1 DRAFT - DO NOT CITE OR QUOTE Selected Sections from the External Peer Review Dra ft IRIS Toxicological Review for Acrylonitrile: Highlighted text showing changes based on recent EPA decisions regarding Ramazzini Institute studies

June 6, 2011

4.2.1.2.8. Maltoni et al. (1988, 1977). In a study conducted at the Ramazzini Institute, Ma ltoni et al. (1988, 1977) exposed Sprague-Dawley rats (40 /sex) to 5 mg/kg AN by gavage in olive oil, 3 days/week for 52 weeks; a control group of 75 rat s/sex received olive oil alone on the same schedule (study designated BT203). After the expos ure period, rats were maintained without
further treatment for the rest of their natural liv es (the study ending on week 131). Rats were examined 3 times daily for their general health sta tus and were subjected to a clinical examination for gross changes every 2 weeks. Rats were weighed every 2 weeks during the first year and monthly thereafter. All rats were examine d by gross necropsy. All tissues with gross lesions and a limited set of 12 tissues/organs from each rat were examined microscopically. No statistically significant increases in tumors we re observed in treated rats in this study; however, decreases in tumor latency or increased in cidence were observed for some tumors types identified in other studies of AN. Papillomas and acanthomas of the forestomach were observed in 1/40 treated males (latency 92 weeks), 4/40 trea ted females (average latency 97.5 weeks), 0/40 control males, and 1/75 control females (latency 54 weeks). Maltoni et al. (1977) considered the
increase in forestomach tumors to be treatment rela ted. Gliomas (a category that includes astrocytomas) appeared in 1/40 treated females (lat ency of 33 weeks), 0/40 treated males, 2/75 control females (average latency 104 weeks), and 1/ 74 control males (latency 98 weeks). Tumors of the mammary gland and Zymbal gland were r eported in treated animals, but the
incidences were not elevated over the control. NTP recently released a memorandum (Malarkey et al., 2010) that discussed differences of opinion between NTP scientists and the Ramazzini Institute in the diagnoses of certain cancers repor ted in a methanol study conducted by the
Ramazzini Institute. See Section 5.4.4.3 for addit ional information on the use of the Maltoni study in this assessment.

4.2.2.2.2. Maltoni et al. (1988, 1977). In studies conducted at the Ramazzini Institute, Ma ltoni et al. (1988, 1977) reported the results of three c ancer bioassays in Sprague-Dawley rats exposed to AN by inhalation. In the first (designated BT20 1 by the authors), 30 rats/sex/group were exposed to 0, 5, 10, 20, and 40 ppm AN for 4 hours/ day, 5 days/week for 52 weeks; the animals then were allowed to complete their natural life sp ans, with the final deaths occurring in week 136 (Maltoni et al., 1977). Rats were examined 3 t imes weekly for general health status and2 DRAFT - DO NOT CITE OR QUOTE

subjected to a clinical examination for gross chang es every 2 weeks. Rats were weighed every 2 weeks during the exposure period and monthly ther eafter. All rats were subjected to gross necropsy. Histopathologic examinations were conduc ted on all gross lesions and a selection of about 12 organs and tissues, including the Zymbal g lands, interscapular brown fat, salivary glands, tongue, lungs, liver, kidneys, spleen, stom ach, intestine, bladder, and brain. Only the incidence of neoplastic lesions was reported. Exposure to AN had no significant effect on surviv al or BWs in male or female rats. Increased incidences of gliomas, forestomach tumors , Zymbal gland carcinomas, and mammary tumors were reported in the treated group; however, increases in the incidences of these tumors in treated rats were not statistically significant. Gliomas were found in 1/30 and 2/30 males at 20 and 40 ppm, respectively, but not in controls and o ther exposed groups of male and female rats. The average latency of the gliomas was shorter at t he higher concentration (84 weeks in 20-ppm males vs. 63.5 weeks in 40-ppm males). Forestomach papillomas and acanthomas were found in 0/30, 1/30, 2/30, 0/30 and 3/30 male rats at 0, 5, 10, 20, and 40 ppm, respectively. In females, the incidence of forestomach tumors was 0/30, 1/30, 2/30, 1/30, and 0/30 at 0, 5, 10, 20, and 40 ppm, respectively. The average latency of forestom ach tumors ranged from 103 to 124 weeks. Zymbal gland carcinomas were found in 1/30 males in the 10-ppm group and 1/30 females in the 20-ppm group. No Zymbal gland carcinomas were foun d in the control and other dose groups. The incidence of benign and malignant mammary gland tumors in treated females rats was increased over controls, but the increase was not d ose related (5/30 [controls], 10/30 [5 ppm], 7/30 [10 ppm], 10/30 [20 ppm], and 7/30 [40 ppm]) ( Maltoni et al., 1977). 1 In two additional cancer bioassays, Maltoni et al. (1988) exposed Sprague-Dawley rats to AN by inhalation beginning in gestation. In the fi rst bioassay (designated BT4003), 54 adult pregnant females, beginning on gestation day 12, we re exposed to 60 ppm AN for 4 hours/day, 5 days/week for 7 weeks and then 7 hours/day, 5 day s/week for 97 weeks. A group of 60 unexposed adult females served as controls. Gestat ion was permitted to proceed normally and the offspring were exposed on the same schedule as the dams. The exposed offspring included 67 males and 54 females; the controls included 158 males and 149 females. Overall, there was a statistically significant trea tment-related increase in the percentage of dams with malignant tumors at all sites (37 vs. 15%). I ncreased incidences in exposed dams compared with controls were observed for several sites (Zymb al gland carcinomas, mammary gland

1 Discrepancies were noted in the incidence of mamma ry gland tumors for controls and 40-ppm females as reported in a subsequent report of this study (Maltoni et al ., 1988). In the 1988 publication, the incidence o f mammary gland tumors was reported as 20% (6/30) in control female s and 26.7% (8/30) in 40-ppm females. Also in the 1988 publication, encephalic gliomas were reported in bo th male and female rats at 20- and 40-ppm, whereas the 1977 publication reported gliomas in male rats only; gli oma incidences reported in Maltoni et al. (1988) we re: 20-ppm females—3.3% (1/30); 40-ppm females—3.3% (1/30); 20 -ppm males—3.3% (1/30); 40-ppm males—6.7% (2/30)..3 DRAFT - DO NOT CITE OR QUOTE

carcinomas, malignant mammary gland tumors, extrahe patic angiosarcomas, and encephalic gliomas), but none of these was statistically signi ficantly different from controls. No hepatomas were observed in exposed or unexposed dams. These tumor results are summarized in Table 4-
40.

Table 4-40. Comparison of carcinogenic effects of chronic exposure to AN at 60 ppm starting either in utero or in adulthood, in Sprague-Dawley rats

Stage during exposure Exposure

protocol Sex a Number

of rats at start b Percent with tumor

Brain tumors (encephalic gliomas) Zymbal

gland carcinomas Hepatomas Malignant
mammary tumors Extrahepaticangiosarcomas

Adult only Chronic c F 54 5.5 5.5 0.0 5.5 1.8

Unexposed controls F 60 0.0 1.7 0.0 3.3 0.0

Starting at GD 12 Chronic M F M+F 67

54 121 16.4

18.5 17.3 14.9 c

1.8 9.1 7.5 d

1.8 4.9 0.0

16.7 d 7.4 4.4

5.5 d 4.9

Subchronic e M

F M+F 60

60 120 5.0

3.3 4.2 6.7

1.7 4.2 1.7

0.0 0.8 0.0

6.7 3.3 5.0

1.7 3.3

Unexposed controls M

F M+F 158

149 307 1.3

1.3 1.3 1.3

0.0 0.7 0.6

0.0 0.3 1.9

5.4 3.6 0.6

0.0 0.3

a F = female; M = male.
b Animals were allowed to live until spontaneous deat h.

c Chronic: 60 ppm AN for 4 hrs/d, 5 d/wk for 7 wks ( starting during gestation), followed by 7 hrs/d for 97 wks.
d Statistically significantly higher than correspondi ng control incidence, p < 0.05.

e Subchronic: 60 ppm AN for 4 hrs/d, 5 d/wk for 7 wk s (starting during gestation), followed by 7 hrs/d for 8 wks.

Source: Maltoni et al. (1988).

In contrast, chronically exposed male and female o ffspring showed statistically significant increases in the incidences of malignan t tumors of the mammary gland in females, extrahepatic angiosarcomas in females, hepatomas in males, Zymbal gland carcinomas in males, and encephalic gliomas (see Table 4-40). In the second bioassay (designated BT4006), Sprague -Dawley rats were initially exposed to AN under the same exposure conditions as bioassa y BT4003; however, exposure of the offspring (127 males and 114 females) ended after 1 5 weeks (Maltoni et al., 1988). This group of offspring was exposed for 4 hours/day, 5 days/we ek for 7 weeks starting on GD 12, followed by exposure for 7 hours/day, 5 days/week for 8 week s. All animals were kept under observation until spontaneous death, at which time they were ex amined for the presence of tumors. The4 DRAFT - DO NOT CITE OR QUOTE

control group of offspring was the one used in expe riment BT4003 (158 males and 149 females). There was a statistically significant increase in t he total incidence of malignant tumors in exposed offspring compared with controls for both m ales (31.7 vs. 17.1%, p < 0.05) and females (35.0 vs. 17.4%, p < 0.01). Increased incidences of the following tum ors were observed when compared with controls, although the incidences wer e not statistically significantly different: Zymbal gland tumors in males and females combined; extrahepatic angiosarcomas in males; encephalic gliomas in males and females combined; a nd hepatomas in males (see Table 4-40). NTP recently released a memorandum (Malarkey et al. , 2010) that discussed differences of opinion between NTP scientists and the Ramazzini Institute in the diagnoses of certain cancers reported in a methanol study conducted by the Ramaz zini Institute. See Section 5.4.4.3 for
additional information on the use of the Maltoni et al. studies in this assessment.

5.4.4.3. Application of Age-Dependent Adjustment F actors (ADAFs) AN is determined to be carcinogenic by a mutagenic mode of action (see Section 4.7.3.1), which raises concern for increased early-life susce ptibility to cancer. Consistent with this possibility, two studies in Sprague-Dawley rats pro vide some evidence of increased cancer susceptibility associated with chronic AN exposure that begins in early periods of development—a chronic-duration inhalation cancer bi oassay (Maltoni et al., 1988) and a threegeneration drinking water reproductive toxicity stu dy (Friedman and Beliles, 2002). According
to the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (“ Supplemental Guidance ”) (U.S. EPA, 2005b), individuals exposed during ea rly life to carcinogens with a mutagenic mode of action are assumed to be at increased risk for cancer. In these situations, the Supplemental Guidance recommends that age dependent adjustment factors (ADAFs) be applied in estimating cancer risk. The guidance further
recommends that when data are available for a susce ptible lifestage, those data should be used directly to evaluate risks for that chemical and th at lifestage. In the Maltoni et al. (1988) study conducted at the Ramazzini Institute, pregnant SpragueDawley rats were exposed to 60 ppm AN by inhalation for 104 weeks (see Section 4.2.2.2.2 for a complete study description). In addition, offsprin g of these dams were exposed to AN starting at GD 12 for a total of 104 weeks. Female rats expose d from GD 12 had a higher incidence of malignant mammary tumors, encephalic gliomas, and e xtrahepatic angiosarcomas than female rats exposed as adults. The drinking water bioassa y by Friedman and Beliles (2002) involved 48-week exposures of each of three generations of f emale breeder Sprague-Dawley rats (see Section 4.2.1.2.9 for a complete study description) . Compared with the 500-ppm F0 breeders that were exposed starting in adulthood, there was an increase in Zymbal gland and brain tumor incidence in the 500-ppm F1b female rats exposed st arting in utero. Increases in tumor incidence5 DRAFT - DO NOT CITE OR QUOTE

in the F2 generation were not statistically signifi cantly different from F0 breeders. The Maltoni et al. (1988) and Friedman and Beliles (2002) studies were considered for use in deriving chemical-specific ADAFs for early-l ife exposure to AN. The Maltoni et al. (1988) tumor results were not used to develop chemi cal-specific ADAFs because of concerns raised by a memorandum from NTP (Malarkey et al., 2 010) that discussed differences of opinion
between NTP scientists and the Ramazzini Institute in the diagnoses of certain cancers reported in a methanol study. While these data are consider ed qualitatively as support for early-life susceptibility, EPA decided not to rely on these da ta for quantitative purposes. The Friedman and Beliles (2002) study was also not considered fu rther for the derivation of chemical-specific ADAFs. This study had several limitations, includi ng a small number of animals per treatment group (20) and, therefore, limited power to detect tumor increases, and a lower tumor response in the F2b generation than the F1b generation. Therefo re, although limited data are available supporting early-life susceptibility to carcinogeni city from AN exposure, the available information was not considered suitable for develop ing data-specific ADAFs. Accordingly, it is recommended that default ADAFs be applied to cancer risk values (OSF and IUR) for AN.
The Supplemental Guidance establishes default ADAFs for three specific age gr oups. These ADAFs and their corresponding age groups are: 10 for exposed individuals <2 years; 3 for exposed individuals 2 to <16 years; and 1 for expos ed individuals ≥ 16 years. The 10- and 3-fold adjustments are combined with age-specific exposure estimates when estimating cancer risks from early life (<16 years of age) exposures to AN. Example calculations for estimating cancer risks based on age at exposure are provided in Sect ion 6 of the Supplemental Guidance (U.S. EPA, 2005b).

6.2.3. Oral Slope Factor

... Because a mutagenic mode of action for acrylonitril e carcinogenicity is sufficiently supported in laboratory animals and relevant to hum ans, and in the absence of adequate chemical-specific data to evaluate differences in s usceptibility, increased early-life susceptibility is assumed. Accordingly, early-life susceptibility factors or ADAFs should be applied to the OSF when assessing cancer risk associated with earl y-life exposures (i.e., birth to 16 years) in
accordance with the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 2005b) (see Section 5.4.4.3).

6.2.4. Cancer Inhalation Unit Risk6 DRAFT - DO NOT CITE OR QUOTE

... As described for the OSF, early-life susceptibility factors or ADAFs should be applied to the IUR when assessing cancer risks associated with early-life exposures (i.e., birth to 16 years)
in accordance with the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 2005b) (see Section 5.4.4.3).