SRC TR-10-018

HPV4 CHEMICAL DATA ADEQUACY REVIEW

2,5-Furandione, 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.

7502 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, 2010
HPV4 Chemical Data Adequacy Review

2,5-Furandione, 3-(dodecenyl)dihydro-, CAS No. 25377-73-5

February 2010

DRAFT

Submitted to:

High Production Volume Chemicals Branch 

Risk Assessment Division 

Office of Pollution Prevention and Toxics 

Environmental Protection Agency 

WAM: Meena Sonawane

Alternate 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 22202
Draft HPV4 Chemical Data Adequacy Review CAS No. 25377-73-5

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A. PREPARATION OF THE DATA ADEQUACY ASSESSMENTS

A-1. Source material used for assessment

For this unsponsored chemical, a comprehensive literature search was 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

Developmental 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 sources were searched for toxicity data:

PubMed - Bibliographic database that contains references to journal articles in the life 

sciences with a concentration on biomedicine

TOXNET - National Library of Medicine 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, peerreviewed toxicology data for about 5000 chemicals;

• Genetic Toxicology Data Bank (GENE-TOX) - Peer-reviewed genetic 

toxicology test data for over 3000 chemicals.

TSCATS - Unpublished 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 database
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EPA’s Integrated 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 INCHEM 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 retrieved. Original 

sources were obtained for any relevant toxicity data found for the assigned chemicals. 

While IUCLID Datasets 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 obtained and reviewed.

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 summaries 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-assigned 

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 endpoints 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 the top of the first page of summaries for that endpoint. If 

the data available for the endpoint 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.
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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 fish 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: Dodecenylsuccinic anhydride, CAS No. 25377-73-5

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 test, Method No. 231.

Test type: Acute Fish Toxicity – Static

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

Exposure 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 analysis. Based on the average fish weight 
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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 water. The analysis was:

Cl- = 65 mg/L

NO2

- = 0 mg/L

NO3

- = 4 mg/L

SO4

2- = 35 mg/L

PO4

3- = 0.15 mg/L

HCO3

- = 25 mg/L

SiO2 = 25 mg/L

NH4

+ = 0 mg/L

Fe = 0.05 mg/L

Mn = 0 mg/L

Ca2+ = 100 mg/L

Mg2+ = 8 mg/L 

Na+ = 30 mg/L

pH = 7.8

The LC50 was obtained by interpolation 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 LC50 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-preferred 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.
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REFERENCES

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. 

TSCATS OTS0206206; Doc ID: 878210130. Shell Oil Company (12/10/82).
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B-2. ACUTE TOXICITY

Summary of endpoint: Inadequate

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, Toxline, HSDB, or TSCATS.

TEST SUBSTANCE 

Identity: Dodecenylsuccinic anhydride, CAS No. 25377-73-5

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 observation 
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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

Endpoint value(s): LD50 = 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 observed 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. Body weight 

data were not available at higher dose levels due to the observed mortality. No test material 

related macroscopic findings were evident.

CONCLUSIONS

LD50 (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 Anhydrides category, received by EPA from 

Albemarle Corporation on April 5, 2006; HPV submission references ACC HERTG panel 

Robust Summary “Alkenyl Succinic Anhydrides”, updated Nov. 22, 2002
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Study 2: Acute inhalation toxicity study – limit test

Title: Acute inhalation study

The study is: Adequate Not Adequate

The study is not adequate because it represents a limit test and it produced compound-related 

mortalities.

TEST SUBSTANCE 

Identity: Dodecenylsuccinic anhydride, CAS No. 25377-73-5

Remarks: Purity not specified; Appearance: Amber liquid; Sponsor’s identification code: 127-

2638, Dodecenyl Succinic Anhydride; FDRL identification code: 80-1168 

METHOD

Method/guideline followed: Similar to OECD limit test

Test type: Acute 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. Industrial 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 inhalation 

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 geometric mean 

particle size of 2.3 μm (S.D. = 2.2 μm). To serve as a control, additional animals were exposed 

to an atmosphere containing a liquid droplet aerosol of ethanol and observed a required by the 
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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 necropsy. 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 actual 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 correcting for dilution (by 

multiplying by 0.90).

RESULTS

Endpoint value(s): LC50 > 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 15th day 

post-exposure. Thus, the LC50 for 4-hour inhalation exposure is greater than 1.2 mg/L. 

Significant observations included labored breathing, nasal discharge, urinary incontinence, and 

eye irritation. 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 eyes (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. Urinary 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 to 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 upon 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 post mortem congestion within the tissues. 

CONCLUSIONS

Four-hour exposure to liquid 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. Labored 
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breathing, transient urinary incontinence, alopecia, eye irritation, and body weight loss were 

observed following exposure to the test article.

STUDY RELIABILITY

Klimisch code = [3] – Not reliable. The study is not adequate for the endpoint as it describes a 

limit test in which compound-related mortalities occurred.

REFERENCE

This summary is based 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

Letter Dated 04/08/82. (Study produced on March 24, 1981) 
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B-3. GENETIC TOXICITY – GENE MUTATION

Summary of endpoint: Adequate

Study 1: In vitro Salmonella mutagenicity assay

Title: Ames test

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Dodecenylsuccinic anhydride, CAS No. 25377-73-5

Remarks: Purity not specified

METHOD

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

incorporation method.

Test type: In vitro Ames Salmonella mutagenicity assay

GLP compliant?: Not specified

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 SpragueDawley 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 the 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 appropriate 
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concurrent solvent (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 the 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, TA100, 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 dose 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 responded 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] – 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 not provided, the primary data from the study were 

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

REFERENCE

Seifried et al. (2006) A compilation of two decades of mutagenicity test results with the Ames 

Salmonella typhimurium and L5178Y mouse lymphoma cell mutation assays. Chemical 

Research in Toxicology 19: 627-644.
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Study 2: Mouse lymphoma mutagenicity assay

Title: Mouse lymphoma mutagenicity assay

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Dodecenylsuccinic anhydride, CAS No. 25377-73-5

Remarks: Purity not specified

METHOD

Method/guideline followed: Clive and Spector (1975) Laboratory procedure for assessing 

specific locus mutations at the TK locus in cultured L5178Y TK+/- mouse lymphoma cells

Test type: In vitro mouse lymphoma cell mutagenicity assay

GLP compliant?: Not specified

Year study performed: Not stated

Test species: L5178Y TK+/- 3.7.C mouse lymphoma cells

Metabolic activation: With (rat S9) and without 

Concentrations tested: 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 with 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 

105

/mL (6 x 106 cells total) were exposed for 4 h to a range of concentrations from 0.0005 to 

10,000 μg/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 solvent 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 methanesulfonate at 10 – 20 μg/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 μg/mL) for the test with metabolic activation.
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The mutagenicity assay was performed according to the procedure described by Clive and 

Spector (1975). A total of 1.2 x 107 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, and 

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 105/mL at 24 h intervals. They were then cloned (1 x 

106 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 adding TFT (final concentration, 3 μg/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% CO2

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 106

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 solventtreated control value as an indication of a positive effect, 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. Doses 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 step 

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 activation. 
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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 not provided, the primary data from the study were 

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

REFERENCE

Seifried et al. (2006) A compilation of two decades of mutagenicity test results with the Ames 

Salmonella typhimurium and L5178Y mouse lymphoma cell mutation assays. Chemical 

Research in Toxicology 19: 627-644.
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B-3. SKIN IRRITATION

Summary of endpoint: Inadequate

Study 1: Skin irritation study

Title: Skin 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. 25377-73-5

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

Doses/concentrations tested: Undiluted and 10% solutions

Exposure Period: 24 hours

Total Volume applied: 0.4 mL
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Vehicle: Corn Oil

Post-Exposure Period: 14 days

Test method/conditions remarks: Patch tests were performed on female, albino guinea pigs 

weighing 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 skin 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 response 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; data are from a secondary source; test substance 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-64-28.
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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. 25377-73-5

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: 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 days. When damage occurred, observations were 

continued until healing was completed. 
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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 appeared to be no effects 

persisting beyond the 2nd post-exposure day. 

CONCLUSIONS

The test substance was slightly irritating to guinea pig eyes.

STUDY RELIABILITY

Klimisch code = [4] – Not assignable; data are from a secondary source; test substance 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-64-28.
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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. 25377-73-5

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 induction 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 cages. Patches moistened 

with test article were applied to the skin in the same manner once a week for three applications. 
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The same site was clipped on the day before application, and the restraint periods were six hours 

on each occasion.

For primary challenge 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 and 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 greater 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 reactions 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 treated 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 sensitization 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 more 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.
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REFERENCES

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

Albemarle Corporation 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 Research, 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.
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B-6. CAS No. 25377-73-5: Summary of Endpoints

Endpoint Study Information 

Available

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

Chromosomal Aberrations No - Yes

Reproductive Toxicity No - Yes

Developmental Toxicity No - Yes