformly throughout the body (EHC 

1993).

EPA Response III-B-CH3-Q1-17(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 inhalation 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 14C in the methyl group indicated that radioactivity was 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 spleen, stomach, and skin (U.S. EPA, 

1999).”

Comment III-B-CH3-Q1-17(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 molecule and its 

cyanide metabolite (Hartung 1982; EHC 1993). Further, animal studies do not indicate that 
16

either acetonitrile or cyanide has the potential for bioaccumulation (EHC 1993; ATSDR 1999).

EPA Response III-B-CH3-Q1-17(b):

The EPA has included the 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-B-CH3-Q1-17(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-order kinetics relative to cyanide; the concentration of sulfurcontaining donor molecules is the rate-limiting factor. 

EPA Response III-B-CH3-Q1-17(c):

The reviewers comments have been incorporated into section 3.2.3., Metabolism, of the 

Technical Review of 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 mediated 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 concentrations. 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 thiocyanate following both 

oral and inhalation exposure (Sylvester and Sander, 1990).”

Comment III-B-CH3-Q1-17(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, 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 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 
17

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 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 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 excessive 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 acetonitrile 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 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 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 deprivation, which is 

met by increased conversion of stored T4 to T3. 

EPA Response III-B-CH3-Q1-17(d):

This reviewer’s comments have been considered and incorporated throughout Chapter 3 of the 

Technical Review of Acetonitrile (Methyl Cyanide). The potential effects of extended 

thiocyanate exposure have been discussed in EPA’s response to comment III-B-CH3-Q1-6 and 

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

The Technical Review of Acetonitrile (Methyl Cyanide) 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 concern 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-B-CH3-Q1-17(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 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 biologically 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 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 knowledge 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-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 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 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-B-CH3-Q1-17(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 Technical 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”. The 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 addition, 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 that 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 developed solely and specifically to support listing/delisting 

decisions for EPCRA section 313. 

Comment III-B-CH3-Q1-17(f): 

The pattern of dosing would also predict mortality as an acutely toxic response, not as a chronic 

one.

The dosing regime was 6 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 deprivation in tissues. 

EPA Response III-B-CH3-Q1-17(f):

It should first be noted that chronic studies often implement a repeated-dose structure 

incorporating a non-exposed period in the interim. The study 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 level, 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 NTP (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 is 
20

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 the 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 the 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-B-CH3-Q1-18: Commenter Five

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 there 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 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-B-CH3-Q1-18:

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 toxicokinetic parameters include 

binding affinity (Km) and metabolic rate (Vmax) for cyanide metabolism. These toxicokinetic 

parameters dictate the ability of species to detoxify acetonitrile and its metabolites, particularly 

cyanide moieties. Variability between species is further compounded by the tissue specific 

inducible nature of the enzyme responsible for converting acetonitrile to hydrogen cyanide 
21

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 (Km) 

for cyanide and a lower rate (Vmax) for cyanide metabolism than rhodanese from rat nasal tissue 

(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, the 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-B-CH3-Q1-19: 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 

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. 

EPA Response III-B-CH3-Q1-19:

The 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 health 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 program, 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-B-CH3-Q1-20: Commenter Five

There is a lack of rigor in assessing and interpreting individual study findings.
22

EPA Response to III-B-CH3-Q1-20:

See specific subsections (a) through (d).

Comment III-B-CH3-Q1-20(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-B-CH3-Q1-20(a):

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 

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-B-CH3-Q1-20(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 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. 

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 toxicologically 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 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 of 
23

1600 ppm for rats and 800 ppm for mice, not 800 ppm for rats and 400 ppm for mice.

EPA Response III-B-CH3-Q1-20(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 be 

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 observations, including statistical 

significance has been extended.

Comment III-B-CH3-Q1-20(c):

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 addition 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 neurotoxicity 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 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 than target inhalation concentrations in animal studies. 

EPA Response III-B-CH3-Q1-20(c):

The Technical Review of Acetonitrile (Methyl Cyanide) has been revised to incorporate a more 

complete discussion of adverse effects observed in the NTP (1996) 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 inhalation 

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-B-CH3-Q1-20(d):

Other adverse effects: 
24

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 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 occurred 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 absence 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 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 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, 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 (WHO 
25

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, 

developmental toxicity at that dose level would be attributed to maternal toxicity. Many 

compounds induce some degree of mortality at the highest dose tested.

EPA Response III-B-CH3-Q1-20(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 

identified 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, Charge 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 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.

Comment III-B-CH3-Q2-1: 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 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 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 would 
26

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, 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 metabolites, 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 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. 

CH3CN ------------ CH2OHCN --------- CH2O + HCN

Cyanide ions had been implicated in most aliphatic nitriles’ toxicity including that of ACN. The 

other metabolite, formaldehyde (CH2O) 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 

formation of Schiff’s base with the amino groups in the enzymatic reactive sites. Also, CH2O 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 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 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 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-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 rabbits 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 CH2O, 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 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 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.

EPA Response III-B-CH3-Q2-1:

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 inhibition 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 to 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 known 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 cardiovascular, 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, subsequent 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 metabolized 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 unknown, 
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 cyanide 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 

in 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 observed 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 for 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-B-CH3-Q2-2: 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 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 increased 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 these 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 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 20th 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.

EPA Response III-B-CH3-Q2-2:

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 does not have a vehicle for modifying the assessment to 

account for implied data needs. 
29

Comment III-B-CH3-Q2-3: 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 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 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.

EPA Response III-B-CH3-Q2-3:

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-CH3-

Q1-17.(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 exposure.

Comment III-B-CH3-Q2-4: 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 interpretation 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. Hence, 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 effective 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-B-CH3-Q2-4:

The EPA agrees with these comments and has included this reasoning in the Technical Review of 

Acetonitrile (Methyl Cyanide). 

Comment III-B-CH3-Q2-5: 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 lessened 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-B-CH3-Q2-5:

An understanding 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, the mode of action of noncancer 

toxicity is unknown; although limited information is available to support different hypotheses. 
30

Comment III-B-CH3-Q2-6: Commenter Three

Another important issue for CERCLA 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 

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. 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 humans 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 associated 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) cytochrome 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 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 T3 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 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 acetonitrile exposure to humans.

EPA Response III-B-CH3-Q2-6:

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 chronic exposure is addressed. 

However, the proposed mode of action of cyanide accumulation through repeated acetonitrile 

exposures has been removed from the document. 

Comment III-B-CH3-Q2-7: 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 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, Wilhite (1981) describes 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 
31

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 support for same mode of 

action (i.e., cyanide inhibition of CNS cytochrome oxidase) of lethality in the three protocols.

EPA Response III-B-CH3-Q2-7:

The Technical Review of Acetonitrile (Methyl Cyanide) details the human relevance (Section 

3.10.) of effects 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 referenced 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 identification of the critical effect.

Comment III-B-CH3-Q2-8: 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 from 

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 the 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 critical 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 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 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 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.

EPA Response III-B-CH3-Q2-8:

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/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 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 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 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.

EPA 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 mortality 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 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 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 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 correspond to cumulative acetonitrile exposure of only 3,000 
33

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 signs, 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 cause 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-ofaction 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-weeks 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 addition, 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 detailed 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 

toxicity 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. Unmetabolized 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 critical 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 subsections)

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 beginning 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 the 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 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 

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, 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 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 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 (1996) 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 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 implausible, given the lack of mortality at later times in the 
35

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 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):

Cyanide-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 mode 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 possible 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, cyanide, 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 

treatment 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 exposures 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 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 co-administration of acetone 

with acetonitrile delays 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 that the rate of cyanide formation from acetonitrile is not 
36

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 that 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 Section 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 mode 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 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).

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. Unfortunately, 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 the 
37

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 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.

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 toxicity 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 exposure (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, viable 

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 somite pairs of the 

developing embryo. All of the nitriles tested, including acetonitrile, produced concentrationdependent decreases in growth and differentiation, including decreased yolk sac diameter, 

crown-rump length, head length, and number of somites, 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 biotransformation system to the culture medium did not change acetonitrileinduced 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-

14C-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 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 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 14C of the 

radiolabeled acetonitrile. Further, methods used by Ahmed et al. (1992) distinguished 

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 
38

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:

The 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. 

Comment 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 acetonitrile. 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 certain 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 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).”

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 speculative, 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 to 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 III-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).

Comment 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/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 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 CHEMISTRY 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 fate 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 wells. This is beyond the scope 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 (Koc) value of 5 has 

been calculated for acetonitrile using its experimental log Kow value of -0.34 (Hansch et al., 

1995) and a regression-derived equation (HSDB, 2002; Lyman et al., 1990). Based on this Koc

value, acetonitrile 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 solubility 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, acetonitrile 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 groundwater pH, 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 land 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 Anaerobic Biodegradation Studies: Limited literature was identified concerning the 

anaerobic biodegradation of acetonitrile. 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 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 initial concentrations of 1 and 2 g/L under both aerobic and 

anoxic conditions in a synthetic 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 

faster 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 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 concentration 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 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 

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% reduction of 

acetonitrile was observed after a 90-day incubation period at 37o

C using an initial acetonitrile 

concentration 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 ecological receptors/aquatic species - no literature was readily identified on 

mechanism(s) of acetonitrile toxicity. Vertebrate biotransformation toxicity mechanisms can be 

found in section 3.2 - Kinetics and Metabolism in the report. No changes 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 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. 

EPA Response IV-A-3:

An elaboration on toxicity exposure levels and the degree of 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 to 
42

levels 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 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) (see for example: 

http://www.epa.gov/oppefed1/ecorisk_ders/toera_analysis_eco.htm#Ecotox). Acute toxicity 

values for freshwater fish and aquatic invertebrates ranged from 100 to 3600 mg/L. The 96 hour 

LC50/EC50 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 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 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 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 radical 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 620 

days based upon an experimental rate constant for the reaction of acetonitrile with hydroxyl 

radicals of 2.63E-14 cm3/molecule-sec and an atmospheric concentration of 5X10+5 hydroxyl 

radicals per cu cm. Limited literature was available addressing the atmospheric 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 hydrogen abstraction reactions involving 

hydrocarbon side chains, oxygenated species are expected but no literature was identified 
43

stating specific abiotic degradation products. However, photochemical smog chamber studies 

show that acetonitrile is unreactive 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 hydrolysis 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 report, in 2009 some 17.1 million pounds of acetonitrile were 

reported to the TRI as being 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 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).”

“2.6.3.2 Aerobic Biodegradation Studies. In review of the studies available on the 

aerobic biodegradation of acetonitrile, several of the studies 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 several factors including media, temperature, available 

oxygen, acetonitrile concentration, specific microorganisms 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 media 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 acclimated 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 Ludzack 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 activated 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 days 

using an activated sludge inoculum (OECD Test Method 301C).

 The biodegradability of acetonitrile was also observed with river water; the 12-day 
44

ThOD (theoretical oxygen demand) test measured the generation of CO2 from Ohio River water 

spiked with acetonitrile at concentrations ranging from 0.1-25 mg/L. After 12 days, a total 

generation of CO2 was observed equivalent to 40% of the ThOD estimated for acetonitrile 

(Ludzack et al., 1959). Acclimation of the microorganisms was examined by re-dosing; the 

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 

acetonitrile 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 applicable 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 acetonitrile, 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 reduction 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 acetonitrile is expected to be slower 

in seawater than in freshwater.”

2.6.3.3 Anaerobic Biodegradation Studies. Limited literature was identified concerning the 

anaerobic biodegradation of acetonitrile. 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 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 initial concentrations of 1 and 2 g/L under both aerobic and 

anoxic conditions in a synthetic 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 

faster 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 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 concentration 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 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 
45

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% reduction of 

acetonitrile was observed after a 90-day incubation period at 37oC 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 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).

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 constants 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). 

Concerning the biodegradation of acetamide, Urano and Kato (1986b) using 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 approximately 4 

days based upon the BOD. In an earlier study, reported acetamide 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 acetonitrile under aerobic conditions peaked at 1800 mg/L about 8 hours after 

the initial exposure of acetonitrile to acclimated bacteria. Within 30 hours, concentration levels 

of acetamide were reduced to non-detectable levels. 

As reported earlier in this report, some 17.1 million 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 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 combustion 

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).”

Comment IV-A-5: Commenter Seven
46

Use of the EPIWIN 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. 

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 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. 

EPA Response IV-A-7:

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 

concerning the fate of acetonitrile relevant to groundwater contamination. Actual exposure 

concentrations relative to people or the environment based upon groundwater contamination 

scenarios are beyond the scope of this technical report concerning the hazard of acetonitrile.

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, some 17.1 million 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 acetonitrile will have decomposed (Britt P. 2002). The ignition of acetonitrile in 
47

excess oxygen results in the production of CO, CO2, H2O, N2 and NO as the major combustion 

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-Q1-1: 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-Q1-1:

No experimental information 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 found in EPA’s response to Comment IV-A-1 above.

Comment IV-B-Ch2-Q1-2: 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-Q1-2:

Section 2.6.5, Toxic Transformation 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-Q1-3: 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-Q1-3:

The additional 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 Valuea Conditions Reference

Molecular Formulae C2H3N

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) 1x106 25°C Riddick et al., 1986

Log Kow -0.34 - Hansch et al., 1995

Henry’s Law constant (atmm3

/mol)

3.45x10-5 25°C Gaffney et al., 1987

Dissociation constant, pKa -4.30 - Perrin, 1965

Hydroxyl radical reaction 

rate constant (cm3/molecsec)

2.63x10-14 25°C Atkinson, 1994

Density (g/cm3) 0.786 20°C McKinney and DeVito, 

1998

Vapor density 1.42 Air = 1 Clayton and Clayton, 1982

Koc 5 (HSDB, 2002; Lyman et al., 

1990)

Comment IV-B-Ch2-Q1-4: 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-Q1-4:

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 injection 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 expanded. However, there is 

Chapter Two, Chemistry 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 limited and theoretical 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 IVA-1, sections “2.6.1Environmental Transport” 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 byproducts of combustion)?

EPA Response IV-B-Ch2-Q2-2:

Additional information on the incineration 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 responses 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 byproducts? (I would think not but some information to this effect, if available, would be helpful.)

EPA Response IV-B-Ch2-Q2-4:

See EPA response 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 problems 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 groundwater 
50

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 Response 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 half-life 

rate is 150,000 years. The section of the report on abiotic degradation has been expanded in 

response to the peer reviewer’s comment and is duplicated in the EPA response to Comment IVA-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 appears to be widespread 

over several genera of prokaryotes (organisms that lack a cell nucleus or any other membranebound organelles), and eukaryotes (organisms whose cells are internally organized into complex 

structures enclosed within membranes) . Experimentally observed degradation rates vary based 

upon several factors including media type, temperature, acetonitrile concentration , available 

oxygen, specific microorganisms involved and the degree of acclimation. Within this section 

additional information is provided on specific microorganisms 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 below are 

from studies that used test conditions likely representative of aerobic or anaerobic digestion 
51

within a sewage treatment plant. These conditions are not necessarily reflective to relevant 

environmental conditions, in particular those conditions that 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 text 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 elucidated 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 ammonia 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., 1989 

,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 

H3C C N

H2O

Nitrilase

H3C

OH

O

NH3 +

H3C C N

H2O

Nitrile hydratase

H3C

NH2

O H2O

Amidase

H3C

OH

O

NH3 +
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 innoculum was obtained from the low-solids anaerobic digesters at the 

Denver Municipal Waste Water Reclamation Plant in Denver, Colorado. The following has been 

added to the technical report.

2.6.3.3 Anaerobic Biodegradation Studies “...An inoculum obtained from low-solids 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% reduction of acetonitrile was observed after a 90-day 

incubation period at 37oC 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. 

Deleted---“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 important 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 

straightforward.

EPA Response IV-B-Ch2-Q2-14:

The model was rerun using values for releases analogous to those being reported as a part of the 
53

TRI. The model inputs will be stated in the report. The regenerated results based upon TRI 

reported releases for 2009 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 analysis. 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 1.54x103

Water 16.7 360

Soil 3.5 720

Sediment 0.0329 3.24x103

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 

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 differing outcomes or is Table 2-2 always the same regardless of release point?

EPA Response IV-B-Ch2-Q2-15:

Different relative releases can have a moderate impact on the predicted environmental 

distribution percentages masses. However, for this report, the model was rerun using releases 

reflective of what was reported to the TRI for 2009. 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 fugacity model data has been revised based upon the overall TRI

reported releases to soil, air and water for TRI reporting year 2009. 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 previously, this leaves open the 
54

question of the transport and eventual fate of methyl cyanide in groundwater.

EPA Response IV-B-Ch2-Q2-18:

No information is available that indicates that acetonitrile is leaking out of underground injection 

wells. To create potential 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 transport 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 and 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 be 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 for relatively low concentrations of the 

chemical.” 

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 mechanism of decomposition. Hydrogen cyanide was not reported in 

the literature reviewed as a bacterial decomposition product hydrogen 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-Q1-2. 

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 responses to Comments IV-BCh2-Q2-1 and IV-A-1.
55

Comment IV-B-Ch2-Q2-22: Commenter Eight

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.

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 revised based upon the overall relative release percentages (air, land, and water) 

reported to TRI. For details see the response to Comment IV-B-Ch2-Q2-14 above.

Comment IV-B-Ch4-Q1-1: 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 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?

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. These 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 supported 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 vertebrates is 

provided in section 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 

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?

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 nonstandard, 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.

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 are given below in Point 13 of my 
57

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 this 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. Information 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 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. 

EPA Response IV-B-Ch4-Q2-1:

The text in question has been revised and is presented below. Context to the phrase “indicating 

relatively low acute aquatic toxicity” 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 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_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 ecological 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 Response 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 the 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 chronic 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-Ch4-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 conclusion 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 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). Acute toxicity 

values for freshwater fish and aquatic invertebrates ranged from 100 to 3600 mg/L. The 96 hour 

LC50/EC50 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 invertebrates, 

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 statements 

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 invertebrates ranged from 100 to 3600 mg/L. The 96 hour 

LC50 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 μg/g soil and 200 μL/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 Kow: -0.34).”
59

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 daysdays

indicates that approximately 69 mg/L would be expected to remain 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.

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 level 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 widespread 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 scope 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 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.” 

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 the 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 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; 
60

• 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);

• 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 inhalation 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 Chemistry 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 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-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 acetonitrile 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 relatively 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 may 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, 1982 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 result in cyanide levels in tissues that 

exceed the capacity of enzymes to metabolize cyanide to isocyanate.”

EPA Response 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 Six
63

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 acetonitrile 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 

relationship between metabolic differences and the LD50s for rats and mice is unavailable for 

acetonitrile.

Comment IV-C-17a: Commenter Six

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 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 consideration 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 the 

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-17b:

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 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).” 

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 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 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 acetonitrile 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 

sections 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 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). In 2006 the aggregated U.S. productions levels 

reported to EPA were between 50 to less than 100 million pounds 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 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 recovery by the manufacturers.”

Section 2.4.... “In 2006 the aggregated U.S. productions levels reported to EPA were between 

50 to less than 100 million pounds 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 

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 recovery by 

the manufacturers.”

Comment IV-C-20: Commenter Six

Pg. 10; Lines 24-25: The text indicates that 30 million pounds of acetonitrile were burned onsite 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 
65

this process?

EPA Response IV-C-20:

The following text has been added to the report in an effort to address information known 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 has 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 IIV-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 report.

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 byproducts).
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

Pg. 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. 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 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 question 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 been 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 deepwell 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. 

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 conditions 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 limiting 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 Six
69

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 IIV-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., 1973).” 

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 single 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 

that 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-generated 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 Six
70

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 potential 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 Summary 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., 

Page 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 conclusion 
71

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 invertebrates ranged from 100 to 3600 mg/L. The 96 hour 

LC50 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 earthworms reported 

100% survival at exposure concentrations of 500 μg/g soil and 200 μL/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 Kow: -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 μg/g soil for 14 days and 200 μL/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 mg/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 warranted. 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 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.

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. Based 
72

upon this hazard assessment, a decision will be rendered by EPA on whether to honor the request 

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 paragraph. Perhaps this sentence could be moved to the 

second paragraph of Section 2.1?

EPA Response IV-C-54:

This paragraph of the report 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 text 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 Studies and now reads as 

follows:

“Earlier studies reported that the biodegradation of acetonitrile using activated sludge or 

sewage proceeds 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 17, 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 range 
73

of conditions related to possible environmental scenarios. The environmental distribution 

section of the report is being updated using a fugacity level III model along with TRI reported 

releases to air, land and water. For further details see the EPA response to comment 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 reported by manufacturers as being disposed of on-site in underground injection 

wells”. 

Comment IV-C-60: Commenter Eight

Page 24, 1st 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 sections of the report.

Comment IV-C-64: Commenter Eight
74

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 acetonitrile. 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 original 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 

CaCO3. Fifth, it appears that nominal concentrations were used. Preference 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 used 
75

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.

EPA Response IV-C-67:

While the nonparametric tests may have been overly conservative 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-68: 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 table.

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 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.

EPA Response IV-C-69:

Agree, but 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 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)

1

. Acute toxicity 

values for freshwater fish and aquatic invertebrates ranged from 100 to 3600 mg/L. The 96 hour 

LC50 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 μg/g soil and 200 μL/g soil. No data were 
76

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 Kow: -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 69 mg/L would be expected to remain 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, 2008
External Peer Review of the Technical Review of Acetonitrile (Methyl Cyanide)

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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 ECOTOXICOLOGY 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: Peer 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-1
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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-toKnow Act (EPCRA) of 1986. Section 313 of EPCRA requires certain facilities manufacturing, 

processing or otherwise 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 chemical 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 beyond 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 Office of Information Analysis and Access (OIAA)

conducted 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 hazard 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-ofthe-science. 
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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 animals 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 result 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 including 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 health hazards, the IRIS (1999) Toxicological Review of Acetonitrile was 

the predominant source of information. 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 

executive 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 Acetonitrile (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. Specific 

charge questions for this assignment are presented below. 

Charge Questions for Human Health Toxicologists

Chapter Three, 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.
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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 years 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 chronic 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 approximately 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 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 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.

Charge Questions for Ecotoxicologists

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., 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?
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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 

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 interpretation 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 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 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 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 (OGWDW) 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 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 acetonitrile) 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 of 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 toxicity of acetonitrile is attributable to its conversion to cyanide.

The document was generally well written and the review of data that has been directly associated 
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with studies of acetonitrile were largely accurate. Below, there are further comments on whether 

the document adequately explored potential thyroid effects of this chemical. 

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 primary 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 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 recycling of detoxication product thiocyanate 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 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 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. 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, reviewed, 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, 
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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 

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 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.

Commenter 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 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 even 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 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 presented separately.

Human relevance is inadequately characterized.
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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 such 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 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 second 

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 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 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 discussion should come 

to a judgment 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 described? Are there any additional studies that 

should be included?
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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 discussion 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 thiocyanate 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 cyanide 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 

allow 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 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 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 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 conversion 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).

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 Review 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 presented 
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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 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) observation, expressed 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 metabolism, 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 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.

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, the 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 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 cyanide 

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, 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 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 
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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 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 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 was 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.

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. 

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-ofevidence narrative is incomplete. 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-action 

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 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” – 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 

indicate bioaccumulation. This study should be added, as should others referenced in 
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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.

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 Perspect. 86:93-98.

U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. [See section on writing a weightof-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 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. Toxicokinetic 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 poisoned 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 likely 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 1999). 

(c) Released cyanide is metabolized to thiocyanate by rhodanese, a sulfur transferase enzyme, 
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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-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 (detoxification). 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 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 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 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 excessive 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 acetonitrile studies. 
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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 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 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 deprivation, 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 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 biologically 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 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 knowledge 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-hour 
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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 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 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. 

(f) The pattern of dosing would also predict mortality as an acutely toxic 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 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 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 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 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? 

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-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 

for (1) strength of association; (2) consistency of association; (3) specificity of association; (4) 
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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 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 toxicologically 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 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 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 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. 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.
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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 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 

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 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 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 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 addition 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 neurotoxicity 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 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 
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+ 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 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 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 occurred 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 absence 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 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 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, a 
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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 (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, 

developmental toxicity at that dose level would be attributed to maternal toxicity. Many 

compounds induce some degree of mortality at the highest dose tested. 
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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 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 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, 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 metabolites, 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 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. 

CH3CN ------------ CH2OHCN --------- CH2O + HCN

Cyanide ions had been implicated in most aliphatic nitriles’ toxicity including that of ACN. The 

other metabolite, formaldehyde (CH2O) 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 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.
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formation of Schiff’s base with the amino groups in the enzymatic reactive sites. Also, CH2O 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 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 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 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-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 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 years 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 CH2O, 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 

at 
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low levels of exposure. 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 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 increased 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 these 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 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 20th 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 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 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 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 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 effective dose level of the subchronic 
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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. 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 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. 

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 

ppm) approximates the lowest reported, historical inhalation LC50 (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 over 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 are 10-fold less than the 

historical LC50. 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 CERCLA 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 

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. 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 humans 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 associated 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) cytochrome 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 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 T3 was reported in high dose rats of the 13-wk study. Also, one lesion characteristic of 
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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 

mortality 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 same 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, Wilhite (1981) describes 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 (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 support for same mode of 

action (i.e., cyanide inhibition of CNS cytochrome oxidase) of lethality in the three protocols.

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 from 

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 the accumulated 

tissue dose of cyanide can approach levels of disposition associated with high 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 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 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 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 
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available. 

Commenter Four

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 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 conducted 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 correspond to cumulative acetonitrile exposure of only 3,000 and 6000 ppmdays, 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 ppmdays), but also in the mice exposed to 100 ppm (13,036 ppm-days) and 50 ppm (6,518 ppmdays). Yet, there were no effects on clinical signs, 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 cause mortality as a result of chronic exposure has no scientific support 

whatsoever.

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. 

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 

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 
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more detail above). Second, based on what is known 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 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, 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 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 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 (1996) 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 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 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 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.
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C. Specific Observations

Commenter One

The 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 sensitive 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 CH3CN 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 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 main reason for using this designation is to distinguish these experiments from noseonly 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 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 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 aa3. 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 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 
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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 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 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 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: 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-

14C-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 daily dose. The Technical Review sentence describing Ahmed et al. (1992) could be 
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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 14C of the 

radiolabeled acetonitrile. Further, methods used by Ahmed et al. (1992) distinguished 

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 from 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. Eugene 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 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.

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.

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 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 

clearance.

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 
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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 mortality for shortterm 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!

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.

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 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 report.

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 acetonitrile than with the majority of 

these other cyanogenic compounds”?
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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 note 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 moribund and death is 

imminent. Please clarify or modify. 
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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 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 

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 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). 

Use of the EPIWIN 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 this 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.
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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 and 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 description is clear and concise.

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?
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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 

injection wells into groundwater and the fate and transport of contaminants if they are 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 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)?

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? It is there 

any information on whether the combustion of methyl cyanide produces toxic byproducts? (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 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 environment. 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. 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?
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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 degradation 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 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 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 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 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 produce 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 its 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 

significantly slower...”?
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• 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, 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.
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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.

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 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 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 

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?
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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 are 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 this very surprising. However, I did not find much more information during a brief 

journal search.
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Commenter Six

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 

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 some 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, impairment 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 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?

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 level 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 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 the environment” supported by the data/information 

presented?
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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, 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);

• 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.

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).”

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.”
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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 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: 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 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 

exceed the capacity of enzymes to metabolize cyanide to isocyanate.”

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 . . .”

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 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 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: Revise 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. In 
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comparison with other chemicals, acetonitrile has a relatively low aquatic toxicity.”

Pg. 8; Line 4: Delete “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 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 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 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 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 onsite 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?

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 acrylonitrile with acetonitrile and hydrogen cyanide as principal byproducts).

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 model 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? 
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Pg. 16; Line 13: Insert “enzyme” before “transforms the nitriles.”

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.”

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 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).”

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, 

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 deepwell 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 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? 
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3. HUMAN HEALTH HAZARD ASSESSMENT (not 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 relatively 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 

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 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.

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 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.

• 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.

• 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 
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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., 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 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 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 presentation.

8. Page 18, Line 20. Change to “... were disposed on-site in ...”?

9. Page 24, 1st 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 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.
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13. 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.

14. 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. 

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 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 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 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 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. 
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APPENDIX A

PEER REVIEW COMMENTS FROM COMMENTER ONE.
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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

June 15, 2008
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I. GENERAL COMMENTS:

The document “Technical review of Acetonitrile,” 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 interpretation 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 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.

 

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 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 such as formaldehyde (see below). 

2. The USEPA has determined that ........................ Based on the totality of available 

evidence, is it reasonable to anticipate that ACN 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 which was administered in the 

two year studies to mice and rats. Due to the similarities in metabolic enzymes between 
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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 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, 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 metabolites, 

particularly cyanide moieties.

The 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 

equilibrium with equal molar concentrations of cyanide ions and formaldehyde. 

CH3CN ------------ CH2OHCN --------- CH2O + HCN

Cyanide ions had been implicated in most aliphatic nitriles’ toxicity including that of 

ACN. The other metabolite, formaldehyde (CH2O) 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 formation of Schiff’s base with the amino groups in 

the enzymatic reactive sites. Also, CH2O 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 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 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 processes of damage accumulation may take a while longer and when 

enough damage accumulates, organ malfunction may occur leading to death as indicated 
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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 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 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 CH2O, 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 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 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: 

The 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 sensitive 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 CH3CN production.

Page 34, line 8: On GD 29 surviving does were sacrificed? 
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APPENDIX B

PEER REVIEW COMMENTS FROM COMMENTER TWO
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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 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 (OGWDW) 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 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 acetonitrile) 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 of 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 toxicity of acetonitrile is attributable to its conversion to cyanide.

The document was generally well written and the review of data that has been directly 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 responses 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 second 
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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 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 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 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 discussion 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 thiocyanate 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 cyanide 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 

allow 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 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 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 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 conversion 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).
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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. 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 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 increased 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 these 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 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 20th 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 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. 

III. SPECIFIC OBSERVATIONS

Whole body inhalation – not sure how this is accomplished? This terminology is used 
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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 main reason for using this designation is to distinguish these experiments from noseonly 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 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 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 aa3. 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 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 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 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 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 acetonitrile with 

those that are produced by direct treatment with cyanide? This requires some exploration of the 
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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.
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APPENDIX C

PEER REVIEW COMMENTS FROM COMMENTER SIX
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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 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. 

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 chemical 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. 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). 

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?

Comments: 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 

injection wells into groundwater and the fate and transport of contaminants if they are released 

into groundwater. Also, the text indicates that 30 million tons of acetonitrile are burned on-site 
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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, “Acetonitrile 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 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.

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 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, 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);
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• 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.

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).

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 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: 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 will 
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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 concentrations 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 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 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 bioassays (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 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.”

Pg. 8; Line 4: Delete “experimental.” 

2. CHEMISTRY AND ENVIRONMENTAL FATE

2.1 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 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 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 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 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 onsite 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?
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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 acrylonitrile with acetonitrile and hydrogen cyanide as principal byproducts).

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 model 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: 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.”

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 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).”

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.” 

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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 deepwell 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 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? 

3. HUMAN HEALTH HAZARD ASSESSMENT (not 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 relatively 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 

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 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.
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APPENDIX D

PEER REVIEW COMMENTS FROM COMMENTER SEVEN
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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 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 EPIWIN 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 this chemical suffers from the lack of available information 

on chronic effects or on population-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. 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 

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.

• 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.

• 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 
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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., 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 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.

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?

Comments:

• 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? 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 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 environment. 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. 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.
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• 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 degradation 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 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 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 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 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 produce 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 halflives 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 its 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 

significantly 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 Assessment
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1. Has the available information on the ecological toxicity of acetonitrile 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 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 performed on embryos of a nonstandard, 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 

adverse 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 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, impairment of 

reproductive ability due to chronic exposures to low levels (e.g., from a groundwater seep to 

surface water).
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• 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 

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?

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.
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APPENDIX E

PEER REVIEW COMMENTS FROM COMMENTER THREE
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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 relevant 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 recycling of detoxication product thiocyanate 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 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 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. Also, semicolons and commas are used inconsistently to separate items of a series in 

the last 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 databases. 

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.
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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 presented 

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 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) observation, 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 metabolism, 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 thiocyanate (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 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.

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 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 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 effective 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. 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 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. 

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 

ppm) approximates the lowest reported, historical inhalation LC50 (1-2 hr, 2300 ppm; p. 6, line 

6). However, this reviewer is unaware of any examples of independent factors (eg., age, 
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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 over 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 are 10-fold less than the 

historical LC50. 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 CERCLA 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 

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. 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 humans 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 associated 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) cytochrome 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 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 T3 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 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 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 same 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, Wilhite (1981) describes clinical symptoms of acute 
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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 (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 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 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 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 from 

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 the accumulated 

tissue dose of cyanide can approach levels of disposition associated with high 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 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 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 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-

14C-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 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 tissue 
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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 14C of 

the radiolabeled acetonitrile. Further, methods used by Ahmed et al. distinguished 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 from a systemic source after inhalation exposure. That 

wording may give incorrect emphasis to the present context.
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APPENDIX F

PEER REVIEW COMMENTS FROM COMMENTER EIGHT
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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, clear, 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 environment 

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 

earlier 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 EPA 
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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 III. 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.

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 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 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 presentation.

8. Page 18, Line 20. Change to “... were disposed on-site in ...”?

9. Page 24, 1st 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.
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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 

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 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. 

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 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 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 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 rudimentary extrapolation to estimate LC50 
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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. 
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APPENDIX G

PEER REVIEW COMMENTS FROM COMMENTER FOUR
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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. General 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 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, reviewed, 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, 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 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 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 consistent 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 acetonitrile 

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 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 
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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 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 cyanide 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, 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 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 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 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 was 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 Acetonitrile” (1999). Mortality 
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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.

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 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 conducted 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 correspond to cumulative 

acetonitrile exposure of only 3,000 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 signs, 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 cause mortality as a result of chronic exposure has no scientific support 

whatsoever.

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. 
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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.

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 concerns ought to identified explicitly.

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 les 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 possibility, but so is enzyme induction, which 

could lead to more rapid metabolism and clearance.

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 mortality 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!
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APPENDIX H

PEER REVIEW COMMENTS FROM COMMENTER FIVE
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Cyanide)” produced by EPA’s Office of Environmental Information

Prepared for:

Versar, Inc.,

Springfield, VA.

Prepared by:

Commenter Five

May, 2008
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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, complete 

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 even 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 at 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 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. 

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-ofevidence narrative is incomplete. 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-action 

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 6 
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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” – 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 indicate 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 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 Perspect. 86:93-98.

U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. [See section on writing a weightof-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 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. Toxicokinetic and mode of toxic action data are incompletely summarized. 

Data which are omitted or inadequately summarized include the following:
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(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 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 is likely 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 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 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-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 (detoxification). 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 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 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). 
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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 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 excessive 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 acetonitrile 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 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 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 deprivation, 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 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 biologically 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 degradation in 

function or an overwhelming of key biologic processes would predict that mortality would 
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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 cyanidecontaining 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-ofevidence 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 knowledge 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 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 further 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 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 toxic 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 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 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 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 the 
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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? 

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 and 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 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 toxicologically 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 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 
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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 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. 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 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 

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 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 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 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 
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encountered in early-death animals in other NTP studies (in addition 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 neurotoxicity 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 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 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 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 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 occurred 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 absence 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 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). 
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Based on these effects, the critical effects 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 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, 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 (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, 

developmental 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 

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.

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-dose 
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exposure to acetonitrile causes mortality is not scientifically defensible. 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). Second, based on what is known 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 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, 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 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 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 (1996) 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 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 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 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.
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III. SPECIFIC OBSERVATIONS

Provide specific observations, corrections, or comments on the document, mentioning 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 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 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 report.

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 acetonitrile than with the majority of 

these other cyanogenic compounds”?

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.”
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Beginning page 39, under Developmental and Reproductive Toxicity. Please note 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 moribund and death is 

imminent. Please clarify or modify.