# TOXICOLOGICAL REVIEW OF 

# ACETONITRILE (CAS No. 75-05-8) In Suppor tof Summar y Infor m ati o n on the Inte gr ate d Ri sk Infor m ati o n Sy ste m (IRIS)

January 1999

U.S. Environmental Protection Ag ency W a shing t on, DCDISCLAIM ER

Thi s docum enthas been revi ewed i n accordance wi th U.S

. Envi ronm ent a l P r ot ect i on Ag ency policy

. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Note: This document may underg o revisions in the future. The most up-to-date version will be made available electronically via the I R I S Home P a g e at http://www.epa.g ov/iris.

iiCONTENTS --TOXICOLOG ICAL REVIEW OF ACETONITRILE (CAS No. 75-05-8)

F O R E W O R D

................................................................ i v AUTHORS , CONTRI BUTORS , AND REVIEW ERS .................................v 1. IN TRODUCTI ON

......................................................... 1
2. CHEMI C AL AND P HYS I C AL IN FORMATI ON RELE VANT TO AS S E S S M ENTS .....2 3. TOXI COKINETI CS RELE VANT TO AS S E S S M ENTS ............................2
4

. H A Z A R D ID E N T IF I C A T I O N ................................................4 4.1. STUDIES IN HUMANS .................................................4 4.2. PRE-CHRONI C AND CHRONI C STUDIES AND CANCER B I OASSAYS IN ANIMAL S —ORAL AND INHALATI ON ..................................5 4.3. REPRODUCTIVE/DEVEL OPMENTAL STUDIES - ORAL AND INHALATI O N

. 10 4.4. OTHER STUDIES ....................................................1 3 4.4.1. Acute Data ....................................................1 3 4.4.2. Genotox icity ...................................................1 4 4.5. SYNTHESI S AND EVALUATI ON OF MAJ O R NONCANCER EFFECTS AND MODE OF ACTI ON—ORAL AND INHALATI O N ..........................1 5 4.6. W E I GHT OF EVID ENCE EVALUATI ON AND CANCER CLASSIFI CATI ON ...1 6 4.7. SUSCEPTI B LE POPULA TI ONS .........................................1 7 4.7.1. Possible Childhood Susceptibility ..................................1 7 4.7.2. Possible Gender Differences .......................................1 7 5

. D O S E R E S P O N S E A S S E S S M E N T S ..........................................1 7 5.1. ORAL REF E RENCE DOSE (RfD) .......................................1 7 5.2. INHALATI ON REFERENCE CONCENTRATI ON (RfC) .....................1 8 5.2.1. Choice of Principal Study and Critical Effect ..........................1 8 5.2.2. Methods of Analy s is .............................................1 9 5.2.3. RfC Derivation - In cluding Application of Uncertainty F actors and M o d i f yi n g F a c t o r s ...............................................1 9 5.3. Cancer Assessment ....................................................2 0
6. MAJ O R CONCLU S I ONS IN THE CHARACTERIZ ATI ON OF HAZ ARD AND DOS E R E S P O N S E ..............................................................2 0 6.1. HUMAN HAZ ARD POTENTIAL ........................................2 0 6.2. DOSE RESPONSE ....................................................2 1
7

. R E F E R E N C E S ...........................................................2 2
AP P E NDIX A: EXTERNAL P EER REVIEW —S UMMARY OF COMMENTS AND DI S P O S I TI ON ..........................................................2 8
iiiF O REWORD
The purpose of this Tox i colog i cal Review is to provide scientific support and rationale for the haz ard and dose-response assessment in I R I S pertaining to chronic ex posure to acet oni tri l e . It i s not i nt e nded t o be a comp rehensi v e tr eat i s e on th e chem i cal or t ox i col og i cal natu re of acet oni tri l e . In Se c tion 6, EPA ha s c har a c t e rize dits ove ra ll c onfide nc e in the qua ntitative a nd qualitative aspects of hazard and dose response. Matters considered in this characterization include knowledg e g aps, uncertainties, quality of data, and scientific controversies. This c har a c t e rization is pre s e n te d in a n e ffort to make appare nt the limitations of the a sse ssme n t a nd to aid and g u ide the risk assessor in the ensuing steps of the risk assessment process.
F or other g e neral information about this assessment or other questions relating to I R I S , the reader is referred to EPA’s Risk In formation Hotline at 202 -56 6

- 1676

.

ivAUTHORS , CONTRIBUTORS , AND REVIEWERS

Ch em i c al Man ager/ Au th or Mark Greenberg National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Ag ency Research Triang le Park, NC Revi ew ers Thi s docum entand sum m ary i n form at i on on I R I S have recei ved peer revi ew both by EP A scientists and by independent scientists ex ternal to EPA. Subsequent to ex ternal review and incorporation of comments, this assessment has underg one an Ag ency -wide review process whereby the I R I S Prog ram Director has achieved a consensus approval with EPA’s Prog ram Offices (the Offices for Air and Radiation, Planning and Evaluation, Prevention, Pesticides, and Tox i c Substances, Research and Development, Solid W a ste and Emerg e ncy Response, and W ater) and the ten Reg i onal Offices.
In te rn al E P

A R evi ew ers Kathe rine Anitole , Ph.D. Risk Assessment Division O PPTS David L a i, Ph.D. Risk Assessment Division O PPTS
E

x te rn al Peer R evi ew ers Shayne C. Gad, Ph.D. Ga d Consulting Se rvic e s Ra le ig h, NC C.C. Conaway , Ph.D. American Health F oundation Va lha lla , NY Edward J

. Sowinski, Ph.D. Environmental Health Manag e ment and Science, In c.

vAUTHORS , CONTRIBUTORS , AND REVIEWERS (con tin u e d )

Summaries of the ex ternal peer reviewers’ comments and the disposition of their recommendations are in Appendix A.

vi1. INTRODUCTION
This document presents backg r ound and justification for the haz ard and dose-response assessment summaries in EPA’s In teg r ated Risk In formation Sy stem (I RI S). I R I S summaries may include an oral reference dose (RfD), inhalation reference concentration (RfC) and a carci nog eni c i t y assessm ent

. The RfD and RfC provide quantitative information for noncancer dose-response a sse ssme n ts. The RfD is ba se d on the a ssumption thatthre sholds e x ist for c e rta in tox i c e ffe c t s such as cel l u l ar necrosi s but m a y notex i s tfor other t o x i c effect s such as som e carci nog eni c responses. Itis ex pressed in units of mg /kg

- day

. In g e neral, the RfD is an estimate (with uncertainty spanning perhaps an order of mag n itude) of a daily ex posure to the human population (including sensitive subg roups) that is likely to be without an appreciable risk of deleterious nonc a n c e r e ffe c t s during a life time

. The inha lation RfC is a n a l og ous to the ora l RfD, but provides a continuous inhalation ex posure estimate. The inhalation RfC considers tox i c effects for both the respiratory sy stem (portal-of-entry ) and for effects peripheral to the respiratory sy stem (ex trarespiratory or sy stemic effects). Itis g e nerally ex pressed in units of mg /m 3

. The carcinog enicity assessment provides information on the carcinog enic haz ard potential of the substa nc e in que stion a nd qua ntitative e s timate s of risk from ora l e x posure a nd inha lation ex posure. The information includes a weig ht-of-evidence judg ment of the likelihood that the ag ent is a human carcinog en and the conditions under which the carcinog enic effects may be e x pre sse d. Qua n titative risk e s timate s ar e pre s e n te d in thre e wa y s . The slope factor is the re sult of application of a low-dose ex trapolation procedure and is presented as the risk per mg /kg / day

. The unit risk is the qua ntitative e s timate in te rms of e ithe r risk pe r μ g /L drinking wate r or risk per � g/m 3 air breathed. Another form in which risk is presented is a drinking water or air concentration providing cancer risks of 1 in 10,000; 1 in 100,000; or 1 in 1,000,000. Development of these haz ard identification and dose-response assessments for acet oni tri l e has fol l o wed th e g e neral g u i d el i n es for ri sk assessm entas setforth by th e Nat i onal Research Council (1983). EPA g u idelines that were used in the development of this assessment ma y inc l ude the following : the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986a), Guide line s for the He alth Risk Asse ssme n t of Che m ic al Mix t ure s(U.S. EPA, 1986b), Guide line s for Mutagenicity Risk Assessment (U.S. EPA, 1986c), Gui del i n es f o r Devel opmenta l T oxi ci t y Risk Assessment (U.S. EPA, 1991), Guide line s for Ne urotox ic ity Risk Asse ssme n t (U.S. EPA, 1998a), Proposed Guidelines for Carcinogen Risk Assessment (1996a), and Reproductive Tox i c ity Risk Asse ssme n t Guide line s (U.S. EPA, 1996b); Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S. EPA, 1988); (proposed) Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity (U.S. EPA, 1994a); Methods for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b); Peer Review and Peer Involvement at the U.S. Envi ronmenta lProt ect i on Agency(U.S. EPA, 1994c); Use of the Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995); Science Policy Council Handbook: Peer Review (U.S. EPA, 1998b); and memorandum from EPA Administrator, Carol B r owner, dated March 21, 1995, Subject: Guidance on Risk Characteriz ation.
1Literature search strateg y employ ed for this compound were based on the CASRN and at le a s t one c o mmon na me . At a minimum, the following databa se s we re se ar c h e d : RTECS, HSDB , TSCATS, CCRI S, GENETOX, EMI C , EMI C B A CK, DART, ETI CB ACK, TOXLIN E, CANCERLINE, MEDLIN E, a nd MEDLIN E ba c k file s. Any pe rtine n t sc ie ntific information submitted by the public to the I R I S Submission Desk was also considered in the development of this document.

2. CHEMICAL AND P H YSICAL INF O RMATION RELEVANT TO ASSESSMENTS Acetonitrile (ACN) is also known as cy anomethane or methy l cy anide. Some relevant phy sical and chemical properties of ACN are listed below (W HO, 1993): CAS Reg i stry number: 75-05-8 Empirical formula: C H 3 CN Molecular weig ht: 41.05 Vapor pressure: 74 mmHg at 20 � C Wate r solubility : i nfinite ly soluble Lo g K OW : - 0.34 Conversion factor: 1 ppm = 1.68 mg /m 3 , 1 mg /m 3 = 0.595 ppm (25 � C, 760 mmHg ) At room temperature, ACN is a volatile, colorless liquid with etherlike odor. Itis one of the most stable nitriles. Acetonitrile has a TLV-TW A of 40 ppm (67 mg /m 3 ), with a short-te rm ex posure limit (STEL ) of 60 ppm (101 mg /m 3 ), recommended to protect ag ainst org a nic cy anide poisoning and injury to the respiratory tract (ACGI H , 1991). Althoug h nitriles are widely used to synthesiz e amines, amides, ketones, aldehy des, and a variety of other compounds (Kirk-Othmer Concise Ency clopedia of Chemical Technolog y , 1985), ACN is used primarily as a solvent. Nitriles in g e neral are hy droly z ed in the presence of acidic conditions to form amides. Althoug h ACN is one of the more stable nitriles, acidic hy droly s is would be ex pected to yi eld hy drog en cy anide. Hy droly s is in water has been reported as ex tremely slow (WHO, 1993). Willhite (1983) found that freshly prepared solutions of ACN in distilled water did not underg o any sig n ificant hy droly s is upon incubation at 37 � C for 2.5 hours.

3. TOXICOK I NETICS RELEVANT TO AS S E S S M ENTS
Inorg a nic cy anide has long been known to react with trivalent iron of cytochrome ox idase in mitochondria and block the reduction of ox y g en needed for cellular respiration, thus leading to cytotox i c anox ia (Albaum et al., 1946). The tox i city of ACN is believed to be mediated, in part, throug h this mechanism. ACN is metaboliz ed to inorg a nic cy anide, but the conversion occurs slowly compared to other nitriles (which may ex plain the delay in onset of acute symptoms). Freeman and Hay e s’ (1988) data sug g e st that the conversion to cy anide is ox y g en- and NADPHdependent, possibly mediated by P450 isoz ym e (2E1 or P-450j). Some investig ators sug g e st that ACN produces cy anohy drin by a P 450 reaction, which is then decomposed by catalase to release
2cy anide (Ahmed et al., 1992; F e ierman and Cederbaum, 1989; Willhite and Smith, 1981). F o rmaldehy de and formic acid are also postulated to be by -products of ACN metabolism (Ahmed et al., 1992). Cy anide can be further ox idiz ed to thiocy anate, a less tox i c compound that is ex creted in urine, but one that may interfere with thy r oid function (Hartung , 1981). Conversion is mediated by rhodanese, a sulfurtransferase found in liver and human nasal respiratory mucosa (L ewis et al., 1991). A minor urinary metabolite that has been detected after administration of ACN in drinking water to rats is 2-aminothiolaz ine-4-carbox y lic acid (Swenne et al., 1996). Cy anide also can be ox idiz ed to cy anate ion with further ox idation to formic acid (McMahon and B i rnbaum, 1990).
Li ke hy drog en cy ani d e (HC N ), AC N i s readi l y absorbed fromth e l ung s and g a strointestinal tract, and is distributed throug hout the body in both humans and laboratory animals. In a g r oup of male and female test subjects (16), 74% of inhaled ACN was absorbed when cig arette smoke was held in the mouth for 2 seconds (and not inhaled), and 91% was absorbed when smoke was inhaled (Dalhamn et al., 1968a,b). Autopsy of an individual who died 2 day s fol l o wi ng i nhal at i on of AC N vapors showed th atcy ani d e reaches th e spl een, l ung s, and kidney s , but was not detected in the liver (W HO, 1993). Oral ex posure of animals resulted in metabolites found primarily in the spleen, stomach, and skin (U.S. EPA, 1985a). ACN serum concentrations were hig h er than cy anide levels in an individual hospitaliz ed after oral ACN ing e stion (Michaelis et al., 1991); elimination half-lives were 32 hours for ACN and 15 hours for cy anide. Cy anide was not detected in the blood of three human subjects ex posed to concentrations of 40, 80, or 160 ppm for 4 hours; a slig ht increase in thiocy anate levels was detected in urine (Poz z a ni et al., 1959). Hy drocy a nic acid was found in brain, heart, kidney , and spleen of rats after inhalation of ACN vapors (Hag uenoer et al., 1975). Cy anide and thiocy anate were measured in blood, liver, brain, and kidney of hamsters following oral ex posure (Willhite, 1983); thiocy anate in blood, kidney , and liver was up to 10-fold hig h er than in the brain, while cy anide in blood and liver was g e nerally hig h er than in the brain and kidney 2.5 hours after ex posure to a sing le dose of 100, 200, or 400 mg /kg ACN. Ex posure of rats to ACN in drinking water indicated that the rat has a hig h capacity for cy anide detox i fication on both normal and low-protein diets. On a low-protein diet, cy anide detox i fication is at the ex pense of protein catabolism (Swenne et al., 1996). Absorption of ACN is rapid in beag le dog s ex posed to 16,000 ppm ACN (26,880 mg /m 3 ) vapors for 4 hours, based on blood cy anide concentrations peaking and reaching steady

- state concentrations of 305-433 μ g /100 mL after approx imately 3 hours (Poz z a ni et al., 1959). W ith long er-term ex posure in monkey s inhaling 350 ppm for approx imately 99 day s , blood samples on the 35th day of ex posure, after a 2-day rest period (i.e., a weekend), did not detect cy anide ion, but 7.6-9.2 μ g cy anide ion/100 mL was measured after the 39th day of ex posure (i.e., following 5 consecut i v e day s of ex posure). Thi o cy anat e was det ect ed i n th e uri n e aft e r th e 2-day break, and accumulated over the 5-day ex posure week (P oz z a ni et al., 1959). Rats ex posed to 166 or 300 ppm, 7 hours/day , 5 day s /week, for 90 day s had almost complete urinary ex cretion of thiocy anate after the 2.5-day rest period each week (P oz z a ni et al., 1959). ACN conversion to cy anide appears to be about 10-fold g r eater in rat nasal ethmoturbinate microsomes than in liver microsomes (Dahl and W arusz ewski, 1989), possibly
3because of hig h P 450 content. The nasal cavity , in particular the olfactory reg i on, has a hig h concentration of rhodanese (Dahl and W arusz ewski, 1989; Dahl, 1989). Rhodanese has also been found in the respiratory epithelium of human nasal tissue (L ewis et al., 1991); these in vitro studies sug g e st that rhodanese in smokers may have reduced affinity for cy anide. L e wis and c o lle a g ue s a l so c a l c u late thatthe c ap a c ity of rat na sa l tissue is suffic i e n t to me tabolize a max imum of 2,800 ppm inhaled HCN. The hig h concentration of rhodanese in rat nasal tissue sug g e sts thatc y a n ide ma y be de tox i fie dmore rapidly to thioc y a n at e tha n in a n ima l spe c i e s with lower amounts of the enz yme (Dahl and W arusz ewski, 1989). Aminlari et al. (1994) demonstrated that dog s had a g r eater rhodanese activity in the nasal cavity than in the lower respi r at ory tr act

. W hole body autoradiog raphy in male mice injected intravenously with ACN radiolabeled with 14 C in the methy l g r oup indicated that radioactivity was widely distributed throug hout the body (e.g ., liver, thym us, and reproductive org a ns). In teresting l y , nonvolatile radioactivity was also observed in nasal secretions, mouth cavity , esophag u s, and stomach contents (Ahmed et al., 1992). One could infer from these observations that ACN could also distribute to the stomach upon inhalation ex posure.

4. HAZARD IDENTIF I CATION 4.1. STUDIES IN HUMANS Other than one case-referent study , there are no epidemiolog i cal studies of effects of ACN ex posure in humans. Individuals who were acutely ex posed to ACN developed effects g e nerally attributed to metabolism of ACN to cy anide (U.S. EPA, 1985a). Case reports of acute occupational ex posure to ACN indicate that workers ex hibited nausea, shallow and/or irreg u lar respiration, and impaired motor activity

. An autopsy of a worker who died shortly after ex posure revealed cerebral, thy r oid, liver, splenic, and renal cong estion (W HO, 1993). Gastric erosion has been reported in individuals who ing e sted ACN (W ay , 1981; B a llantyn e, 1983).
In a clinical study by Poz z a ni et al. (1959), two men who inhaled 40 ppm ACN (67 mg /m 3 ) for 4 hours did not develop any adverse subjective effects, and blood cy anide and urinary thiocy anate were not appreciably increased. A third subject, also ex posed to the same conditions, ex perienced slig ht chest tig htness the evening after ex posure, and cooling sensation in the lung s th e fol l o wi ng day

. Onl y an i n crease i n th i o cy anat e i n th e uri n e was evi d ent ; no cy ani d e was detected in the blood. “Olfactory fatig ue” was reported during ex posure for all subjects. Nine day s later, two of the subjects inhaled 160 ppm ACN vapor (269 mg /m3 ) for 4 hours. F l ushing of face and chest t i g ht ness were report e d 5 day s aft e r ex posure, but th ere were no chang e s i n uri n ary thiocy anate and blood cy anide from pre-ex posure values.
W HO (1993) has reported on several cases of children or adults who ing e sted larg e amounts of ACN ( � 250 to 4,000 mg /kg ) ; symptoms included vomiting , respiratory distress, confusion, convulsions, seiz ures, and pulmonary edema, and in some instances, death occurred. Cy anide was detected in the blood of these individuals.

4In a case-referent study of medical records of F i nnish women employ ed in state laboratories that was desig n ed to determine if there was an association between spontaneous abortion and ex posure, the odds ratio for seven cases involving ex posure to ACN was close to unity (Taskinen et al., 1994). However, the small number of cases and other limitations (e.g ., ex posure was self-reported) of this study preclude any definitive conclusions.

4.2. P R E-CHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN ANIMALS —ORAL AND INHALATION

The National Tox i colog y Prog ram (NTP, 1996) evaluated the tox i city of ACN to the rat and mouse in both subchronic and chronic inhalation studies. In the 13-week subchronic study that served to set ex posure levels for the chronic study , F 344/N rats and B 6 C3F 1 mice (10/sex / g r oup) were ex posed whole-body to ACN concentrations of 0, 100, 200, 400, 800, or 1,600 ppm (0, 168, 336, 672, 1,343, or 2,686 mg /m 3 ), 6 hours/day , 5 day s /week (durationadjusted concentrations were 0, 30, 60, 120, 240, and 480 mg /m3 ). Purity of ACN was 99% or g r eater and actual concentrations were within 10% of targ et concentrations. Clinical observation and body weig hts were recorded weekly

. At necropsy , brain, heart, kidney , liver, lung s, testis, and thym us weig hts were measured, hematolog y was performed, and thy r oid hormone assay s were conducted. All org a ns of animals ex posed to 0, 800 ppm (males only ) , and 1,600 ppm were ex amined by histopatholog y . Selected org a ns of 800-ppm females (bone marrow, brain, lung , lymph node, ovary , spleen, thym us) and 400-ppm males (bone marrow, testes, thym us) were subjected to histopatholog ical ex amination; additional org a ns were ex amined if they ex hibited g r oss lesions.
In rats, deaths occurred at 800 ppm (1 male) and 1,600 ppm (6 males and 3 females). All but one of the deaths occurred during the first 2 weeks of ex posure. There was a sig n ificant decrease in body weig ht g a in and final body weig ht at 1,600 ppm (81% of control for males; 91% for females); no chang e occurred in the other g r oups. Clinical sig n s at the two hig h -concentration g r oups included hy poactivity and ruffled fur during the first week. Atax ia, abnormal posture, and clonic convulsions occurred in the 1,600-ppm males that died. The other g r oups did not ex hibit any treatment-related sig n s. Thym us weig hts were sig n ificantly lower in 800- and 1,600-ppm rats (both sex e s), compared to those of the controls. Sig n ificant decreases in red blood cell count, hemog l obin concentration, and hematocrit occurred in the 800 and 1,600-ppm females and 1,600-ppm males. The investig ators reported that these alterations were sug g e stive of anemia (characteriz ed as nonresponsive, normocytic, and normochromic) since reticulocyt e counts, mean cell volume, and mean cell hemog l obin concentration were similar to controls. The 1,600-ppm females also ex hibited a decrease in triiodothy ronine (T3) concentration, without chang e s in thyr ox ine (T4) and thyr oid-stimulating hormone (TSH) concentrations. Histopatholog ic effects were limited to rats that died at 800 and 1,600 ppm; effects included cong estion, edema, and hemorrhag e in alveoli observed in lung s (no incidence data were provided). B ecause of the one death at 800 ppm (in week 1), coupled with mortality in the mouse (see below) at a lower concentrations, it is prudent to reg ard 800 ppm as a F E L (F EL [ ADJ ] = 239 mg /m 3 ) for the rat. B ecause only limited histopatholog y (i.e., bone marrow, thym us, and testes) was conducted in animals ex posed to 400 ppm (males only ) , the results are insufficient to identify a no-observed-adverse-effect-level (NOAEL )

. 5In mice, deaths were observed at concentrations of 400 ppm and g r eater (0/10, 0/10, 0/10, 0/10, 1/10, 10/10 in males; 0/10, 0/10, 0/10, 1/10, 4/10, 10/10 in females). All animals in the 1,600-ppm g r oups died by week 4 of the study

. F i nal body weig ht (92% of control) and body weig ht g a in were sig n ificantly reduced at 400 ppm in males, but are not considered tox i c o log i c a lly sig n ific a n t. He matolog i c a l para me te rs we re not e v a l uate d in mic e . Ma le s e x hibite d a sig n ific a n t c onc e n tration-re late d inc r e a s e in ab solute ( � 200 ppm; p � 0.05) and re lative ( � 100 ppm; p � 0.01) liver weig ht. Sig n ificant increased relative lung and kidney weig hts in some male g r oups were also observed, but chang e s were not concentration-related. F e males had a sig n ificant increase in absolute liver weig ht at 800 ppm and in relative weig ht at � 400 ppm. Histopatholog y revealed an increased incidence of hepatocellular vacuolation, with sig n ificance at 400 and 800 ppm ( p � 0.01) (0/10, N/A, 0/10, 8/10, 7/9, and 0/10 in males; 0/10, N/A, 0/10, 7/10, 6/10, and 0/10 in females; 100 ppm males and females were not ex amined). No other hepatic effects were observed. Vacuoliz ation was considered to represent increased g l y c og en st orag e by di st ensi on of previ ousl y ex i s t i n g cl ear spaces. S e veri t y of vacuol i z at i on was characterized as moderate in the 800-ppm female g r oup and mild in males. No hepatocellular vacuoliz ation was observed in the 1,600-ppm animals that died during the study

. The absence of this chang e in the 1,600-ppm animals may be indicative of an increased utilization of g l y c og en stores by the animals that died. In cidences of forestomach squamous epithelial hy perplasia were sig n ificantly increased in 800-ppm males and in females ex posed to � 200 ppm. The incidences were 0/10, 0/10, 3/10, 6/9, and 1/9 in males (100-ppm males were not ex amined) and 0/10, 0/10, 7/10, 8/10, 7/10, and 5/10 in females; severity was not concentration-related. Hyperkeratosis and infla mmatory c e ll infiltrat e (e ffe c t s a ssoc i at e dwith hype rpla sia ) a l so oc c u rre d in the forest om ach. A si g n i f i cant i n crease i n focal ul cers of th e forest om ach was al so observed i n 1,600-ppm female mice. There were no effects reported for the lung s. The study identified a NOAEL of 200 ppm (NOAEL [ ADJ ] = 60 mg /m 3 ) based on mortality

. The level of 400 ppm is considered a F E L g i ven the early death (week 2) of one female mouse in the 400 ppm g r oup and increased mortality (one male and four females) at 800 ppm. Neither a NOAEL nor lowest-observed-adverse-effect level (L OAEL ) can be identified for fore stoma c h le sions ina s muc h a s g r ooming of c onta minat e dfur a nd/or muc o c iliary c l e ar a n c e l i k el y was th e pri m ary cause of th e i n creased i n ci dence i n hy perpl a si a of th e forest om ach. Hy perplasia is considered adverse because it was associated with infiltration of inflammatory cells and, at the hig h est concentration in females, focal ulcers.
B a sed on the finding s in the 13-week study , the two-y ear study was initiated with F 344/N rats (56/sex / g r oup) ex posed to actual ACN concentrations of 0, 100, 200, or 400 ppm (0, 168, 336, or 672 mg /m 3 ), 6 hours/day , 5 day s /week, for 103 weeks (duration adjusted to 30, 60, and 120 mg /m 3 ) and B 6 C3F 1 mice ex posed to concentrations of 0, 50, 100, or 200 ppm (0, 84, 168, or 336 mg /m 3 ) for 111 weeks (duration adjusted to 15, 30, and 60 mg /m3 ). An interim necropsy at 15 months involved 8 rats (each sex ) and 10 mice (each sex )

. Complete histopatholog ical ex aminations were conducted on all animals at this time and hematolog i cal parameters and liver, kidney , and lung weig hts were measured. Clinical sig n s and body weig ht were assessed throug hout the study

. After 2 y ears, animals were necropsied and ex amined for g r oss and mic rosc opic a lte rations.

6At the 15-month interim necropsy for rats, hematolog i cal alterations were observed, but we re sig n ific a n t ( p � 0.05) only at the hig h concentration. Chang e s included decreased mean cell volume and mean cell hemog l obin (in both sex e s), increased red cell count (males), and decreased hematocrit and hemog l obin (females); none of these effects was concentration-related. At this time, there were no neoplastic or nonneoplastic lesions observed in tissues ex amined that were attributable to ex posure. Hematolog i cal parameters were not measured at study end. No sig n ificant chang e s in survival, body weig hts, or clinical appearance were observed in rats following 103-week ex posure to ACN. Histopatholog ical ex amination revealed no neoplastic or nonneoplastic lesions in any org a ns of ex posed females. In male rats, a statistically sig n ificant increase in the incidence of basophilic hepatic foci was observed in the 200- and 400- ppm g r oups (15/48, 22/47, 25/48, and 31/48), but the foci were not aty p ical in appearance. Thus, it is uncertain if these lesions are preneoplastic. The incidences of eosinophilic and mix ed cell foci were marg inally elevated in 400-ppm males, but were not statistically sig n ificant. Althoug h the r e wa s a marg ina lly sig n ific a n t positive tre nd in the inc i de nc e of a d e noma , c ar c i noma , or adenoma and carcinoma (combined) in liver of male rats, no sig n ificant dose-related trend was presentaft e r i n ci dences were adj u st ed for survi v al usi n g th e l i f e t abl e t e st

. The i n ci dences of he patoc e llular a d e noma s a nd c ar c i noma s in ma le rats we re not sig n ific a n tly inc r e a s e din the treated animals based on pairwise comparison with incidences in control animals. Also, the tumor incidences at 400 ppm were only slig htly hig h er than the historical control rang e. Other e ffe c t s obse r ve d in ma le rats inc l ude d marg ina l (not c onc e n tration-re late d) inc r e a s e s in tumors in the adrenal medulla and pancreatic islets; incidences observed were within historical control rang e. Keratoacanthoma was observed in the skin of 400-ppm males (0/48, 1/47, 0/48, and 4/48), but was not considered treatment-related; the incidence was within the historical control rang e. Althoug h an increased incidence of basophilic foci is g e nerally considered a possible preneoplastic effect and, thus, appropriate for discussion of potential carcinog enicity , the incidence is not considered evidence of a hepatotox i c effect nor a precursor to an hepatotox i c effect. As support for this view, hy drog en cy anide (U.S. EPA, 1985b) has not been found to cause adverse l i v er effect s i n ratfeedi n g studi es nor has i tbeen associ at ed wi thl i v er effect s i n human occupational studies. Thus, a NOAEL of 400 ppm (NOAEL [ ADJ ] = 120 mg /m 3 ) was ide n tifie d for the rat.
In m i ce, no chang e s i n th e survi v al of th e tr eat ed ani m a l s were observed, comp ared t o th e survival in control animals. B ody weig hts were similar for all g r oups, and treatment-related clinical sig n s were not evident. In contrast to the 13-week study , there were no concentration-related effects of liver weig ht, sug g e sting that the chang e s observed in the 13-week study were adaptive. At the 15-month interim sacrifice, the only nonneoplastic chang e observed was a si g n i f i cant i n crease i n th e i n ci dence of squam ous hyperpl a si a i n th e forest om ach of 200-ppm females (incidences were 0/10, 1/10, 0/10, and 6/10). At terminal sacrifice, the i n ci dence of al veol ar/ b ronchi ol ar adenom as was si g n i f i cant l y i n creased i n m a l e m i ce fol l o wi ng a d ministration of the hig h c onc e n tration ( p = 0.011) (6/50, 9/50, 8/48, and 18/50). Combined i n ci dences of al veol ar/ b ronchi ol ar adenom as or carci nom as were al so si g n i f i cant l y i n creased i n 200-ppm males ( p = 0.042) (10/50, 14/50, 14/48, and 21/50). The 100-ppm males ex hibited a statistically sig n ificant increased incidence of hepatocellular carcinoma (7/50, 11/50, 13/49, and 7/50) ( p = 0.038) and combined adenoma or carcinoma (19/50, 21/50, 30/49, and 15/50)

7( p = 0.013), with incidences g r eater than those observed in historical controls. Because the incidence of this lesion did not increase with increasing concentration, it was considered a spora d ic finding

. Unlike the rat study , ba sophilic live r c e ll foc i we re not obse r ve d in the mouse . The inc i de nc e s of he patoc e llular a d e noma or c ar c i noma (c ombine d) in fe ma le s we re similar to controls. No increases in the incidence of lung tumors were observed in female mice. F o restomach squamous hy perplasia was sig n ificantly increased in 200-ppm males (3/49, 3/50, 6/48, and 12/50) and in 100- and 200-ppm females (2/49, 7/50, 9/50, and 19/48); however, severity of the effect was not concentration-related. The incidence at 200 ppm equaled the hig h e s t va lue s obse r ve d in historic a l c ontrols. The inc i de nc e of squa mous c e ll papilloma s in the forestomach was slig htly increased after 2 y ears (incidences were 0/49, 0/50, 1/48, and 2/5 in males; 1/49, 0/50, 1/50, and 3/48 in females); however, these increases were not statistically sig n ificant and were within the rang e of historical control values. Althoug h forestomach hy perplasia in mice is clearly associated with ex posure to ACN, the role of inhaled concentrations in eliciting these lesions is not known. Itis likely that preening a c tivitie s a nd/or muc o c iliary c l e ar a n c e , re sulting in ora l ing e stion of ACN, pla y a

c e n tra l role

. Thus, it is not possible to identify either a NOAEL or L OAEL attributable to inhalation ex posure in the chronic study

. The absence of these lesions in the rat study is puz z ling

. In a study by W o lff et al. (1982), whole-body ex posure versus nose-only ex posure of rats to radiolabeled fine particles indicated that 60% of the pelt burden was calculated to be ing e sted following whole-body ex posure. Subchronic studies (Poz z a ni et al., 1959) were performed on Carworth F arms-W i star rats (15/sex / g r oup) ex posed to 0, 166, 330, or 655 ppm ACN vapors (0, 278, 554, or 1,100 mg /m 3 ), 7 hours/day , 5 day s /week, for 90 day s (duration-adjusted to 0, 58, 115, and 229 mg /m 3 ). The purity of the ACN was not reported. B ody , liver, and kidney weig hts were determined, and histopatholog y was performed on liver and lung s (any effects in these org a ns resulted in ex amination of brain, pancreas, spleen, trachea, and testis). Hematocrit and hemog l obin values were measured in 5 rats in the 655 ppm g r oup and controls 4 day s prior to ex posure and on the 53 rd , 72 nd , and 89 thday s . The values with ex posed animals were no different from controls. Ex posure to ACN did not affect body or org a n weig hts in ex posed rats, and no deaths attributable to ex posure were reported. Patholog ical effects were limited primarily to the 655-ppm g r oup; alveolar capillary cong estion, focal edema, bronchial inflammation, desquamation, and hypersecretion of mucus occurred in lung s (10/27; p = 0.001), tubular swelling in kidney s (8/27; p = 0.05), and central cloudy swelling in liver (7/27; p = 0.04). No lesions were reported for other org a ns at 655 ppm. In the other g r oups, histiocyte clumps in alveoli or atelectasis (2/28 at 166 ppm), as well as bronchitis and pneumonia (3/26 at 330 ppm) were reported. Hematocrit and hemog l obin values for five female rats (males not evaluated) were similar to controls at 655 ppm. No treatment-related tumors developed in any g r oups. In terpretation of study results was limited by incomplete histopatholog y (e.g ., stomach and thym us) and a lack of details about protocol. An unambig uous NOAEL and L OAEL could not be identified. The investig ators also ex amined effects of 350 ppm ACN (588 mg /m 3 ) on thre e ma le rhesus monkey s and three male dog s (2 B a senji-Cocker hy brid and one B a senji-Chow × Spring er spaniel hy brid), 7 hours/day , 5 day s /week, for 91 day s (duration-adjusted to 123 mg /m 3 ). The purity of the ACN was not reported. Controls consisted of two male B a senji-Cocker hy brid
8dog s; there was no control g r oup for monkey s . In dog s and monkey s , focal emphy sema and diffuse proliferation of alveolar septa were ex hibited. Monkey s also showed hemosiderin accumulation in lung s and swelling of convoluted tubules in kidney s . In terpretation of this e x pe rime nt wa s limite d by ina d e quate me a s ure m e n t of c h a mbe r c onc e n trations a nd the la c k of a control g r oup for monkey s . In a separate inhalation study (7 hours/day , 5 day s /week) with 4 rhesus monkey s , the one monkey (female) ex posed to 2,510 ppm died on the second day , the two female monkey s ex posed at 660 ppm died by day 51, and the one male monkey ex posed to 330 ppm wa s sa c rific e datda y 99 of e x posure . At thattime it e x hibite d c onside r ab le e x c itability

. Each of three monkey s in the 2,510, 660, and 330 concentration g r oups was found to have dural and subdural hemorrhag es. The two monkey s in the 660-ppm g r oup had focal areas of emphy sema and cloudy swelling of the prox imal and convoluted tubules of the kidney

. An unpublished 90-day inhalation study in the B 6 C3F 1 mouse was conducted by Haz e lton L aboratories (1983a) for the National Tox i colog y Prog ram (NTP). In this study , male and female mice (10/sex / g r oup) were ex posed by inhalation to ACN concentrations (purity >99%) of 0, 25, 50, 100, 200, and 400 ppm (0, 42, 84, 168, 336, and 672 mg /m 3 ) for 6.5 hours/day , 5 day s /week for a total of 65 ex posures during a 92-day period. The duration-adjusted concentrations were 0, 8.1, 16.2, 32.5, 65, and 130 mg /m3

. Chamber atmospheres were monitored every 30 minutes using infrared spectroscopic methods. Actual mean concentrations were all within ± 15% of nominal concentrations. Histopatholog ical ex amination at necropsy included all major tissues and org a ns, including thym us, testes, ovaries, and lung s from controls and 400-ppm g r oup mice. Three sections of the nasal turbinates were ex amined from all animals in all g r oups. Li vers were ex amined from mice in 100- and 200-ppm g r oups as well. Clinical chemistry and hematolog i cal parameters were also ex amined. All animals from control, 100-, 200-, and 400-ppm g r oups at terminal necropsy were subjected to ex amination of sperm motility , count, and sperm head staining

. Separate g r oups of females were ex posed in the same study to 0, 100, 200, and 400 ppm ACN for 6.5 hours/day , 5 day s /week for a total of 10 ex posures and used for immunotox icolog y studies. Three male mice died during the course of the study (one in each of the 50, 200, and 400- ppm g r oups). Mortality was not considered to be ex posure-related. In contrast to the finding s from a 14-day study (ImmuQuest L aboratories In c., 1984; see Section 4.4) in the same strain of mice, there were no reported histopatholog ical effects on the thym us. Thym us/body weig ht ratios were somewhat lower in the 200- and 400-ppm g r oups compared to controls, but were not sig n ificantly decreased. Other altered terminal org a n/body weig ht and terminal org a n/brain weig ht ratios were mentioned, but tabular data were not included with the final report. There were no adverse effect s on spermor i n th e nasal tu rbi n at es. There were no adverse l i v er effect s observed upon histopatholog y . Althoug h cytoplasmic vacuoliz ation was observed in all animals, including those in the control g r oup, the vacuoliz ation in animals in the 200- and 400-ppm g r oups was only slig htly g r eater than those in animals of the 100-ppm and control g r oups. In the g r oup of females ex amined for hematolog i c and immunotox ic responses, all ex posure g r oups ex hibited sig n ificant decreases in hematocrit, hemog l obin, and red blood cell counts. They were described as of low mag n itude and of questionable biolog ical sig n ificance. Lymphocyt e counts were decreased only in the 200- and 400-ppm g r oups. I g G was sig n ificantly decreased in all ex posure g r oups in a concentration-related manner. These decreases in I g G are
9consistent with the finding s in the ImmuQuest stud y (see Section 4.4) at these concentrations. Other tests of immune function (e.g ., l ymphoc yt e proliferation, dela y e d h y persensitivit y , host resistance) were unaffected b y e x posure; thus, the depressed I g G is of uncertain sig n ificance. The hematolog i cal and hepatic effects in mice were used as the basis for deriving an oral RfD th atwas previ ousl y pl aced on I R I S

. In asm u ch as (1) th ese hem at o l o g i cal and hepat o cel l u l ar effects were of questionable biolog ical sig n ificance, (2) hematolog i cal parameters were unaffected in the NTP (1996) subchronic and chronic rat stud y (these parameters were not measured in the mouse portion of the stud y ), and (3) hepatic vacuoliz ation was not observed in the chronic mouse stud y , it was recommended that the RfD for ACN be withdrawn from I R I S

. In a stud y with the F 344 rat using the same e x posure and e x amination protocols, there were no adverse g r oss or histopatholog ical effects (Haz e l ton L aboratories, 1983b). As part of a developmental to x i cit y stud y (Mast et al., 1994), nonpreg nant female Sprag u e-Dawle y rats (10/g r oup) were e x posed for 14 consecutive da y s to 0, 100, 400, or 1,200 ppm ACN (168, 672, and 2,015 mg /m 3 ). One ani m a l at th e hi g h concentr at i on di ed; no treatment-related clinical sig n s or bod y weig ht chang e s were evident in e x posed animals. Gross e x am i n at i on di d notreveal an y si g n i f i canteffect s i n th e e x posed ani m a l s . Willhite (1981) e x posed CD-1 mice to ACN for 60 minutes to determine the lethal concentration ( L C). The L C 50 value was determined to be 2,693 ppm (4,524 mg /m 3 ).

4.3. RE P R ODUCTIVE/DEVELO P M ENTAL STUDIES – ORAL AND INHALATION Mast et al. (1994) e x posed positivel y mated female Sprag u e-Dawle y rats (33/g r oup) to 0, 100, 400, or 1,200 ppm (0, 168, 672, and 2,015 mg /m 3 ) ACN, 6 hours/da y , 7 da y s /week during g e station da y s 6-19, and then sacrificed on g e stational da y 20. Controls consisted of 10 nonpreg nant females per e x posure g r oup. The 1,200-ppm dams e x hibited h y poactivit y (14/33) and appeared emaciated (6/33). Deaths occurred in two 1,200-ppm dams and one 400-ppm dam. The death at 400 ppm was sug g e sted b y the investig ators to be the result of a possible spontaneous cerebral hemorrhag e. There was no effect on bod y or org a n weig hts in preg nant da ms. F e rtilit y did not appe ar to be a ffe c t e db y ACN e x posure ; me a n pre g na nc y rate for spe rmpositive fe ma le s wa s 79%. A slig ht inc r e a s e in pe rc e n ta g e of re sorptions pe r litte r (partic ular ly late resorptions) was seen at the hig h concentration, but the effect was not sig n ificant or concentration-related. The percent of live fetuses per litter was not affected for an y g r oup, nor were there treatment-related fetal malformations. The onl y effect on skeletal variations was a sig n ificant increase in percent of supernumerary ribs per litter at 100 ppm, but the effect did not occur for the other g r oups. W h ile ACN was readil y measurable in maternal blood (determined in a separate g r oup of nonpreg nant animals), c y anide was not detectable e x cept in the 1,200 ppm g r oup. Considering that mortalit y was treatment-related at 1,200 ppm and e x posure ma y also have pla y ed a role in the one death at 400 ppm, it is prudent to consider 400 ppm (672 mg /m 3 ) as an FE L

. The NOAE L for developmental effects is 1,200 ppm (2,015 mg /m 3 ). Preg nant Sprag u e-Dawle y rats (20-23/g r oup) were e x posed to 0, 1,000, 1,287, 1,592, or 1,827 ppm ACN (0, 1,679, 2,161, 2,673, or 3,067 mg /m 3 ), 6 hours/da y , on g e station da y s 6 to 20
10(Saillenfait et al., 1993). Dams were sacrificed on g e stational day 21. At 1,827 ppm, mortality occurred in 8/20 dams, and maternal body weig ht g a in was sig n ificantly reduced from g e station day s 6 to 21. There was no mortality at other concentrations. Acetonitrile did not affect fertility (i.e., no differences in number of preg nancies). A markedly increased percentag e of nonlive impla nts pe r litte r (re sorptions a nd de a dfe tuse s) a nd e ar ly re sorptions pe r litte r we re obse r ve d at 1,827 ppm, along with a decrease (not sig n ificant) in the mean number of live fetuses per litter. One litter was completely resorbed at 1,827 ppm. No differences were observed between the other ex posed g r oups and the controls. Acetonitrile ex posure had no sig n ificant effect on the me a n numbe r of impla ntation site s pe r litte r, fe ta l se x ratio, or fe ta l we ig hts pe r litte r. In c i de nc e s of any visceral or skeletal anomalies were not sig n ificantly different between ex posed and control g r oups. A NOAEL of 1,592 ppm (2,673 mg /m 3 ) was determined for maternal and developmental tox i city and a F E L of 1,827 ppm (3,067 mg /m 3 ). B a sed on increased percentag e of nonlive implants per litter, a L OAEL of 1,827 ppm was identified for developmental tox i city

. The la c k of morta lity in a ll but the a n ima ls in the hig h e s t c onc e n tration of the Sa ille nfa i t et al. (1993) study is in sharp contrast with mortality observed in the Mast et al. (1994) study , i n asm u ch as both studi es used S p rag u e-Dawl ey rat s . There i s no cl ear ex pl anat i on for th ese di verg enteffect s. There were no effects on pup birth weig ht, litter size, or pup viability when ACN was administered by intubation to preg nant L ong -Evans Hooded female rats (Smith et al., 1987). In this teratolog y screen, ACN, dissolved in tricapry lin oil, was administered at 0, 50, 150, 300, and 600 mg /kg from day s 7-21 of g e station. Principal parameters evaluated were preg nancy rate, litter resorption, and early neonatal death. The two hig h est dosing levels were maternally tox i c. Preg nant Sprag u e-Dawley rats (25/g r oup) were administered g a vag e doses of 0, 125, 190, or 275 mg /kg

- day of ACN (dissolved in distilled water) on g e station day s 6-19 (J ohannsen et al., 1986; I R DC, 1981; B e rteau et al., 1982). Animals were sacrificed on g e station day 20. Maternal effects were seen with 275 mg /kg

- day ; two dams died and four dams appeared emaciated. B ody weig ht g a in from day s 6 to 20 was slig htly reduced (96% of control) in the 275 mg /kg

- day dams. F e rtility wa s not a ffe c t e d. An inc r e a s e in postimpla ntation loss pe r da m oc c u rre d atthe hig h dose; however, effect was not sig n ificant or dose-related. The number of live fetuses per dam was slig htly less than the control value for the 275 mg /kg

- day g r oup (not sig n ificant), but the effect was not measured for each litter. No differences in total implantation, corpora lutea, fetal sex ratio, or fetal body weig ht were observed in any treated g r oups compared to controls. There was a slig ht, but not sig n ificant, increase in the number of fetuses with unossified sternebrae (5th and 6th) in the hig h -dose g r oup; however, the number of litters with unossified sternebrae was not dose-related. No sig n ificant effects on fetal anomalies occurred. This study identifies a L OAEL of 275 mg /kg / day for maternal and developmental effects and a NOAEL of 190 mg /kg

day

. Inhalation and oral developmental studies were performed on preg nant hamsters ex posed to ACN (Willhite, 1983). Preg nant Syrian g o lden hamsters were ex posed via inhalation to 0, 1,800 (6 animals), 3,800 (6 animals), 5,000 (6 animals), or 8,000 (12 animals) ppm ACN (0, 3,022, 6,380, 8,395, and 13,432 mg /m 3 ) for 1 hour on g e station day 8 and then sacrificed on g e station day 14. Parameters of maternal and developmental tox i city were evaluated. Only those
11dams that died were ex amined for histopatholog ical effects. No hamsters ex posed to 1,800 ppm ex hibited any sig n s of intox i cation and all offspring were normal. Histopatholog y was not performed on this g r oup. One dam at 3,800 ppm died 3 hours after ex posure after ex hibiting dy spnea, tremors, hy persalivation, atax ia, and hy pothermia. All offspring from this g r oup were normal. At 5,000 ppm, all animals ex hibited irritation and ex cessive salivation; one dam in this g r oup died after displayi ng dy spnea, hy pothermia, and tremors. Six abnormal offspring from two litters of this g r oup ex hibited ex encephaly and rib fusions. In the 8,000-ppm g r oup, clinical effects included respiratory difficulty , letharg y , atax ia, hy pothermia, irritation, and g a sping (4/12 dams), followed by tremors, deep coma, and death (3/12 dams). Histopatholog ical ex amination of the liver, kidney s , and lung s from the dams that died in all g r oups did not reveal any sig n ificant treatment-related effects. F e totox i city occurred in offspring of dams ex posed to 8,000 ppm, as evidenced by decreased fetal body weig ht compared to controls (not concentrationrelated). F i ve of the nine surviving litters at 8,000 ppm developed severe ax ial skeletal dy sraphic di sorders; one fetu s ex hi bi t e d ex tr athoraci c ect opi a cordi s wi th accomp anyi ng defect s i n th e sternum. This study identifies a maternal NOAEL of 1,800 ppm (3,022 mg /m 3 ) and an F E L of 3,800 ppm (6,380 mg /m 3 ). The NOAEL (3,800 ppm [ 6,380 mg /m 3 ] ) and L OAEL (5,000 ppm [ 8,395 mg /m 3 ] ) i d ent i f i e d for devel opm ent a l effect s occurred ator ex ceeded th e m at e rnal FEL

. The effects of ing e sted ACN on preg nant hamsters and their fetuses were ex amined (W illhite, 1983). Preg nant Syrian g o lden hamsters (6-12/g r oup) received g a vag e doses of 0, 100, 200, 300, or 400 mg /kg ACN (in distilled water) on g e station day 8, and were sacrificed on g e station day 15. Parameters of maternal and developmental tox i city were evaluated. Maternal effects (not specified) were evident in dams g i ven 300 and 400 mg /kg , and death occurred in 1/6 and 4/12 dams in these g r oups, respectively

. There was a sig n ificant reduction in body weig ht g a in in dams (g estation day s 8-15) g i ven up to 300 mg /kg ACN compared to controls (no incidence data provided); however, this effect was not seen at 400 mg /kg

. Slig ht, but sig n ificant, decrease in fetal body weig ht ( p < 0.05) was seen in all ex posed g r oups; however, the effect was not dose-related. A sig n ificant increase in the number of resorptions per g r oup (not dose-related) was noted in dams ex posed to 200 or 400 mg /kg (incidences were 0, 6%, 12%, 0, and 22%). A sig n ificant increase in the number of malformed offspring per g r oup occurred in dams ex posed to 300 or 400 mg /kg ACN (0, 10%, 0, 19%, and 18%). The most common effect was rib fusions. The reason for the nonlinear effects was not ex plained by the investig ators. A concurrent g r oup was administered 300 mg /kg thiosulfate by intraperitoneal injection 20 minutes prior to inhalation, and repeated every 2 hours for the nex t10 hours. Treatment with thiosulfate prevented both maternal and developmental tox i city , indicating a causal connection between cy anide and the effects observed. This study identifies a NOAEL of 200 mg /kg and L OAEL of 300 mg /kg for maternal tox i city

. The NOAEL for fetotox i city could not be established on the basi s of th e nonl i n ear effect s i n th e fetu ses. Preg nant New Z ealand W h ite SPFrabbits (25/g r oup) were administered 0, 2, 15, or 30 mg /kg

- day of ACN by g a vag e during g e station day s 6-18, and sacrificed on g e station day 29 (Arg us Research L aboratories, In c., 1984). Dams were ex amined for clinical sig n s, body weig ht chang e s, feed consumption, and chang e s in absolute liver weig ht. Ex amination was performed to identify any g r oss lesions, which were then retained for histopatholog y . In the hig h -dose g r oup, 5 dams died ( p = 0.025) between day s 12-19 and 2 aborted on either g e station day 23 or 27 (both effect s at tri but ed t o tr eat m e nt ). C l i n i cal si g n s i n th ese affect ed rabbi t s i n cl uded at ax i a , anorex i a , 12decreased m o t o r act i v i t y , brad y pnea, d y spnea, i mp ai red ri g h t i n g refl e x , and col o red e x udat e i n cag e pan. A decrease in bod y weig ht was observed in 30-mg /kg

- da y dams between g e station da y s 15 and 19 ( p � 0.01), but the weig ht increased sig n ificantl y after end of administration (i.e., g e station da y s 19-24) and was hig h er than control weig hts on da y s 24-29. Necrops y revealed thin stomach walls in the cardiac reg i on in those rabbits that died, and this observation was considered treatment-related. E x posure to ACN did not appear to cause reproductive d y sfunction, althoug h a slig ht, but not sig n ificant, increase in resorptions per litter was observed. De ve lopme n ta l e ffe c t s we re se e n atthe hig h dose

. The a v e r a g e numbe r of live fe tuse s pe r litte r was si g n i f i cant l y decreased ( p = 0.011) at 30 mg /kg

- da y . There was no sig n ificant increase in the incidence of fetal malformations or anomalies with ACN e x posure. B a sed on maternal to x i cit y , this stud y identifies 30 mg /kg / da y as a F E L (mortalit y ). A NOAE L of 15 mg /kg / da y is g i ven for maternal to x i cit y . A NOAE L of 15 mg /kg / da y is identified for developmental to x i cit y (decreased live fe tuse s pe r litte r). Morrisse y et al. (1988) evaluated reproductive endpoints in the male rat and mouse ex posed to nominal concentrations of 100, 200, and 400 ppm in the Haz e l ton (1983a,b) studies. No tre atme nt e ffe c t s we re obse r ve d on the we ig hts of rig h t c auda e p idid ymis, rig h t e p idid ymis, and rig h t testis, nor were an y effects found on sperm motilit y in the mouse. Similarl y , a lack of effect on these endpoints were found in the rat; the weig ht of the rig h t epidid ymis was not evaluated. In neither species were effects on sperm densit y or percent of abnormal sperm eval uat e d.

4.4. OTHER STUDIES 4.4.1. Acute Data The r e ar e limite d a c u te data on the ora l a nd inha lation e x posure of ACN to huma n s a n d animals. In humans, qualitative information is based primaril y on case reports (see Section 4.1). In animals, oral LD 50 s have been reported for the mouse (269-453 mg /kg ) and the rat (2,230-4,050 mg /kg ) , inhalation L C 50 s (1-2 hours) for the mouse (2,300-5,700 ppm), and L C 50 s (4 hours) for the rat (16,000 ppm) (W HO, 1993).
In an unpublished subacute stud y ( ImmuQuest L aboratories, In c., 1984), B 6 C3F 1 female mice were e x posed to 0, 100, 200, or 400 ppm ACN, 6 hours/da y , 5 da y s /week, for 10 da y s during a 14-da y period. Gas chromatog r aphic anal y s is indicated the test compound had a purity e x ceedi n g 99%. C h amber concentr at i ons were m oni t o red b y i n frared spectroscop y every 30 minutes during each 6-hour e x posure. No treatment-related clinical sig n s were evident. S t at i s t i cal l y si g n i f i cantdecreases ( p < 0.05) in red and white blood cell counts, hematocrit, and hemog l obin at the two hig h est concentrations were reported. However, mean values at these two concentrations seemed to be marg inall y lower than controls and ma y be of questionable biolog ical sig n ificance. Necrops y revealed th ymic atroph y in the 200- and 400-ppm g r oups (incidence not stated); the effect corresponded to reduction (not sig n ificant) in th ym us/brain weig ht ratio (absolute org a n weig ht and th ym us-to-bod y -weig h t ratio were not reported). The number of mice e x amined histopatholog icall y was not stated and appears to be no g r eater than six per e x posure g r oup, based on information presented for other endpoints. Serum I g G levels
13were sig n ificantl y decreased in a concentration-related manner (26%, 33%, and 48% decrease, respectivel y , of controls), but linear reg r ession anal y s is indicated no concentration-related trends with I g M and I g A. Tests of B

- cell function were unchang ed from controls.
In the same report, reference was made to a concurrent 90-da y stud y in which a separate g r oup of B 6 C3F 1 mice were e x posed to the same concentrations. No data per se were presented for this stud y . However, th ymic atroph y was mentioned for the 200 and 400 ppm g r oups of the 90-da y stud y as had been for the 14-da y stud y . No incidence or severit y data were reported. A statement referring to slig ht vacuoliz ation of hepatoc ytes and h y dropic deg e neration was the onl y information presented on histopatholog ical finding s in liver; alanine aminotransferase (AST) and aspartate aminotransferase (A LT ) levels were within the normal rang e. Itis likel y that the 90-da y stud y referred to is the Haz e l ton (1983a), since the principal investig ator was a co-author of the ImmuQuest report. Of sig n ificance is the lack of effect of ACN on th ym us in the Haz e l ton (1983a) report, in contrast to the statement in the ImmuQuest report. Thus, the sig n ificance of an effect of ACN on the th ym us without further corroboration is doubtful. 4.4.2. G e notoxicity The overall data indicate that ACN is not a point mutag e n, but does interfere with chromosome seg r eg ation. Acetonitrile at concentrations up to 10,000 μ g /plate was neg ative in Salmonella typhimurium strains TA1535, TA1537, TA97, TA98, and TA100 in the absence of S9 and in the presence of rat or hamster S9 induced with Aroclor 1254 (Mortelmans et al., 1986; NTP, 1996). Neg ative results were also obtained in a preincubatio n S. typhimurium assa y and a re ve rse mutation a ssa y in Saccharomyces cerevi si ae D7, conducted in the presence and absence of S9 from rats induced with ACN or phenobarbitone (Schleg e lmilch et al., 1988), althoug h the ba c t e ria l a ssa y wa s limite d b y the use of stationar y c u lture s. S i st er chrom at i de x chang e s (S C E s) i n C h i n ese ham s t e r ovary (C HO) cel l s were sig n ificantl y increased in the absence of S9 (onl y at 5,000 μ g /m L ), but there was no effect in the presence of S9 (Gallowa y et al., 1987; NTP, 1996). Gene conversions i n S. cerevi si ae were increased in the absence of S9, but not in the presence of S9 (Schleg e lmilch et al., 1988). B o th of these assa y s measure repair of DNA damag e , rather than persistent DNA damag e . Chromosome aberrations were sig n ificantl y elevated in CHO cells at 5,000 μ g /m L in the presence of rat S9 but the trend test was borderline ( p = 0.016); there was no effect in the absence of S9 (Gallowa y et al., 1987; NTP, 1996). Althoug h the t ypes of chromosome aberrations were not reported, Gallowa y et al. (1987) reported that both simple and complex aberrations were elevated at the hig h dose. The abilit y of ACN to induce mutations at the HGPRT g e ne locus in CHO cells in vitro was investig ated both in the presence and absence of rat liver S9 (B ioassay S y stems Corporation, 1984). There were no sig n ificant differences between treated and control cells over a concentration rang e of 0.1 to 30 mg /m L

. Micronucleated normochromatic erythroc yt es (NCEs) were increased in the peripheral blood of female mice, but not in males, in a micronucleus assa y conducted in conjunction with a 13-week inhalation to x i cit y e x periment (NTP, 1996). Althoug h the micronucleus assa y is usuall y conducted in pol y c hromatic erythroc yt es (PCEs), MacGreg o r et al. (1990) showed that micronucleated peripheral blood PCEs and NCEs are at stead y state following dosing for 45-90 da y s . Schleg elmilch et al. (1988) found
14that intraperitoneal injection of mice with ACN at 60% of the oral LD 50 was weakl y cl ast o g e ni c, with micronucleated pol y c hromatic erythroc yt es sig n ificantl y increased at 24 hours. W h en the mice were induced b y injection of low doses of ACN for 7 da y s , and then challeng ed with 60% of the oral LD 50 , an increase in micronucleated PCEs was not observed until 72 hours after the challeng e dose. Positive micronucleus assa y s can indicate either clastog e nic activit y or interference with chromosome seg r eg ation. ACN (131 ppm for up to 70 minutes) also induced aneuploid y (both chromosome g a in and chromosome loss) in treated mature ooc yt es of Drosophila melanogaster females e x posed either as larvae or as adults (Osg ood et al., 1991a,b). To x i cit y and sterilit y were induced b y the 70-minute e x posure. Whe n S. cerevi si ae was e x posed to 5% ACN, it induced mitotic aneuploid y ( Z immerman et al., 1985); the investig ators sug g e sted that the induction of aneuploid y b y ACN in the absence of point mutations or recombination resulted from interference with tubulin assembl y and the formation of microtubules in the spindle apparatus. More recentl y , S e hg al et al. (1990) obtained in vitro evidence that ACN does inhibit microtubule assembl y in tax o l-purifie d Drosophila or mouse microtubules, further indicating that ACN has potential to induce aneuploid y . Althoug h ACN is larg el y neg ative in g e ne mutation assa y s and produces onl y marg inal e ffe c t s in c h romosome ab e rration a ssa y s , the pote n tia l of ACN to inte rfe re with c h romosome seg r eg ation both in vivo and in vitro has been demonstrated i n D. melanogaster

.

4.5 . SYNTHESIS AND EVALUATION O F MAJOR NONCANCER E F F E CTS AND MODE O F ACTION—ORAL AND INHALATION

The database for ACN lacks subchronic and chronic oral to x i cit y studies in animals. Huma n data ar e limite d to c a s e re ports of a c u te ACN e x posure with little information on e x posure level. The noncancer inhalation to x i cit y of ACN in the rat and mouse is overtl y demonstrated in subchronic studies at levels of 400 ppm and hig h er, at which lethalit y takes place (NTP, 1996). The two- y ear studies (NTP, 1996), conducted at lower concentrations, did not demonstrate adverse effects, clinicall y or b y histopatholog y , in an y of the org a n s y stems e x amined in either the rat or mouse. The lethal effects of e x posure to ACN are believed to be associated with the production of c y anide leading to respiratory paral y sis and inhibition of CNS processes. At these lethal levels, clinical sig n s included atax ia, convulsions, and abnormal posture (NTP, 1996). Disturbances in blood chemistry and decreased th ym us weig ht (rats onl y ) were associated with levels of 800 ppm and hig h er. (Concentration-related decreases in hemocrits and red blood cell counts were, however, reported in both the ImmuQuest and Haz e l ton mouse studies at lower concentrations). Abnormal histopatholog y was larg el y releg ated to the lung s and included cong estion, hemorrhag e, and edema (NTP, 1996; Poz z a ni et al., 1959). The absence of th ymic atroph y in the mouse in both the NTP (1996) stud y and the Haz e l ton (1983a) stud y are in direct contrast to a statement (incidence/severit y not reported) in the unpublished stud y b y ImmuQuest L aboratories (1984) that th ymic atroph y was observed in
15B 6 C3F 1 mice e x posed to 200 and 400 ppm ACN for either 14 or 90 da y s . The reference to the latter 90-da y stud y is most likel y to the Haz e lton (1983a) stud y , since one of the authors of the ImmuQuest report was the principal investig ator for the Haz e l ton stud y . Evidence of abnormal histopatholog y in other org a ns at levels below those at which lethalit y occurred was not apparent in the NTP stud y . Thus, the concentration-response re lationship is quite ste e p. L e tha lit y in the rat ma y be stra in-de p e nde nt. In the subc hronic study b y the NTP (1996), 5/6 F 344 rats e x posed to 1,600 ppm died during weeks 1 and 2, whereas there were no deaths in preg nant Sprag u e-Dawle y rats e x posed to 1,592 ppm during a similar time period (Saillenfait et al., 1993). In addition, no deaths were reported in Carworth F armsW i star rats at 655 ppm (Poz z a ni et al., 1959). Developmental endpoints also have been associated with concentrations hig h er than those that caused lethalit y (Mast et al., 1994; Saillenfait et al., 1993; Willhite, 1983). The effects of ACN e x posure on reproductive endpoints in either species prior to mating and throug h parturition has not been e x amined. Mic e ma y be more se nsitive tha n rats to ACN tox i c it y , e v ide n c e db y the inc r e a s e in forestomach h y perplasia and ulcers, effects not observed in the rat (NTP, 1996). The forestomach lesions observed in the subchronic and chronic studies conducted b y the NTP are cri t i cal noncancer effect s g i ven th e case report s of g a stri c erosi on i n hum ans who i n g e st ed AC N (W a y , 1981; B a llant yn e, 1983). However, the role that inhalation e x posure pla y s in the occurrence of the lesions is unknown, and ma y be minor compared to ing e stion as a result of g r ooming of c onta minat e dfur a nd/or muc o c iliar y c l e ar a n c e . More ove r, a pote n tia l role of inhalation can be envisioned g i ven the detection of label as earl y as 5 minutes post-injection in nasal secretions, esophag u s, and stomach contents after intravenous administration of C14-ACN to mice (Ahmed et al., 1992). Because of the uncertainties surrounding the contribution of oral ing e stion versus inhalation, quantifiable levels of ACN via inhalation e x posure cannot be ascertained for this endpoint. The subchronic data (NTP, 1996) for the rat and mouse do indicate a NOAE L of 200 ppm (NOAE L [ AD J ] = 60 mg /m 3 ) and an F E L of 400 ppm (F E L [ AD J ] = 120 mg /m 3 ) for le tha lit y . However, an unambig uous NOAE L pertaining to ACN inhalation cannot be determined because of the possible role of inhalation in causing forestomach lesions.

4.6. WEIG HT-O F - EVIDENCE EVALUATION AND CANCER CLASSI F I CATION The National To x i colog y Prog ram (1996) concluded that the evidence for carcinog enicity via inhalation of ACN in F 344/N rats was equivocal: “a causal relationship between ACN e x posure and liver neoplasia in male rats is uncertain.” Althoug h there was a statistically sig n ific a n t positive tre nd in the inc i de nc e of he patoc e llular a d e noma s , c ar c i noma s , a nd a d e noma s and carcinomas (combined) in male rats onl y , the incidences were not statisticall y sig n ificant by pairwise comparison or b y life table anal y s is. In addition, the incidence of adenomas and carcinomas combined in the 400 ppm g r oup was onl y slig htl y hig h er than the historical rang e for inha lation stud y c ontrols. Ma le rats e x hibite d a n inc r e a s e inc i de nc e of ba sophilic foc i in live r that was statisticall y sig n ificant in the 200- and 400-ppm g r oups. Althoug h the appearance of th ese foci was notat yp i cal , as those m o re cl osel y rel at e d t o th e carci nog eni c process (Harada
16et al., 1989), altered hepatocellular foci are g e nerally considered to be preneoplastic (Williams and Enz m an, 1998; Pitot, 1990). There was no evidence of carcinog enicity in female rats or in e ithe r ma le or fe ma le B 6 C3F 1 mic e .

The data from the NTP study coupled with the overall lack of g e notox icity potential strong ly sug g e st that the carcinog enic potential of ACN in the rat and mouse is low. However, because of uncertainty as to the sig n ificance of (1) positive trend test of hepatocellular carcinomas/adenomas (combined) in male rats compared to controls, (2) the relevance of the basophilic cell foci, and (3) the lack of effect in the mouse, the proposed Guidelines for Carcinog en Risk Assessment (U.S. EPA, 1996) are clear in stating that the carcinog enic potential of a substance (e.g ., ACN) for humans following inhalation, oral or dermal ex posure “cannot be det e rm i n ed because th e ex i s t i n g evi d ence i s composed of confl i c t i n g dat a (e.g ., som e evi d ence i s sug g e stive of carcinog enic effects, but other equally pertinent evidence does not confirm any concern).” Similarly , when these data are assessed under the ex isting Guidelines for Carcinog en Risk Assessment (U.S. EPA, 1987), ACN would be classified as Group D - Not Classifiable as to Hum a n C arci nog eni c i t y . There i s an absence of hum an evi d ence and th e ani m a l evi d ence i s equivocal.

4.7. SUSCEP TIBLE P O P U LATIONS Sensitive populations may include individuals who g e nerate increased concentrations of cy anide because of induction of the cytochrome P 450 isoform, CYP 2 E1. 4.7.1. P o ssible Childhood Susceptibility There are a few case reports of accidental ing e stion of products containing ACN by children or infants (WHO, 1993). The symptoms in these cases are similar to those ex perienced by adults in accidental or intentional ing e stion of ACN-containing products. The few developmental effects seen in oral and inhalation ex posures of laboratory animals below levels thatc au se mate rna l tox i c ity sug g e st thatc h ildre n ar e not like l y to be susc e p tible to developmental effects induced by ACN during prenatal ex posure. 4.7.2. P o ssib l e G e n d er Dif f

eren ces The W o rld Health Org a niz ation (1993) reported two cases in which adult females accidentally ing e sted products containing ACN. The symptoms reported were similar to those reported for males.

5. D O SE- R ESP O N S E A SSESSM EN TS 5.1. ORAL REF E RENCE DOSE (Rf D ) The oral reference dose and supporting information previously on I R I S have been withdrawn. The oral reference dose (RfD), derived via a route-to-route ex trapolation, had been
17based on the judg ement that the observation of decreased red blood cells and hepatic lesions (i.e., vacuoliz ation) in the unpublished Haz e l ton L aboratories (1983) 90-da y inhalation stud y were adverse. The decreases in red blood cells are not considered adverse in the present U.S. EPA assessment. Althoug h blood chemistr y was not evaluated in mice in the current NTP studies (1996) and thus represents a shortcoming in the protocol, the Haz e l ton investig ators had described these effects as being of “low mag n itude and questionable biolog ical sig n ificance.” The vacuoliz ation noted b y these investig ators was described as “slig htl y more pronounced ... as compared to the control mice.” Similar finding s were noted in the NTP (1996) stud y . The vacuoliz ation is not judg ed adverse. Althoug h the available information was inadequate for developing an oral RfD, the derivation of a developmental to x i cit y RfD (RfD DT ) was considered due to developmental to x i cit y reported in oral developmental studies reported in hamsters, rats, and rabbits (Arg us Research L aboratories, In c., 1984; I R DC, 1981; J ohannsen et al., 1986; Willhite, 1983). B a sed on the available developmental studies, the most sensitive developmental endpoint (decreased a v e r a g e numbe r of live fe tuse s pe r litte r), wa s re porte d for rabbits a d ministe re d ACN b y g a va g e during g e stational da y s 6-18 (Arg us Research L aboratories, 1984). This effect occurred at the hig h e s t dose te ste din the stud y ( L OAE L = 30 mg /kg

- da y ) ; howe v e r , the hig h morta lit y (20%) in dam s al so i ndi cat ed a FE L for th i s dose l e vel

. Because th e deaths occurred wi th a shortdurat i o n of e x posure and dosing errors were not reported, it is likel y that the observed effects were chemical-related. The other oral developmental studies in hamsters and rats were not appropriate for the development of an RfD DT . No developmental tox i cit y was seen at an y dose up to 275 mg /kg / da y in the rat stud y ( J ohannsen et al., 1986), and the dose-response data in the hamster stud y (Willhite, 1983) were too inconsistent to determine whether there was an effect, and if so, atwhat l e vel

. In l i g htof th ese i n consi s t e nci e s, and because no stud y i d ent i f i e d a L OAE L i n th e absence of a maternal F E L , the derivation of an RfD DT wa s not a tte mpte d. The NTP (1996) 2- y ear inhalation stud y in the mouse was not used for route-to-route e x trapolation to an oral scenario because the quantitative contribution of inhalation and ing e stion of ACN to the occurrence of forestomach lesions could not be delineated. Therefore, no oral dose-response assessment was performed for this compound.

5.2. INHALATION RE F E RENCE CONCENTRATION (RfC ) 5.2.1. Choice of P r incipal Study and Critical Ef f

ect
Inhalation data on subchronic and chronic to x i cit y of ACN are available from several studies: (1) the 1996 NTP stud y on F 344 rats and B 6 C3F 1 mice, (2) the 1984 ImmuQuest L aboratories stud y on mice, and (3) the 1983 Haz e l ton studies on the rat and mouse. The subchronic inhalation stud y b y Poz z a ni et al. (1959) reported respiratory effects in dog s and mice at hig h er concentrations, and interpretation was limited because of inadequate stud y details. The developmental tox i cit y endpoints (Mast et al., 1994; Saillenfait et al., 1993; Willhite, 1983) also occurred at much hig h er inhalation concentrations ( L OAE L s � 1,200 ppm) than those of the subchronic and chronic studies.

18The NTP subchronic stud y in the mouse, supported b y the results of the chronic stud y , was chosen as the principal stud y , with mortalit y of 1/10 females ex posed to 400 ppm (duration-adjusted concentration = 120 mg /m 3 ) as th e cri t i cal effect

. The i n creases i n absol u t e and relative liver weig hts in males and the incidence of hepatic vacuoliz ation are not considered biolog icall y sig n ificant hepatocellular adverse finding s. Hepatic vacuoliz ation also was not considered a biolog icall y sig n ificant finding in either the ImmuQuest stud y or in the Haz e l ton studi es. Because th e i n ci dence of forest om ach squam ous h yperpl a si a i n fem a l e s was i n creased above controls at 100 and 200 ppm (chronic) and at 200 ppm and above (subchronic) coupled with uncertaint y as to the role of inhalation in the development of this lesion, there is no unambig uous NOAE L in either inhalation stud y conducted b y the NTP. The absence of forestomach lesions in the rat cannot be e x plained. There is no information to sug g e st that differences between species in g r ooming behavior account for the forestomach lesions. W o lff et al. (1982) found that rats e x posed whole-bod y ing e sted 60% of the pelt burden of a radiolabeled aerosol throug h preening

. Althoug h an F E L would ordinaril y not be chosen as a point of departure for RfC derivation, the choice of mortalit y in this instance appears appropriate. In both se x e s of two laboratory animal species, there were no observed nonneoplastic adverse effects clearly associated with inhalation e x posure at three e x posure levels (chronic stud y ) below those (400 ppm) that resulted in mortalit y . This steep ex posure-response relationship is consistent with e x posure-response data for other c y anide-containing chemicals. In most cases, abnormal lung a nd bra i n patholog y a nd c linic a l sig n s of re spirat or y distre ss ar e fe ature s c onc omita nt with morta lit y . Th ymic atroph y cited in the ImmuQuest L aboratories (1984) report was not selected as a critical effect because it was not corroborated in an y of the other studies with the mouse. 5.2.2. Methods of Analysis The RfC was derived according to procedures identified in U.S

. EP A (1994b) using the reg i onal deposi t e d g a s rat i o (R DGR ). Acet oni tri l e i s consi d ered t o be a cat eg ory 2 g a s because (1) it has hig h water solubilit y , (2) is metaboliz ed to reactive c y anide in the liver, but ma y be rapi dl y det o x i fi ed t o th i o c y anat e, and (3) does notreactdi rect l y wi th respi r at ory tr act t i ssues. The RDGR (e x trarespiratory effects) for categ ory 2 g a ses is equivalent to 1 (U.S. EPA, 1994b). The RDGR, when multiplied with the NOAE L for mortalit y adjusted for duration, served to derive a human equivalent concentration (HEC). A benchmark concentration anal y s is (U.S. EP A, 1995) was not applied because there were onl y two data points. 5.2.3 . Rf C Derivation - In clu d i n g Ap p lication of Un certain t y F a ctors (UF ) an d Mod i f y in g F a c t o r s ( M F)

The following uncertaint y factors (UFs) are applied to the NOAE L ( AD J ) of 60 mg /m 3 : 3 for interspecies variation, because dosimetric adjustments were applied; 10 for intraspecies variation; and 3 for database insufficiencies. The total UF= 3 × 3 × 10 = 100. No uncertainty factor was applied to the use of a subchronic stud y because lethalit y did not occur in the long er-term mouse or rat stud y at lower levels. Therefore, althoug h this endpoint is of concern
19based on the subchronic study , increased ex posure would not be ex pected to increase the sensitivity to this endpoint g i ven the known metabolism of cy anide-containing compounds. A partial UFof 10 1/2 (3), instead of a full factor of 10, was used for database insufficiencies because of the lack of data on reproductive endpoints involving ex posure of laboratory animals before and during mating throug h parturition. A full factor of 10 was not consi d ered necessary because (1) th ere i s no evi d ence t o sug g e st th atAC N accum u l at e s i n th e body , (2) the developmental effects observed seem to be marg inal, and (3) these effects occur at concentrations lethal to dams. However, a modifying factor (MF ) of 10 was applied to account for the uncertain role that inhalation may play in causing forestomach lesions. These lesions were found in mice subchronically and chronically ex posed via inhalation and are likely to be a result of g r ooming of contaminated fur althoug h inhalation cannot be ruled out.
Rf C = 60 mg /m 3 × RDGR (1) = 60 mg /m 3 (HEC ) 60 mg /m 3 ÷ [ U F× MF ] = 60 ÷ [ 100 × 10] = 6E-2 mg /m 3

.
5.3. CANCER ASSESSMENT
Because th e evi d ence of carci nog eni c pot ent i a l of AC N i s equi vocal i n l aborat ory ani m a l s (see Section 4.6) and there are no data for humans, a quantitative dose-response assessment cannot be made.

6. MAJOR CONCLUS IONS IN THE CHARACTERIZATION OF HAZARD AND DOS E RES P ONS E 6.1. HUMAN HAZ ARD P O TENTIAL ACN is principally used in manufacturing operations as an intermediate in closed sy stems to produce other org a nic chemicals. B ecause it is sometimes used in noncaptive processes, it does have th e pot ent i a l t o reach ambi entai r and wat e r. In wat e r, i ti s onl y sl owl y hy drol y z ed at neutral pH. At acidic pH, it would be ex pected to yi eld hy drog en cy anide. It s half-life in air depends on the ex tent of its reaction with hy drox y l radicals, the principal scaveng i ng mechanism (NTP , 1996). Italso is one component of tobacco smoke. ACN is readily absorbed in the respiratory tract and distributes throug hout the body , where it is metaboliz ed in the liver. Metabolism is known to result in formation of cy anide and thioc y a n at e , with forma l de hy de a nd formic a c i d a s a dditiona l postulate d me tabolite s. Hy drocy a nic acid has been detected in various org a ns (e.g ., brain and heart) of the rat upon inhalation. Cy anide and thiocy anate have been detected in various org a ns and the blood of hamsters upon oral ex posure. However, in clinical studies of three individuals who inhaled ACN for 4 hours, no cy anide was detected in blood. The r e is little dire c tinformation c onc e rning the nature of e ffe c t s thatACN c au se s in humans. Case reports of adults or children who ing e sted ACN indicate that symptoms included
20respiratory distress (e.g ., pulmonary edema), vomiting , confusion, convulsions, g a stric erosion, and seizures. Case reports involving occupational ex posure involved a similar spectrum of symptoms. Autopsy finding s from an occupationally ex posed individual revealed brain, kidney , thyr oid, and liver involvement. Fr om these reports alone, it is clear that the respiratory tract, the central nervous sy stem, and other org a ns can be adversely affected. Some of the effects observed in the human case reports have also been noted in studies with laboratory animals. Inhalation ex posure of rats over 13 weeks indicated that ACN at hig h concentration causes pulmonary edema, cong estion, and hemorrhag e leading to death. At these concentrations, atax ia and clonic convulsions were observed as well as decreases in hemog l obin and thym us weig ht. W h en e x pose dove r 2 y e ar s to lowe r c onc e n trations, ma le rats (only ) e x hibite d a positive tre nd in live r tumors, but the incidence was not sig n ificant when compared pairwise to controls or after adjustment for survival. In female mice ex posed to hig h concentrations for 13 weeks, the principal finding was focal ulcers in the forestomach. F o restomach hy perplasia was observed in males. Other than increases in liver weig ht, there were no histopatholog ical effects in the liver, nor were there adverse lung effects. Effects on hematolog i cal parameters were not studied. In a life time study in the mouse atlowe r c onc e n trations, the r e we re no c linic a l e ffe c t s or e ffe c t s in the liver. F o restomach hy perplasia was increased in incidence, but severity of the lesion was not concentration-related. The incidence of pulmonary adenomas/carcinomas observed in males was not considered to be treatment-related. Oral and inhalation ex posure of preg nant rats and hamsters during g e station to ACN reveal ed th atdevel opm ent a l effect s occurred onl y at l e vel s ator ex ceedi n g those atwhi c h maternal adverse effects were observed. In one study that ex amined the effect of inhalation ex posure on male rat and mouse reproductive parameters, there were no observed effects on sperm motility or sperm density

. Althoug h two-g e neration studies have not been performed, and this represents a database deficiency , there is no indication that ACN at levels ex pected in the environment poses a risk of developmental effects. Because the carcinog enic potential in both the rat and mouse is low, coupled with an overall lack of g e notox icity potential, the carcinog enic potential in humans is ex pected to be low. Howe ve r, the r e is unc e rta inty in this ar e a ina s muc h a s the r e wa s a positive tre nd for hepat o cel l u l ar adenom as/ carci nom as (combi ned) i n th e m a l e ratas wel l as an i n crease i n th e inc i de nc e of ba sophilic live r foc i , of whic h the latte r ma y re pre s e n t a pre n e opla s tic e ffe c t

. 6.2. DOSE RESP ONSE Qua n titative e s timate s of huma n risk a s a re sult of e x posure to low le ve ls of ACN ar e based on ex posures of l aborat ory ani m a l s because no hum an dat a are avai l abl e. The hum an chronic concentration of inhaled ACN considered to be safe (the RfC) is 6E-2 mg /m 3

. The RfC is based on subchronic and chronic inhalation studies in the rat (NTP, 1996) in which thoroug h histopatholog ical analy s es were performed. B ecause only limited evaluation of hematolog i cal endpoints was carried out in the rat and none in the mouse, coupled with no ex amination of the effects on ventilatory parameters or CNS endpoints, the scientific quality of these studies is considered medium. The confidence in the overall database also is medium inasmuch as twog e neration studies were not performed. Althoug h acceptable developmental studies were carried out (via inhalation) in two species, rat and rabbit, with adverse effects occurring at levels at or
21ex ceeding the level that caused severe maternal effects, reproductive endpoints have not been thoroug hly evaluated. This represents a database deficiency

. In addition, the potential of ACN inhalation to induce g a stric lesions is unknown, but is a consideration g i ven the possible role of inhaled ACN in causing forestomach lesions in the mouse. The neurolog ical symptoms and respiratory distress in humans after acute hig h -level ex posures sug g e st that effects observed in laboratory animals are consistent with those observed in humans. No dose-response assessment was performed for oral ex posure to ACN. The database la c k e dsubc hronic a nd c h ronic studie s for laborat ory a n ima ls a nd the r e wa s only limite d information from case reports of human ing e stion. The NTP (1996) inhalation study was not used i n a rout e-t o -rout e ex tr apol at i on because th e contri but i ons of i nhal at i on and i n g e st i on t o th e oc c u rre nc e of fore stoma c h le sions c ould not be qua ntifie d. Similarly , a route

- to-route ex trapolation from inhalation to other endpoints could not be conducted because of lack of me c h a n istic data

.

7. REF E RENCES ACGI H. (1991) Document of the threshold limit values and biolog ical ex posure indices. 6th ed. Cincinnati, OH: American Conference of Governmental Industrial Hy g i enists. Ahmed, AE; L oh, J P ; Ghanay em, B; et al. (1992) S tudies on the mechanism of acetonitrile tox i city : I

. W hole body autoradiog raphic distribution and macromolecular interaction of 2 14 Cacetonitrile in mice. P h armacol Tox i col 70:322-330. Albaum, HG; Tepperman, J ; B odansky , O. (1946) The in vivo inactivation by cy anide of cytochrome ox idase and its effects on hig h energ y phosphorous compounds in the brain. J B i ol Chem 64:45-51. Aminlari, M; Vaseg h i, T; Karg ar, MA. (1994) The cy anide-metaboliz ing enz yme rhodanese in different parts of the respiratory sy stems of sheep and dog . Tox i col Appl Pharmacol 124:67-71. Arg u s Research L aboratories, In c. (1984) Embry o fetal tox i city and teratog e nicity study of acetonitrile in New Z ealand W h ite rabbits (S eg ment I I evaluation). W a shing t on, DC: Office of Tox i c Substances submission. Microfiche No. OTS 507279.
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B i oassay Sy stems Corporation. (1984) In vitro g e ne mutation assay (HGPRT locus) in cultured chinese hamster ovary (CHO) cells on acetonitrile. EP A Document No. 40-8446070. Dahl, AR. (1989) The cy anide-metaboliz ing enz yme rhodanese in rat nasal respiratory and olfactory mucosa. Tox i col L e tt 45:199-205.
22Dahl, AR; W arusz ewski, B A . (1989) Metabolism of org a nonitriles to cy anide by rat nasal tissue enz ymes. Xenobiotica 19:1201-1205. Dalhamn, T; Edfors, ML , Ry lander, R. (1968a) Mouth adsorption of various compounds in cig arette smoke. Arch Environ Health 16:831-835. Dalhamn, T; Edfors, ML ; Ry lander, R. (1968b) Retention of cig arette smoke components in human being s . Arch Environ Health 17:746-748.
F e ierman, DE; Cederbaum, AI . (1989) Role of cytochrome P-450 I IE 1 and catalase in the ox idation of acetonitrile to cy anide. Chem Res Tox i col 2:359-66. Freeman, J J ; Hay e s, EP . (1985) Acetone potentiation of acute acetonitrile tox i city in rats. J Tox i col Environ Health 15:609-622. Freeman, J J ; Hay e s, EP . (1988) Microsomal metabolism of acetonitrile to cy anide. Effects of acetone on and other compounds. Biochem P h armacol 37:1153-1159. Galloway , SM; Armstrong , MJ ; Reuben, C; et al. (1987) Chromosome aberrations and sister chromatid ex chang e s in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ Mol Mutag e n 10(Suppl):1-175. Hag u enoer, J M ; Dequidt, J ; J acquemont, MC. (1975) In tox i cations ex périmentales par l’acétonitrile 2ème note: intox i cations aig u ës par voie pulmonaire. Eur J Tox i col 8:102-106. Harada, T; Maronpot, RR; Morris, RW ; et al. (1989) Observations on altered hepatocellular foci in National Tox i colog y Prog ram two-y ear carcinog enicity studies in rats. Tox i col Pathol 17:690- 708. Hartung , R. (1981) Cy anides and nitriles. In : Patty ’s industrial hy g i ene and tox i colog y , 3rd rev. ed. Patty , F A ; Clayton, GD; Clayton, F E ; et al., eds. New York: W iley , pp. 4845-4900.
Haz e lton L aboratories. (1983a) 90-day subchronic tox i city study of acetonitrile in B6C3F1 mice. F i na l Re port (Re v ise d ). Pre p ar e dfor the Nationa l Tox i c o log y Prog ra m (NTP).
Haz e lton L aboratories. (1983b) 90-day subchronic tox i city study of acetonitrile in Fischer 344 rats. F i na l Re port (Re v ise d ).
ImmuQuest L aboratories, In c. (1984) Limited tox i city of inhaled acetonitrile on the immune sy stem of mice. OTS F Y I submission. Microfiche No. F Y I

- AX-0284-0292.
I R DC (International Research and Development Corporation). (1981) Acetonitrile (I R-79-162). Teratolog y study in rats. Unpublished study sponsored by Monsanto Company

. (Cited from U.S. EPA, 1985) (as cited in W HO, 1993).

23J ohannsen, F R ; L e vinskas, GJ ; B e rteau, PE; et al. (1986) Evaluation of the teratog e nic potential of three aliphatic nitriles in the rat. F undam Appl Tox i col 7:33-40. Kirk-Othmer Concise Ency clopedia of Chemical Technolog y . (1985) New York: W iley

. L e wis, J L ; Rhoades, CE; Gervasi, P-G; et al. (1991) The cy anide-metaboliz ing enz yme rhodanese in human nasal respiratory mucosa. Tox i col Appl Pharmacol 108:114-120. MacGreg o r, J T ; W e hr, CM; Henika, PR; et al. (1990) The in vivo erythrocyt e micronucleus test: Me a s ure m e n t atste a d y stat e inc r e a s e s a ssa y e ffic i e n c y a nd pe rmits inte g r ation with tox i c ity studies. F undam Appl Tox i col 14:513-522. Mast, TJ ; W e ig el, RJ ; W e sterberg , RB ; et al. (1994) Inhalation development tox i colog y studies: acetonitrile in rats. Battelle L aboratory for NIEHS , NTP

. P N L

- 9401. McMahon, TF ; B i rnbaum, L S . (1990) Ag e-related chang e s in tox i city and biotransformation of potassium cy anide in male C57B 1/6N mice. Tox i col Appl Pharmacol 105:305-314. Michaelis, HC; Clemens, C; Kijewski, H; et al. (1991) Acetonitrile serum concentrations and cy anide blood levels in a case of suicidal oral acetonitrile ing e stion. Clin Tox i col 29:447-458. Morrissey , RE; Schwetz , B A ; L a mb, J C IV ; et al. (1988) Evaluation of rodent sperm, vag i nal cytolog y , and reproductive org a n weig ht data from National Tox i colog y Prog ram 13-week studies. F undam Appl Tox i col 11:343-358. Mortelmans, K; Haworth, S; L a wlor, T; et al. (1986) Salmonella mutag e nicity tests: I I

. Results from the testing of 270 chemicals. Environ Mutag e n 8(Suppl 7):1-119. National Research Council. (1983) Risk assessment in the F e deral Government: manag i ng the process. W a shing t on, DC: National Academy Press. National Tox i colog y P r og ram. (1996) Tox i colog y and carcinog enesis studies of acetonitrile (CAS NO. 75-05-8) in F 344/N rats and B 6 C3F 1 mice (inhalation studies). NTP TR 447. Osg ood, C; B l oomfield, M; Z immering , S. (1991a) Aneuploidy in Drosophila, IV . Inhalation studies on the induction of aneuploidy by nitriles. Mutat Res 259:165-76. Osg ood, C; Z immering , S; Mason, J M . (1991b) Aneuploidy in Drosophila, I I

. F u rther validation of the F IX and Z E STE g e netic test sy stems employing female Drosophila melanogaster . Mutat Res 259:147-63. Pitot, H. (1990) Altered hepatic foci: their role in murine hepatocarcinog enesis. Ann Rev Pharmacol Tox i col 30:465-500.
Poz z a ni, UC; Carpenter, CP; Palm, PE; et al. (1959) An investig ation of the mammalian tox i city of acetonitrile. J Occup Med 1:634-642.
24Saillenfait, AM; B onnet, P; Guenier, J P ; et al. (1993) Relative developmental tox i cities of inhaled aliphatic mononitriles in rats. F undam Appl Tox i col 20:365-75. Schleg elmich, R; Krug , A; W o lf, HU. (1988) Mutag e nic activity of acetonitrile and fumaronitrile in three short term assay s with special reference to autoinduction. J Appl Tox i col 8:201-209. Sehg al, A; Osg ood, C; Z immering , S. (1990) Aneuploidy in Drosophila. III: A n e u p l o i d o g e n s inhibit in vitro assembly of tax o l-purified Drosophila microtubules. Environ Mol Mutag e n 16:217-224. Smith, MK; Georg e , EL ; Z e nick, H; et al. (1987) Developmental tox i city of halog e nated acetonitriles: drinking water by -products of chlorine disinfection. Tox i colog y 46:83-93. Swenne, I ; Eriksson, UJ ; Christoffersson, R; et al. (1996) Cy anide detox i fication in rats ex posed to acetonitrile and fed a low protein diet. Fundam Appl Tox i col 32:66-71. Taskinen, H; Kyyr onen, P; Hemminki, K; et al. (1994) L aboratory work and preg nancy outcome. J Occup Med 36:311-319. U.S. Environmental Protection Ag ency (EPA). (1985a) Health and environmental effects profile for acetonitrile. Environmental Criteria and Assessment Office. ECAO-CIN-P 137. U.S. EPA. (1985b) Reference dose for chronic oral ex posure (hy d rog e n cy anide). In teg r ated Risk In formation S y stem. U.S. EPA. (1986a) Guidelines for carcinog en risk assessment. F e deral Reg i ster 51 (185):33992- 34003. U.S. EPA. (1986b) Guidelines for the health risk assessment of chemical mix tures. F e deral Reg i ster 51(185):34014-34025. U.S. EPA. (1986c) Guidelines for mutag e nicity risk assessment. F e deral Reg i ster 51(185):34006- 34012. U.S. EPA. (1987) Risk assessment g u idelines of 1986. Office of Research and Development. EPA/600/8-87/045. U.S. EPA. (1988) Recommendations for and documentation of biolog ical values for use in risk assessment. EPA 600/6-87/800, NTI S PB 88-179874/AS. U.S. EPA. (1991, Dec. 5) Guidelines for developmental tox i city risk assessment. F e deral Reg i ster 56:63798-63826. U.S. EPA. (1994a, Oct. 26) In terim policy for particle size and limit concentration issues in inhalation tox i city : notice of availability . F e deral Reg i ster 59:53799.

25U.S. EPA. (1994b) Methods for derivation of inhalation reference concentrations and application of inhalation dosimetry

. EPA/600/8-90/066F

. U.S. EPA. (1994c) Peer review and peer involvement at the U.S. Environmental Protection Ag ency . Sig n ed by the U.S. EPA Administrator, Carol A. B r owner, J une 7.U.S. EPA. (1995) Use of the benchmark dose approach in health risk assessment. EPA/630/R94/007. U.S. EPA. (1996a) Proposed g u idelines for carcinog en risk assessment. W a shing t on, DC: National Center for Environmental Assessment. EPA/600/P-92/003C. U.S. EPA. (1996b) Reproductive tox i city risk assessment g u idelines. F e deral Reg i ster 61(212):56274-56322. U.S. EPA. (1998a) Guidelines for neurotox icity risk assessment. F e deral Reg i ster 63(93):26926- 26954. U.S. EPA. (1998b) Science policy council handbook: peer review. Prepared by the Office of Science Policy , Office of Research and Development, W a shing t on, DC. EPA/100/B

- 98/001.
W a y , J L . (1981) Pharmacolog i c aspects of cy anide and its antag onism. In : Cy anide in biolog y . Vennesland, B ; Conn, EE; Knowles, CJ ; et al., eds. New York: Academic Press, pp. 29-49.
W HO. (1993) Environmental health criteria 154: Acetonitrile. In ternational Prog ramme on Chemical Safety . Geneva: W o rld Health Org a niz ation. Willhite, CC. (1981) Inhalation tox i colog y of acute ex posure to aliphatic nitriles. Clin Tox i col 18:991-1003.
W illhite, CC. (1983) Developmental tox i colog y of acetonitrile in the Sy rian g o lden hamster. Teratolog y 27:313-325. Willhite, CC; Smith, RP. (1981) The role of cy anide liberation in the acute tox i city of aliphatic nitriles. Tox i col Appl Pharmacol 59:559-602. Williams, GM; Enzman, H. (1998) Rat liver hepatocellular-altered, focus-limited bioassay for c h e mic a l s with c ar c i nog e n ic a c tivity . In : Carc inog e n ic ity : te sting , pre d ic ting , a nd inte rpre ting chemical effects. Kitchin, KT, ed. New York: Marcel Dekker, In c. Wolff, RK; Griffis, L C ; Hobbs, CH; et al. (1982) Deposition and retention of 0.1 μ m 67 Ga 2 O 3 ag g r eg ate aerosols in rats following whole body ex posures. F undam Appl Tox i col 2:195-200.
Z immermann, FK; May e r, VW ; Scheel, I ; (1985) Acetone, methy l ethy l ketone, ethy l acetate, acetonitrile and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevi si ae . Mutat Res 149:339-351.
26AP P E NDIX A. EXTERNAL P EER REVIEW--S UMMARY OF COMMENTS AND DIS P OSITION The support document and I R I S summary for Acetonitrile have underg one both internal peer review performed b y scientists within EPA and a more formal e x ternal peer review perform ed b y sci e nt i s t s perform ed accordance wi th EP A g u i d ance on peer revi ew (U.S . EP A, 1992). Comments made b y the internal reviewers were addressed prior to submitting the documents for e x ternal peer review and are not part of this appendi x . The e x ternal peer re vie w e r s we re ta ske dwith providing writte n a n swe r s to g e ne ra l que stions on the ove ra ll assessment and on chemical-specific questions in areas of scientific controvers y or uncertaint y . A summary of sig n ificant comments made b y the e x ternal reviewers and EPA’s response to these c o mme nts follows. (1) Gen e ral C o m m e n t s A. The carci nog eni c pot ent i a l of AC N C o mme n t : Al l th ree revi ewers ag reed th at th e charact eri z at i on of carci nog eni c pot ent i a l i s appropriate. All reviewers ag reed that “equivocal” is the appropriate desig n ation. Two of the reviewers were of the opinion that the sig n ificance of liver tumors in rodents is of uncertain re le va nc e to huma n risk. One re vie w e r intimate d thata be tte r disc ussion of the sig n ific a n c e of basophilic liver foci could be presented and cited a recent article b y W illiams and Enz m an (1998) as support for an association of these foci with carcinog enicit y . Response to Co m m e nt: In formation pertaining to this reference and to the publication of Harada et al. (1989) was added to both the To x i colog i cal Review and cancer summary to indicate thatba sophilic live r foc i ar e g e ne ra ll y c onside r e dto be pre n e opla s tic (Willia ms a nd Enzma n , 1998), but that the foci in the NTP mouse stud y are not at y p ical in appearance, a characteristic that is often associated with prog ression to cancer (Harada et al., 1989).
B. S e l ect i on of th e m o stappropri at e cri t i cal effectfor R f C cal cul at i o n C o mme n t : All thre e re vie w e r s a g re e dthatmorta lit y is the most appropriate c hoic e for the cri t i cal effect g i ven th e dat a present e d. C. Discussion pertaining to “Supporting /Additional or Other” studies C o mme n t : Two reviewers thoug ht the Haz e l ton (1983a) and ImmuQuest L aboratories (1984) should be discussed more full y . In particular, the Haz e l ton (1983a) should not be discounted without presenting a clear rationale. One reviewer sug g e sted that a discussion of this study appear in the summary document under I

. B .4. Response to Co m m e nt: A full discussion of the unpublished Haz e l ton (1983a) 90-day inhalation stud y was included and the results, in part, were contrasted with those of the unpublished 14-da y ImmuQuest L aboratories (1984) stud y , presented under “Other Studies.”

2 7The shortcoming s of the ImmuQuest stud y were presented. A discussion of Haz e l ton (1983a) was also presented in Section I

. B .4 of the summary document.
D. Data that should be considered in developing uncertaint y and modif ying factors C o mme n t : Two reviewers ag reed that the selection of uncertaint y and modif ying factors was appropriate. One reviewer was uncomfortable with the identification of 400 ppm as an F E L considering there was onl y one mouse death at this concentration. Itwas proposed that 800 ppm be identified as the F E L for risk assessment purposes. Response to Co m m e nt: Itis possible that the one death at 400 ppm was unrelated to treatment. However, g i ven the steep concentration-response characteristics of ACN and the sudden onset of mortalit y in both the rat and mouse, it was considered appropriate to reg ard 400 ppm as treatment-related.
E. The rat i onal e for th e wei g ht -of-evi dence and confi d ence st at em ent s C o mme n t : One re vie w e r sug g e ste dthatthe re le va nc e of live r tumors in the rat to huma n s should be presented here and that the sig n ificance of the preneoplastic lesions should be addressed more full y . A second reviewer indicated that g e noto x icit y data should be included. The third reviewer stated that the underl yi ng assumptions and limitations are sufficiently apparent. Response to Co m m e nt: EPA recog n iz es the ong oing scientific debate reg arding the relevance of rodent tumors to humans. However, in this case the overall weig ht of evidence is i n adequat e for det e rm i n i n g th e hum an cancer pot ent i a l for acet oni tri l e , g i ven th e l ack of evi d ence seen i n th e ratand th e m arg i n al effect s seen i n th e m ouse. Therefore, EP A bel i e ves th atan e x panded discussion of the sig n ificance of preneoplastic basophilic foci is unneeded. (2) C o m m e n t s on C h e m i c al -S

peci fi c Qu esti o n s Question 1: I s it appropriate to use morta lit y a s a point-of-de p arture in the de rivation of the Rf C? All reviewers ag reed that there are no more sensitive endpoints to use in RfC derivation. Question 2: I s the rationale g i ven for discounting the Haz e l ton (1983a) stud y sufficient? All reviewers ag reed that a complete discussion of this stud y should be presented. Response to Co m m e nt: In the draft presented to the reviewers a discussion of the results of this unpublished stud y was not presented because NTP had elected not to use or cite the stud y , but repeated it. Therefore, a comment was included to sug g e st that the stud y did not meet NTP acceptance criteria. In view of the fact that the reasons NTP did not choose to utiliz e this study were not e x pressed, EPA adopted the reviewers’ sug g e stion to include a discussion of the conduct of the stud y and its results.
28Question 3: Do y ou ag ree that forestomach lesions in mice cannot be used as the critical effect because of uncert a i n t i e s pert ai ni ng t o oral i n g e st i on? One reviewer stated that “the discussion provided is a g ood e x ample of the use of scientific knowledg e in the risk assessment process.” This reviewer indicated that the lesions ha ve little re le va nc e to huma n e ffe c t s re porte d a f te r inha lation e x posure . The othe r two revi ewers ag reed th atth e di scussi on present e d on forest om ach l e si ons cl earl y i ndi cat es wh y th ese lesions cannot be used in derivation of the RfC. Question 4: W a s it appropriate to discount the ImmuQuest L aboratories (1984) results in the choice of the principal stud y ? All reviewers were of the opinion that a more complete discussion of the streng ths and limitations of this stud y should be presented. One indicated more weig ht should be g i ven. Response to Co m m e nt: A full discussion of this stud y is now presented in the Tox i colog i cal Review and in the RfC summar y document. In these discussions, it is indicated that the lack of stud y results to support the statement of th ymic atroph y in the mouse and the lack of such histopatholog ical results in either the subchronic NTP (1996) or Haz e l ton (1983a) studies limit the sig n ificance of this undocumented and uncorroborated finding

. Question 5: W ould y ou ag ree that the NTP chronic stud y cannot be used to derive an oral RfD? All reviewers were in ag reement that an oral RfD cannot be derived because of the uncertainties in the role of inhalation and oral ing e stion in causing forestomach lesions.

29