SRC TR

- 10

- 018

HPV4 CHEMICAL DATA ADEQUA CY REVIEW

2,5

- Fu randione, 3

- (dodecenyl)dihydro

- , CAS No. 25377

- 73

- 5

Prepared by:

Justine Von Runnen, Teresa Manyin, William L. Richards, and Joan D. Garey

Environmental Science Center
SRC, Inc.

75 02 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027

Task FG007.0.009.002

Submitted to:

OPPT/RAD/HPVCB (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Elizabeth Margosches , Project Officer Meena Sonawane, Work Assignment Manager David Brooks, Alternate Work Assignment Manager

February 5, 2010HPV4 Chemical Data Adequacy Review

2,5

- Furandione, 3

- (dodecenyl)dihydro

- , CAS No. 25377

- 73

- 5

February 2010

DRAFT

Submitted to: High Pr oduction Volume Chemicals Branch

Risk Assessment Division

Office of P ollution Prevention and Toxics Environmental Protection Agency

WAM: Meena Sonawane

Altern ate WAM: David Brooks 1200 Pennsylvania Avenue, NW

Washington, DC 20460

- 0001

Prepared By:

SRC , Inc.

Environmental Science Center 2451 Crystal Drive, Suite 804 Arlington, VA 22202Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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1 A. PREPARATION OF THE DATA ADEQUACY ASSESSMENTS

A

- 1. Source material used for assessment

For this unsponsored chemical, a comprehensive literature search w as performed to identify available toxicity data for the following SIDS endpoints:

Acute toxicity to fish Acute toxicity to Daphnia Toxicity to aquatic plants

Acute (oral, inhalation ,dermal ) toxicity Repeated

- dose toxicity Reproductive toxicity Developmen tal toxicity

Gene mutation

Chromosomal aberrations

Studies relevant to non

- SIDS endpoints (e.g., eye and skin irritation; neurotoxicity) conducted via oral, inhalation, or dermal routes were also identified through literature searching.

The following sou rces were searched for toxicity data:

PubMed

- B ibliographic database that contains references to journal articles in the life

sciences with a concentration on biomedicine

T OXNET

- N ational L ibrary of M edicine databases on toxicology, hazardous chemicals

, environmental health, and toxic releases; includes:

**•** TOXLINE with BIOSIS and NTIS ;

**•** Development and Reproductive Toxicology File (DART) ;

**•** Chemical Carcinogenesis Research Information System (CCRIS)

- Carcinogenicity and mutagenicity test results for over 8000

**•** chemicals) ;

**•** Hazardous Substances Data Bank (HSDB)

- Comprehensive, peer

reviewed toxicology data for about 5000 chemicals ;

**•** Genetic Toxicology Data Bank (GENE

- TOX)

- Peer

- reviewed genetic toxicology test data for over 3000 chemicals.

TSCATS

- U npu blished technical reports submitted by industry to the EPA under the

Toxic Substances Control Act

Registry of toxic effects of chemical substances (RTECs)

Environmental Fate Database (EFDB)

ECOTOX

- EPA’s peer

- reviewed ecotoxicology databaseDraft HPV4 Chemical Data Adequacy Review CAS No. 25377

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2 EPA’s Inte grated Risk Information System (IRIS) website

Agency for Toxic Substances and Disease Registry (ATSDR) website

National Toxicology Program (NTP) website

International Program on Chemical Safety Concise International Chemical Assessment Documents (IPCS I NC HE M or CICADs)

European Chemical Substances Information System (ESIS)

- source of IUCLID Datasets

Any available IUCLID Datasets found through the ESIS website and robust summaries submitted to EPA by former sponsors on the assigned chemicals were retr ieved. Original sources were obtained for any relevant toxicity data found for the assigned chemicals.

While IUCLID D ataset s sometimes contained studies of interest, certain study descriptions contained insufficient detail to assess adequacy. In these cases, the primary source of the study, if available and accessible, was obtain ed and review ed.

A

- 2. Evaluating data adequacy

Using standard OECD testing guidelines and professional judgment as guides, available toxicity studies for the unsponsored chemical were reviewed for adequacy. Summaries were prepared for all relevant data found, even if little or no study detail was available. Each study was summarized according to a standard format and assigned a Klimisch reliability code; all study summari es for the chemical were combined into a single summary document. For studies already assigned Klimisch codes (e.g., studies summarized in IUCLID datasets), an explanation was provided if the reviewer’s assessment of data adequacy differed from the pre

- as signed Klimisch code. A table summarizing data availability/adequacy for each SIDS endpoint is

included at the end of the summary document.

B. SPECIFIC INFORMATION ON THE AVAILABLE TOXICITY DATA

The available data for each of the SIDS toxicity endp oints are evaluated on the following pages. Studies for a common endpoint are grouped together in the document and an endpoint adequacy assessment is provided near t he top of the first page of summaries for that endpoint. If

the data available for the en dpoint is determined to be inadequate, a basis for that conclusion is supplied. NOTE: If no relevant studies were identified for the chemical , only the data availability/adequacy summary table was completed for the report

.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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3 B

- 1. ACUTE TOXICITY TO FISH

Summary of endpoint: Inadequate

Study 1: Acute toxicity to fish study

Title: Acute toxicity to fish

This study is: Adequate Not Adequate

This study is inadequate because it tested a non

- preferred species, had a f ish loading rate that exceeds the recommended value, and is missing study details, including water hardness and total organic carbon, test substance purity, nominal and measured test concentrations, and mortality at

each concentration.

TEST SUBSTANCE

Identity: Dodecenyls uccinic a nhydride , CAS No. 25377

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Remarks: Purity not specified

METHOD

Method/guideline followed: Standard Methods for the Examination of Water and Wastewater (1971) American Public Health Association, Static acute toxicity te st, Method No. 231.

Test type: Acute Fish T oxicity

– S tatic

GLP compliant?: Not stated

Year study was performed: 1979

Test species: Goldfish ( Carassius auratus ) ; average length: 6.2 ± 0.7 cm; average weight: 3.3 ± 1.0 g

Analytical monitoring: Yes

Exp osure duration: 24 or 96 hours

Concentrations tested: Not specified

Test method/conditions remarks: Ten fish per test concentration were exposed at 20 ± 1 °C in

25 L of solution contained in all

- glass tanks measuring 42 x 28 x 28 cm. The duration of the test was 24 or 96 hours and the solutions were aerated throughout the test period. The concentration of the chemical in solution was determined before and after each test, either with a total organic carbon analyzer or by extraction and subsequent GC ana lysis. Based on the average fish weightDraft HPV4 Chemical Data Adequacy Review CAS No. 25377

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4 of 3.3 g and the testing of 10 fish/25L, the loading rate was 1.32 g fish/L. The latter value

exceeds the OECD TG 203

- recommended maximum loading of 1g fish/L for static tests.

Solutions were made in local tap wat er. The analysis was:

Cl - = 65 mg/L

NO 2 - = 0 mg/L

NO 3 - = 4 mg/L

SO 4 2

- = 35 mg/L

PO 4 3

- = 0.15 mg/L

HCO 3 - = 25 mg/L

SiO 2 = 25 mg/L

NH 4 + = 0 mg/L Fe = 0.05 mg/L Mn = 0 mg/L

Ca 2+ = 100 mg/L Mg 2+ = 8 mg/L

Na + = 30 mg/L pH = 7.8

The LC 50 was obtained by inter polation from a graph of the logarithm of the concentration versus the percentage mortality.

RESULTS

Endpoint value(s):

24

- hour LC50 (pH = 7.0) = 4 mg/L

96

- hour LC50 (pH = 7.0) = 3 mg/L

24

- hour LC50 (pH = 6.0) = 2 mg/L

96

- hour LC50 (pH = 6.0) = 2 mg/L

Results remarks: None specified

CONCLUSIONS

The LC 50 for fish ranged from 2 to 4 mg/L in static acute exposure studies at a pH of 6.0 or 7.0 and varying in duration from 24 to 96 hours.

STUDY RELIABILITY

Klimisch code = [3]

– Not reliable; non

- preferre d species and fish loading rate that exceeded guideline recommendations; missing study details included water hardness, total organic carbon, test substance purity, nominal and measured test concentrations, and mortality at each

concentration.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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5 REFERENC E S

Bridie et al. (1979) The acute toxicity of some petrochemicals to goldfish. Water Research 13,

623

– 626.

Bridie et al. (1974) Determination of Acute Toxicity to Fish of Shell Chemicals, II.

AMGR.0071.74.

T SCATS OTS0206206; Doc ID : 878210130. She ll Oil Company (12/10/82).Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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6 B

- 2. ACUTE TOXICITY

Summary of endpoint: Ina dequate

Study 1: Acute oral toxicity study

Title: Acute oral study

The study is: Adequate Not Adequate

This study is inadequate because the source document, a robust summary in a submission to the HPV Challenge Program, cites a secondary source ( ACC HERTG panel Robust Summary “Alkenyl Succinic Anhydrides”, updated Nov. 22, 2002 ) and no primary source was identified by searches of PubMed, Toxli ne, HSDB, or TSCATS.

TEST SUBSTANCE

Identity: Dodecenyls uccinic a nhydride , CAS No. 25377

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Remarks: Purity not specified

METHOD

Method/guideline followed: Similar to OECD Guideline 401

Test type: Acute oral toxicity

GLP compliant?: Pre

- GLP

Year study performed: 1978

Test species: Sherman

- Wistar rats

Number of animals/sex/dose: 5 males/dose

Route of administration: Oral (gavage)

Vehicle: None

Doses/concentrations tested: 1, 2, 4, 8, 16 g/kg

Statistical methods used: Not stated

Test method /conditions remarks: Single administration of the test material was given intragastrically to five fasted male rats at each dose level. The animals were observed for signs of toxicity or behavioral changes on the day of treatment and throughout the 14

- day observationDraft HPV4 Chemical Data Adequacy Review CAS No. 25377

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7 period. Individual weights were recorded immediately prior to dosing and prior to termination. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals.

RESULTS

Endpo int value(s): LD 50 = 2.9 g/kg (2

– 4 g/kg )

Results remarks: During the first three days of study all animals treated at the 4, 8 and 16 g/kg

dose levels died. No deaths were observed at the 1 and 2 g/kg dose levels. No clinical signs of toxicity were obs erved at 1 g/kg. At 2 g/kg the animals were lethargic and had an oily appearance for up to 48 hours post dosing. All animals at the 4, 8 and 16 g/kg dose levels were severely depressed prior to death. No body weight effects occurred at 1 or 2 g/kg. Bod y weight

data w ere not available at higher dose levels due to the observed mortality. No test material related macroscopic findings were evident.

CONCLUSIONS

LD 50 (males) = 2.9 g/kg

STUDY RELIABILITY

Klimisch code = [4]

– Not assignable; data are from a secondary source.

REFERENCE

HPV submission for Higher Alkenyl Succinic Anhydride s category, received by EPA from Albemarle Corporation on April 5, 2006 ; HPV submission references ACC HERTG panel Robust Summary “Alkenyl Succinic Anhydrides”, updated No v. 22, 2002Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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8 Study 2: Acute inhalation toxicity study

– limit test

Title: Acute i nhalation study

The study is: Adequate Not Adequate

The study is not adequate because it represents a limit tes t and it produced compo und

- related

mortalities

.

TEST SUBSTANCE

Identity : Dodecenyls uccinic a nhydride , CAS No. 25377

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Remarks: Purity not specified ; A ppearance: Amber liquid; S ponsor’s identification code: 127

- 2638

, Dodecenyl Succinic A nhydride; FDRL i dentification c o de: 80

- 1168

METHOD

Method/guideline followed: S imilar to OECD limit test

Test type: A cute inhalation toxicity, limit test

GLP compliant?: Yes (FDA guidelines for Good Laboratory Practice were followed)

Year study performed: 1981

Test species: Sprague

- Dawley rats obtained from Blue Spruce Farms, Altamont, NY. Group mean body weight (g) on day 1: 285 ± 5 (males) and 247 ± 6 (females)

Number of animals/sex/dose: 5/sex/dose

Route of administration: Inhalation

Vehicle: Ethanol (190 proof, U.S. Industria l Chemicals, New York, NY)

Doses/concentrations tested: 5.3 mg/L (nominal); 1.22 mg/L (time

- weighted gravimetric concentration)

Statistical methods used: Not stated

Test method/conditions remarks: The purpose of the study was to evaluate the acute inh alation toxicity of dodecenyl succinic anhydride (DDSAA) according to the OECD Acute Toxicity Screening Program, Acute Inhalation Studies Protocol, as modified and amplified by the sponsor. The aerosol consisted of 90% DDSAA and 10% ethanol (w/w) with a g eometric mean particle size of 2.3 μ m (S.D. = 2.2 μ m). To serve as a control, additional animals were exposed to an atmosphere containing a liquid droplet aerosol of ethanol and observed a required by theDraft HPV4 Chemical Data Adequacy Review CAS No. 25377

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9 protocol. The animals were exposed to an airborne aerosol of the test article for 4 hours. During exposure, the animals were individually housed in wire mesh cages and denied food and water. Following exposure, the animals remained individually housed and food and water were provided ad libitum

.

The animals were observed for pharmacotoxic signs during exposure (day 1), subsequent to exposure on day one, and daily for 14 additional days (days 2

– 15). Body weights were also recorded from day 1 to 15 of the study. All animals were subject to gross ne cropsy. During necropsy, all major organs were examined for any signs of abnormalities and the following tissues excised and preserved in 10% neutral buffered formalin: whole head with nasal passages, trachea, bronchi, lungs, liver, and kidneys.

The ac tual exposure concentrations were determined by gravimetric analysis; a known volume of chamber air was drawn through a preweighed filter and the actual concentration was calculated by dividing the filter weight gain by the sample volume, and then correcti ng for dilution (by multiplying by 0.90).

RESULTS

Endpoint value(s): LC 50 > 1.2 mg/L (time

- weighted gravimetric concentration)

Results remarks: The observed mortality was 2/5 for females by the second day and 2/5 males by the fifth day of post

- exposure observation. No further deaths were observed by the 15 th day

post

- exposure. Thus, the LC 50 for 4

- hour inhalation exposure is greater than 1.2 mg/ L

.

Significant observations included labored breathing, nasal discharge, urinary incontinence, and eye irri tation. Labored breathing was present in all animals and continued throughout the study. Bloody nasal discharge was also observed in all animals during exposure and generally persisted throughout the observation period. Eye irritation, closing of the e yes (due to swelling of periorbital tissues) and a cloud or film

- covered appearance of the eye was evident. Apparent blood around the eyes may have been porphyrin

- containing secretions of the Harderian glands

and is often seen in debilitated animals. Ur inary incontinence was noted in all males on days two through four, and alopecia (hair loss) was evident in two males and one female subsequent to day seven. Following exposure to the test article, body weight loss occurred in all animals. This was

due t o dodecenyl succinic anhydride exposure, as ethanol treatment caused no comparable effect. By day 15, the three surviving males and one female had regained their original body weights.

No findings attributable to test article exposure were evident upo n gross necropsy. Various color alterations of the lung and other organs (dark, pale, spotting) are routine post mortem observations in the laboratory rat and results from p ost mortem congestion within the tissues.

CONCLUSIONS

Four

- hour exposure to liq uid droplet aerosol of dodecenyl succinic anhydride (90% w/w solution in ethanol) produced mortality in two of five male and two of five female rats. LaboredDraft HPV4 Chemical Data Adequacy Review CAS No. 25377

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10 breathing, transient urinary incontinence, alopecia, eye irritation, and body weight loss were

ob served following exposure to the test article.

STUDY RELIABILITY

Klimisch code = [3]

– Not reliable . The study is not adequate for the e ndpoint as it describes a

limit test in which compound

- related mortalities occurred.

REFERENCE

This summary is base d on a study from a TSCATS submission OTS0505452 (Document ID: 88

- 8100369) conducted by Food and Drug Research Laboratories , for Buffalo Color Corporation: Acute Inhalation Toxicity of Dodecenyl Succinic Anhydride in Sprague

- Dawley Rats with Cover

Lette r Dated 04/08/82 . (Study produced on March 24, 1981)Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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11 B

- 3. GENETIC TOXICITY

– GENE MUTATION

Summary of endpoint: Ad equate

Study 1: In vitro Salmonella mutagenicity assay

Title: Ames test

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Dodecenylsuccinic anhydride , CAS No. 25377

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Remarks : Purity not specified

METHOD

Method/guideline followed: Ames et al. 1975, with and without metabolic activation. Plate incorporation method.

Test typ e: In vitro Ames Salmonella mutagenicity assay

GLP compliant?: Not s pecified

Year study performed: Not stated

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation: With (rat or hamster S9) and without

Concentrations tested: 0.3, 1, 3.3, 10, 100 μg/plate without activation and 100, 333, 1000,
3333, 10 ,000 μg/plate with activation

Statistical methods used: No data

Test method/conditions remarks: Liver S9 homogenate was prepared from male Sprague

Dawle y rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added.

For testing in the absence of S9 mix, 100 μL of the tester strain and 50 μL of t he solvent or test chemical were added to 2.5 mL of molten selective top agar at 45 ± 2 oC. When S9 was used, 0.5 mL of S9 mix, 50 μL of tester strain, and 50 μL of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45 ± 2 oC. After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates

were incubated for 48 h at 37 ± 2 oC. Five doses of test chemical, together with the appropriateDraft HPV4 Chemical Data Adequacy Review CAS No. 25377

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12 concurrent sol vent ( dimethyl sulfoxide; DMSO) and positive controls (not specified), were tested in triplicate on each tester strain without metabolic activation and also with activation by induced rat and hamster liver S9 preparations.

The doses that were tested in th e mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range

- finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of

induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA 100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the

test chemical. If the study showed a do se response with a less than 3

- fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration: None specified

Results remarks: Positive and negative controls res ponded appropriately.

CONCLUSIONS

The test substance did not cause an increase in mutation frequency in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 or TA1538 with or without metabolic activation.

STUDY RELIABILITY

Klimisch code = [2]

– R eliable with restrictions; test substance purity was not specified. Although the findings from this study were presented in a review article and a citation to a previous publication of the data was not provided, the primary data from the study were

present ed in “Supporting Information” documents at http://pubs.acs.org

.

REFERENCE

Seifried et al . (2006) A c ompilation of t wo d ecades of m utagenicity t est r esults with the Ames Salmonella typhimurium and L5178Y m ouse l ympho ma c ell m utation assays. Chem ical Research in Toxicology 19 : 627

- 644.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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13 Study 2: Mouse lymphoma mutagenicity assay

Title: Mouse lymphoma mutagenicity assay

The study is: Adequate Not Adequate

TEST SUBSTANCE

Ident ity: Dodecenylsuccinic anhydride, CAS No. 25377

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Remarks: Purity not specified

METHOD

Method/guideline followed: Clive and Spector (1 975 ) Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y TK +/

- mouse lymph oma cells

Test type: I n vitro m ouse lymphoma cell mutagenicity assay

GLP compliant?: Not s pecified

Year study performed: Not stated

Test species: L5178Y TK +/

- 3.7.C mouse lymphoma cells

Metabolic activation: W ith (rat S9) and without

Concentrations t ested: 0.017 , 0.022, 0.026, 0.031, 0.035 μg/mL without activation and 0.035, 0.08, 0.13, 0.17, 0.22, 0.26, 0.31 μg/ mL with activation

Statistical methods used: No data

Test method/conditions remarks: The toxicity of each chemical was determined both wit h and without liver S9 prepared from Aroclor 1254

- induced male Sprague

- Dawley rats. S9 mix was prepared according to the procedure of Clive et al. (1979). Cells at a concentration of 6 x

10 5 /mL (6 x 10 6 cells total) were exposed for 4 h to a range of con centrations from 0.0005 to

10

, 000 μ g/mL. The cells were then washed, resuspended in growth medium, and incubated at 37

± 1 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solv ent controls. The doses of chemical selected for testing were within the range yielding approximately 0

– 90% cytotoxicity. For each assay, there

were 2

– 4 solvent (DMSO) controls, a positive control of ethyl methylsulfonate at 4.7 x10 - 6 M (or methyl me thanesulfonate at 10

– 20 μ g/mL) for the test without metabolic activation, and a positive control of 3

- methylcholanthrene at 1.86 x 10 - 5 M (or dimethylbenz[ a ]

- anthracene at 0.5

– 4 μ g/mL) for the test with metabolic activation.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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14 The mutagenicity assay was performed according to the procedure described by Clive and Spector (1975) . A total of 1.2 x 10 7 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 °C, washed twice with growth medium, a nd maintained at 37 ± 1 °C for 48 h in log

- phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 x 10 5 /mL at 24 h intervals. They were then cloned (1 x

10 6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F

- 68, and 0.23% granulated agar (BBL, Inc., Cockeysville, MD). Resistance to trifluorothymidine (TFT) was determined by add ing TFT (final concentration, 3 μ g/mL) to the cloning medium for mutant selection. The 100x stock solution of TFT in saline was stored at

- 70 °C and was thawed immediately before use. Plates were incubated at 37 ± 1 °C in 5% CO 2 in air for 10

- 12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than

~ 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10 6
surviving cells.

Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent

treated control value as an indication of a positive ef fect, together with evidence of a dose

- related

increase. Doubling of the mutant frequency was previously reported as representing a positive

effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Do ses yielding less than 10% total growth were used in determining dose

response. The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to st ep sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from

~ 0.2 to 1.1 mm.

RESULTS

Genotoxicity result: Negative with and without metabolic activation using the evaluation criteria that are described above. The Seifried et al. (2006) publication also presented mutagenicity conclusions that were stated to be “new” evaluations based on ‘current international “harmonization” recommendations.’ However, these “new” evaluations were not interpretable and are not considered further because this publication neither presented the criteria used for the “new” evaluations nor cited literature that present such criteria.

Cytotoxic concentration: ≥ 0.031 μg/mL without activation, ≥ 0.13 μg/mL with activation

Results remarks: Positive and negative controls responded appropriately.

CONCLUSIONS

The test substance was negative for mutagenicity in mouse lymphoma cells with and without

metabolic act ivation.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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15 STUDY RELIABILITY

Klimisch code = [2]

– Reliable with restrictions; test substance purity was not specified. Although the findings from this study were presented in a review article and a citation to a previous publication of the data was no t provided, the primary data from the study were presented in “Supporting Information” documents at http://pubs.acs.org

.

REFERENCE

Seifried et al . (2006) A c ompilation of t wo d ecades of m utagenicity t est r esults with the Ames Salmonella typhimurium and L5178Y m ouse l ymphoma c ell m utation assays. Chem ical Research in Toxicology 19 : 627

- 644.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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16 B

- 3. SKIN IRRITATION

Summary of endpoint: Inadequate

Study 1: Skin irritation study

Title: Skin Irritation Study

The st udy is: Adequate Not Adequate

This study is inadequate because data are from a secondary source, test substance purity was not provided and study details were missing.

TEST SUBSTANCE

Identity: Dodecenylsuccinic a nhydride, CAS No. 25377

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Remarks: Light yellow, clear, viscous liquid; MW: 266; Solubility: reacts with water and alcohols, soluble in certain oils and industrial solvents

METHOD

Method/guideline followed: Not Specified

Test type: Dermal Irritation

GLP compliant?: Pre

- GLP

Year study performed: 1964

Test species: Female albino guinea pig (387

– 395 g)

Number of animals/sex/dose: Not Specified

Route of administration: Dermal

Type of Coverage: Occlusive

Preparation of test site: Clipped skin

Dos es/concentrations tested: Undiluted and 10% solutions

Exposure Period: 24 hours

Total Volume applied: 0.4 mLDraft HPV4 Chemical Data Adequacy Review CAS No. 25377

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17 Vehicle: Corn Oil

Post

- Exposure Period: 14 days

Test method/conditions remarks: Patch tests were performed on female, albino guinea pigs weigh ing 387 to 395 g, using 0.4 mL of the indicated concentrations of the test solution.

Undiluted material was applied to closely clipped abdominal skin of albino guinea pigs by means

of cotton

- gauze patches (2 x 2 cm) that were held in contact with the sk in by means of a 3

- in. Elastoplastic bandage. The patches were removed 24 hours after application, and the animals were observed for a period of at least 14 days; when damage occurred, observations were continued until the damaged areas healed. When a re sponse was elicited with the undiluted material, tests were repeated using 10% and 1% solutions.

RESULTS

Results remarks: With the pure compound, there was a slight edema which persisted for 3 days, a scab which was evident on the 8th day, and complete healing on the 14th day. The 10% solution (corn oil) produced no effects.

CONCLUSIONS

The test substance was slightly irritating to guinea pig skin.

STUDY RELIABILITY

Klimisch code = [ 4 ]

– Not assignable; d ata are from a secondary source; test substanc e purity not provided; limited study details

.

REFERENCE

Longley et al. 1964; AMRL (1964) Toxic Hazard Study of Selected Missile Propellants Document Number(s): AD 441 640. AMRL

- TDR

- 64

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18 B

- 4. EYE IRRITATION

Summary of endpoint: Inadequate

Study 1: Eye irritation study

Title: Eye Irritation Study

The study is: Adequate Not Adequate

This study is inadequate because data are from a secondary source, test substance purity was not provided and study details were missing.

TEST SUBSTANCE

Identity: Dodecenylsuccinic anhydride, CAS No. 25377

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Remarks: Light yellow, clear, viscous liquid; MW: 266; Solubility: Reacts with w ater and alcohols, soluble in certain oils and industrial solvents

METHOD

Method/guid eline followed: Not Specified

Test type: Eye Irritation

GLP compliant?: Pre

- GLP

Year study performed: 1964

Test species: Albino Guinea Pig (sex not specified)

Number of animals/sex/dose: Not Specified

Vehicle: None

Doses/concentrations tested: 0. 05 mL, undiluted

Test method/conditions remarks: Undiluted material was instilled into the conjunctival sac of albino guinea pigs. Effects were noted at 1 min, 5 min, 1 hour and 24 hours after exposure, and daily thereafter for a total of at least 14 day s. When damage occurred, observations were continued until healing was completed.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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19 RESULTS

Results remarks: The pure compound produced slight swelling, with a very slight opacity in the cornea and a moderate discharge which lasted for 1 day. There a ppeared to be no effects persisting beyond the 2nd post

- exposure day.

CONCLUSIONS

The test substance was slight ly irritating to guinea pig eyes.

STUDY RELIABILITY

Klimisch code = [ 4 ]

– Not assignable; d ata are from a secondary source; test substance p urity not provided; limited study details

.

REFERENCE

Longley et al . 1964; AMRL (1964) Toxic Hazard Study of Selected Missile Propellants Document Number(s): AD 441 640. AMRL

- TDR

- 64

- 28.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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20 B

- 5. DERMAL SENSITIZATION

Summary of endpoint: Adequate

Study 1: Dermal sensitization in guinea pigs

Title: Delayed contact sensitization in guinea pigs

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Dodecenylsuccinic anhydride, CAS No. 25377

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Remarks: 98% purity; test substance referred to as "C12 ASA"

METHOD

Method/guideline followed: Ritz and Buehler, 1980

Test type: Dermal Sensitization

GLP compliant?: Yes

Year study performed: 1986

Test species: Hartley Albino Guinea Pig

Number of animals/sex/ dose: 10/sex for test article; 5/sex for two naive control groups

Route of administration: Dermal

Doses/concentrations tested:

Induction: 25% w/v Primary challenge: 5% w/v Rechallenge: 3% w/v

Vehicle: Acetone

Test method/conditions remarks:

For ind uction of sensitization, the upper left quadrant of the backs of guinea pigs were clipped of hair. On the following day, moistened patches were applied, to the test group, the animals restrained as previously described, and the animals returned to their ca ges. Patches moistened with test article were applied to the skin in the same manner once a week for three applications.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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21 The same site was clipped on the day before application, and the restraint periods were six hours

on each occasion.

For primary challe nge two weeks after the last of the induction applications, a fresh application site was prepared by clipping the lower left quadrant of the backs of test and naïve control animals. The next day, challenge patch was applied to each guinea pig in the test a nd control groups. Each animal was restrained for 6 hours as before, and the animals returned to their cages. On the next day, sites were depilated and scored for severity of response at least two hours later and for a 48 hour reading. Scores of "1" or gre ater in the test group were considered to be indicative of sensitization providing grades of less than 1 were found in the naïve control group. If the naïve group had grades of "1" or greater, the reactions in the test group that exceeded the

most severe r eactions in the control group were presumed to be indicative of sensitization.

For rechallenge, eight days after the primary challenge, all of the original animals were single patch rechallenged. Ten previously unexposed naïve animals were identically tre ated to serve as a new naïve control group. The right rear quadrant was used for rechallenge. Depilation and observation procedures were the same as described for primary challenge.

Individual weights were recorded immediately prior to dosing and prior to termination. The surviving animals were euthanized at the conclusion of the observation period.

RESULTS

Results remarks: No deaths were observed in the test. The incidence of grade "2" responses or greater in the test group exposed to 25% for induction, and 5% at primary challenge was greater for the test animals (13 of 20) compared to the naïve challenge group (0 of 10). The incidence and severity of responses in the test group were more pronounced than the responses of the naïve group, suggesting sensi tization had occurred.

At rechallenge with 3% test article in acetone, the incidence of grade "1" responses or greater in the test group (17 of 20) was greater than that in the naïve control group (0 of 10). The incidence and severity of responses were mo re pronounced in the test group indicating that a sensitization response had been elicited.

CONCLUSIONS

The test article, when administered to guinea pigs according to the method of Ritz and Buehler (1980) , caused delayed contact hypersensitivity of the skin.

STUDY RELIABILITY

Klimisch code = [2]

– Reliable with restrictions ; study obtained from secondary source; primary

source not available.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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22 REFERENCE S

HPV submission for Higher Alkenyl Succinic Anhydrides category, received by EPA from

Albemarle Co rporation on April 5, 2006; HPV submission references the following:

Buehler, E.V., "Delayed Contact Hypersensitivity Study in Guinea Pigs of Dodecenyl Succinic Anhydride C12 ASA" for Ethyl Corporation, Hill Top Research Project No. 86

- 0873

- 21, Hill Top R esearch, Inc., 1986.

Ritz, H.L, and Buehler, E.V., (1980), In Current Concepts in Cutaneous Toxicity, (V.A. Drill and T.Lazar, eds.) pp. 25

- 40, Academic Press, New York.Draft HPV4 Chemical Data Adequacy Review CAS No. 25377

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23 B

- 6. CAS No. 25377

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- 5: Summary of Endpoints

Endpoint Study Information

Availab le Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish Yes No Yes Acute Toxicity to Daphnia No

- Yes Toxicity to Aquatic Plants No No Yes

Health Effects Acute Toxicity Yes No Yes Repeated

- Dose Toxicity No

- Yes

Gene Mutation Yes Yes No

Chro mosomal Aberrations No

- Yes

Reproductive Toxicity No

- Yes Developmental Toxicity No

- Yes