butanedioic acid, 2-octenyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro GLP guideline genetic toxicity tests are available for the registered substance: bacterial reverse mutation assay (Klimisch 1), in vitro mammalian chromosome aberration test (Klimisch 1) and in vitro mammalian CHO-HGPRT gene mutation assay (Klimisch 1). All three in vitro assays with the registered substance were negative (not mutagenic/genotoxic).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP characterization and neat test material stability of the test material was run concurrently with the study. This had no adverse impact on the integrity of the data or the validity of the study conclusion.

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100 and at tryptophan locus in Escherichia coli WP2uvrA (pKM101)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 homogenate

Test concentrations with justification for top dose:

In the Initial Toxicity-mutation Assay, bacterial cultures were exposed to Octenylsuccinic acid at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (two plates/concentration). Precipitation was not observed up to the concentration of 5000 μg/plate. No inhibition of background lawn was observed up to the tested concentration of 5000 μg/plate in any of the five strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations in the absence or presence of metabolic activation (5% v/v S9 mix) when compared with the concurrent negative control. From the results of the Initial Toxicity-mutation Assay, 5000 μg/plate was selected, being the recommended top dose indicated in the guidelines for' testing in the Confirmatory Mutation Assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Octenylsuccinic acid was found to be insoluble in sterile distilled water, while it was soluble in dimethyl sulfoxide at concentration of 50000 μg/mL. Octenyl Succinic Acid was found to be stable in dimethyl sulfoxide (DMSO) at room temperature for at least 4 hours.

Solubility and Precipitation Test:
Solubility and precipitation tests of the test item were performed prior to the initial toxicity mutation assay. Octenylsuccinic acid was found to be insoluble in sterile distilled water, while it was soluble in dimethyl sulfoxide at concentration of 50000 μg/mL (Stock B). A volume of 0.1 mL of stock B was added to 2 mL of top agar, to assess the precipitation. Precipitation was not observed at the tested concentration of 5000 μg/plate. Therefore, 5000 μg/plate was selected as the highest concentration to be tested for initial toxicity-mutation assay both in the absence and presence (5% v/v S9 mix) of metabolic activation.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 9-Aminoacridine hydrochloride hydrate (CAS N° 52417-22-8) ; 2-aminoanthracene (613-13-8)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation assay

Number of Replicates:
There will be two replicates for the initial toxicity-mutation assay and three replicates for the confirmatory mutation assay.

Plating Procedure"
The initial toxicity-mutation, as well as the confirmatory mutation assay will be conducted using the preincubation assay method.
A. Presence of metabolic activation
a) 50 or 100 μL test dose/vehicle/appropriate positive control
b) 100 μL bacterial culture
c) 500 μL S-9 mix

B. Absence of metabolic activation
a) 50 or 100 μL test dose/vehicle/appropriate positive control
b) 100 μL bacterial culture
c) 500 μL of 0.2 M phosphate buffer

These test constituents will be transferred into sterile test tubes and will be kept in an incubator shaker for approximately 20 ± 2 minutes at 37 ± 1 ºC. After this period, 2 mL of soft agar containing histidine-biotin / tryptophan will be added to each of the tubes. These constituents will be overlaid onto VB agar plates. After the soft agar sets, the plates will be incubated at 37 ± 1°C for 48 to 72 hours.

Justification for Selection of the Test System:
This assay measures the ability of the test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100 and at tryptophan locus in Escherichia coli WP2uvrA (pKM101), which are known for their reliability and reproducibility in a short term mutagenicity assay and are also recommended by the OECD, EPA, EC and other guidelines.

Rationale for test conditions:

In the Initial Toxicity-mutation Assay, bacterial cultures were exposed to Octenylsuccinic acid at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (two plates/concentration). Precipitation was not observed up to the concentration of 5000 μg/plate. No inhibition of background lawn was observed up to the tested concentration of 5000 μg/plate in any of the five strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations in the absence or presence of metabolic activation (5% v/v S9 mix) when compared with the concurrent negative control. From the results of the Initial Toxicity-mutation Assay, 5000 μg/plate was selected, being the recommended top dose indicated in the guidelines for' testing in the Confirmatory Mutation Assay. In the Confirmatory Mutation Assay, bacterial cultures were exposed to Octenylsuccinic acid at concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate (three plates/concentration) in the absence and presence of metabolic activation (10% v/v S9 mix).

Evaluation criteria:

Assay Evaluation Criteria:
Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were that there should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.

Strains TA98, TA1535 and TA1537:
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.

Strains TA100 and Escherichia coli WP2uvrA (pKM101):
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be non-mutagenic.

Statistics:

The following statistical treatments were used in this study: Means, standard deviations and relative standard deviations.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All doses tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All doses tested.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All doses tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All doses tested.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All doses tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All doses tested.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All doses tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All doses tested.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All doses tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All doses tested.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative Controls:
The results of the study indicate that the revertant colony numbers for the negative controls (DMSO) in all strains were within limits of historical range.

Positive Controls:
2-Aminoanthracene was used as the positive control in the presence of metabolic activation for all the tester strains during the Initial Toxicity Mutation Assay and Confirmatory Mutation Assay. Historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation. Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were within the historical ranges.This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.

Initial Toxicity Mutation Assay:
Bacterial cultures were exposed to Octenylsuccinic acid at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (two plates/concentration) both in the absence and presence of metabolic activation system (5% v/v S9 mix). Normal growth was observed up to the tested concentration of 5000 μg/plate in all tester strains. Precipitation was not observed up to the tested concentration of 5000 μg/plate. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control. Hence, 5000 μg Octenylsuccinic acid /plate was selected as the highest concentration to be tested in the Confirmatory Mutation Assay both in the absence and presence of metabolic activation system (10% v/v S9 mix).

Confirmatory Mutation Assay:
In the Confirmatory Mutation Assay, bacterial cultures were exposed to Octenylsuccinic acid at concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate (three plates/concentration) both in the absence and presence of metabolic activation system (10% v/v S9 mix). Normal growth was observed up to the tested concentration of 5000 μg/plate in all tester strains. Precipitation was not observed up to the tested concentration of 5000 μg/plate. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

Dose Formulation Analysis:

The Octenylsuccinic acid stock concentrations were found to be within acceptable range of ± 15% of nominal (% RSD < 10%) during confirmatory mutation assay. The 0 hour concentrations of Octenylsuccinic Acid in the dose formulation were found to be 100%, 103%, 111%, 93.5%, 92.4% and 96.8% of the nominal concentrations of dose level T1 (1562.5 μg/mL), T2 (3125 μg/mL), T3 (6250 μg/mL), T4 (12500 μg/mL), T5 (25000 μg/mL) and T6 (50000 μg/mL), respectively. The concentrations of Octenylsuccinic Acid in the dose formulation after 4 hours were found to be 92.6% and 92.2% of the 0 hour concentrations of dose level T1 (1562.5 μg/mL) and T6 (50000 μg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

Conclusions:

From the results of this study, under the specified experimental conditions, Octenylsuccinic acid was concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).

Executive summary:

The potential of Octenylsuccinic acid to induce reverse mutations in Salmonella typhimurium strains TA1537, TA1535, TA98 and TA100 and a tryptophan deficient strain, Escherichia coli WP2uvrA (pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method.

Octenylsuccinic acid was tested in the absence and presence of metabolic activation using dimethyl sulfoxide (DMSO) as the solvent. In the Initial Toxicity-mutation Assay, bacterial cultures were exposed to Octenylsuccinic acid at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (two plates/concentration). Precipitation was not observed up to the concentration of 5000 μg/plate. No inhibition of background lawn was observed up to the tested concentration of 5000 μg/plate in any of the five strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations in the absence or presence of metabolic activation (5% v/v S9 mix) when compared with the concurrent negative control.

From the results of the Initial Toxicity-mutation Assay, 5000 μg/plate was selected, being the recommended top dose indicated in the guidelines for' testing in the Confirmatory Mutation Assay. In the Confirmatory Mutation Assay, bacterial cultures were exposed to Octenylsuccinic acid at concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate (three plates/concentration) in the absence and presence of metabolic activation (10% v/v S9 mix). Precipitation was not observed up to the concentration of 5000 μg/plate. No inhibition of background lawn was observed at all concentrations tested in any of the five strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations in the absence or presence of metabolic activation when compared with the concurrent negative control.

All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

The Octenylsuccinic acid stock concentrations 1562.5, 3125, 6250, 12500; 25000 and 50000 ug/mL were found to be within acceptable range of +/- 15% of nominal concentrations during the Confirmatory Mutation Assay. The 0 hour concentrations of Octenylsuccinic acid in the dose for Mulation were found to be 100%, 103%, 111%, 93.5%, 92.4% and 96.8% of the nominal concentrations of dose level T1 (1562.5 ug/mL), T2 (3125 ug/mL), T3 (6250 ug/mL), T4 (12500 ug/mL), T5 (25000 ug/mL) and T6 (50000 ug/ mL), respectively. The concentrations of Octenylsuccinic acid in the dose formulation after 4 hours were found to be 92.6% and 92.2% of the 0 hour concentrations of dose level T1 (1562.54 ug/mL) and T6 (50000 ug/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, Octenylsuccinic acid is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coil WP2uvrA (pKM101).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In-vitro chromosomal aberration assay utilizing rat lymphocytes.

Specific details on test material used for the study:

Test Material Name: Octenylsuccinic acid
Chemical Name: 2-(Octen-1-yl) butanedioic acid
Synonyms: DF-20 Acid, OSAC
Lot/Reference/Batch Number: 8515025
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area by high performance liquid chromatography with identification by liquid chromatography mass spectrometry and nuclear magnetic resonance (Megregian, 2016).
Test Material Stability Under Storage Conditions: Octenylsuccinic acid, lot 8515025, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6313. (Megregian and Crispin, 2016).

Target gene:

Chromosomal aberration assay utilizing rat lymphocytes.

Species / strain / cell type:

lymphocytes: Rat

Details on mammalian cell type (if applicable):

Species and Sex: Rats (Male)
Strain and Justification:
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: 10-12 weeks

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin), and from several rats was pooled for sample collection. Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heatinactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 μg/ml), and an additional 2 mM L-glutamine) in addition with 30 μg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C. Treatment medium was the above-mentioned medium without serum and was used during the 4-hour treatment conditions, while complete medium was used for the 24-hour treatment condition.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male Sprague Dawley rats.

Test concentrations with justification for top dose:

The cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 2000 μg octenylsuccinic acid per ml of culture medium. The highest concentration was based on the guideline recommended maximum concentration of 2000 μg/ml and the solubility of the test material in the treatment medium.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO, CAS No. 67-68-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: cyclophosphamide monohydrate

Details on test system and experimental conditions:

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin), and from several rats was pooled for sample collection. Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heatinactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 μg/ml), and an additional 2 mM Lglutamine) in addition with 30 μg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C. Treatment medium was the above-mentioned medium without serum and was used during the 4-hour treatment conditions, while complete medium was used for the 24-hour treatment condition.

In Vitro Metabolic Activation System:
S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male Sprague Dawley rats was purchased from a commercial source, and stored at -100°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a "mix" (O'Neill et al., 1982). The mix consisted of 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the treatment medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the medium was 1/5 of the concentrations stated above.

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be soluble in DMSO up to 228.4 mg/ml. All test material solutions were prepared fresh on the day of treatment and used within two hours of preparation. The test material was dissolved in DMSO and further diluted (1:100) in medium. This technique has been shown to be an effective method for detecting various in vitro clastogens in this test system. All dosing units were expressed in μg/ml. MMC was dissolved in treatment medium, and CP stock was dissolved in distilled water. The pH of the medium was adjusted back to approximately 6.8-7.8, as addition of the test material appreciably altered the pH and the osmolality of the medium was not altered in doing the pH adjustment.

Dose Level Selection:
The cultures were treated with various concentrations of the test material and the selected concentration of the positive control chemicals. Soluble materials were tested up to 10 mM, 2000 μg/ml, or 2 μl/ml whichever was the lowest. Test materials with limited solubility were tested up to or beyond their limit of solubility. In some cases, more than one insoluble concentration was tested to ascertain whether toxicity would occur at higher insoluble concentrations. The other concentrations tested were separated by a factor of 2 to 3.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay A1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by high performance liquid chromatography with mass spectrometry detection (HPLC/MS). Analytical method validation was performed concurrently with sample analysis. Homogeneity analysis was not conducted as dosing solutions were not administered as suspensions.

Identification of the Test System:
All test cultures were identified using self-adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Evaluation criteria:

Evaluation Criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the vehicle controls. The aberration frequency in the vehicle and positive controls should be within the control limits of the laboratory historical control values as calculated using previous laboratory values. A test chemical was considered positive in this assay if it induced a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was outside the control limits of the laboratory historical vehicle control range. A test chemical was considered negative in this assay if it did not induce a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was not outside the control limits of the laboratory historical vehicle control range. If a test chemical did not meet either of the above criteria it may have been considered equivocal.

Statistics:

Statistical Analysis:
The proportions of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two-way contingency table was constructed to analyze the frequencies of aberrant cells. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the global hypothesis of no difference in the average number of cells with aberrations among the dose groups (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If this statistic was found to be significant at alpha = 0.05, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, versus a one-sided alternative. If any of the pairwise tests were significant, a test for linear trend of increasing number of cells with aberrations with increasing dose was performed (Armitage, 1971).
Polyploid cells were analyzed by the Fisher Exact probability test (Siegel, 1956). The number of polyploid cells were pooled across replicates for the analysis and evaluated at alpha = 0.05. The data were analyzed separately based on the presence or absence of S9 and based on the exposure time.

Key result

Species / strain:

lymphocytes: Rat

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Dose levels: 0, 62.5, 250 and 1000 μg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Rat

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Dose levels: 0, 31.3, 500 and 1000 μg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Rat

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Dose levels: 0, 7.8, 31.3, 125 μg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 2284.0 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts), respectively. There was an appreciable change observed in the pH but not in osmolality at this concentration as compared to the treatment medium with solvent alone (treatment medium with the test material, pH = 6.68, osmolality = 414 mOsm/kg H2O; treatment medium with 1% DMSO, pH = 7.37, osmolality = 440 mOsm/kg H2O). Therefore, the pH was adjusted accordingly with sodium hydroxide (NaOH).

Remarks on result:

other:

Remarks:

Assay B1 4-Hour Treatment

Assay A1:

Due to poor lymphocyte growth in the initial cytogenetic assay the slides were not analyzable thus, the study was repeated in a separate assay (Assay B1). Despite the lack of lymphocyte upon visualization of the slides it was evident the top two target concentrations (i.e., 1000.0 and 2000.0 μg/ml) exhibited evidence of excessive toxicity. Therefore, the highest concentration (i.e., 2000.0 μg/ml) was excluded from the repeat cytogenetic assay (Assay B1), and additional lower concentrations (i.e., 15.6 and 7.8) were utilized in the 4 and 24-hour treatment conditions, respectively.

Assay B1:

Cultures were treated with the test material for 4 hours in the absence and presence of S9 activation at concentrations of 0 (vehicle control), 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, and 1000.0 μg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 7.8 μg/ml. Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 107 to 121% of the target and verified that concentrations used for treatment were within the acceptable range.

Short Treatment:

In the absence of S9, the cultures displayed relative mitotic indices ranging from 43.8 to 109.4% compared to the vehicle control values. In the presence of S9, the mitotic indices of the treated cultures ranged from 50.6 to 92.6% as compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 62.5, 250.0, and 1000.0 μg/ml in the absence of S9 and targeted concentrations of 0 (vehicle control), 31.3, 500.0, and 1000.0 μg/ml in presence of S9 activation were chosen for the determination of chromosomal aberrations frequency and incidence of polyploidy.

Among the cultures treated with the positive control chemicals 0.5 μg/ml of MMC was selected for the evaluation of aberrations in the absence of S9. Both concentrations of 2 and 4 μg/ml of CP were selected for evaluation of aberrations in the presence of S9.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

In the 4-hour non-activation assay, the frequency of cells with aberrations in the vehicle control was 0.3% and the corresponding values at treatment levels of 62.5, 250.0, and 1000.0 μg/ml were 0.3, 0.0, and 0.0%, respectively. In the activation assay, cultures treated with the test material at concentrations of 31.3, 500.0, and 1000.0 μg/ml had aberrant cell frequencies of 0.0, 0.0 and 0.0%, respectively, as compared to the vehicle control value of 0.3%. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures without or with S9 activation. The frequencies of aberrant cells observed in the test material treated and vehicle control cultures were within the control limits of the laboratory historical vehicle control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in 0.5 μg/ml MMC (-S9, 4-hour treatment), and 2 and 4 μg/ml CP (+S9, 4-hour treatment) cultures were 17.8%, 6.7%, and 24.0%, respectively. The frequencies of cells with aberrations in 0.5 μg/ml MMC (-S9, 4-hour treatment) and 4 μg/ml CP (+S9, 4-hour treatment) cultures were within the control limits of the laboratory historical positive control range.

Continuous Treatment:

Based upon the negative findings in the 4-hour treatment, slides from the continuous 24-hour treatment were evaluated. Cultures treated continuously for 24 hours in the absence of S9 activation had excessive toxicity at the three highest concentrations (i.e., 250.0, 500.0, and 1000.0 μg/ml) as indicated by the lack of mitotic figures. The remaining concentrations had relative mitotic indices ranging from 39.7 to 79.4% relative to the vehicle control value. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 7.8, 31.3, and 125.0 μg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy. Cultures treated with 0.075 μg/ml MMC were selected to serve as the positive control.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

The frequency of aberrant cells in the vehicle control was 0.0% and the corresponding values at concentration levels of 7.8, 31.3, and 125.0 μg/ml were 1.0, 0.3, and 0.0%, respectively. There were no statistically significant differences between the test material treated cultures and the vehicle control values and all values were within the control limits of the laboratory historical vehicle control range.

A significant increase in the frequency of cells with aberrations was observed in cultures treated with the positive control chemical. Aberrant cell frequency in MMC treated cultures was 21.3%. All values were within the control limits of the laboratory historical positive control range.

Conclusions:

It was concluded that under the experimental conditions used, octenylsuccinic acid was negative in this in vitro chromosomal aberration test.

Executive summary:

Octenylsuccinic acid (2-(Octen-1-yl) butanedioic acid) was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 2000 μg octenylsuccinic acid per ml of culture medium. The highest concentration was based on the guideline recommended maximum concentration of 2000 μg/ml and the solubility of the test material in the treatment medium. The analytically determined concentrations of octenylsuccinic acid in the dose preparations ranged from 107 to 121% of the targeted values. The duration of treatment was 4 hours without and with S9 and 24 hours without S9. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon cytotoxicity. In this study cultures treated for 4 hours with targeted concentrations of 0

(vehicle control), 62.5, 250.0, and 1000.0 μg/ml in the absence of S9 and 0 (vehicle control), 31.3, 500.0, and 1000.0 μg/ml in the presence of S9 and cultures treated for 24 hours with 0

(vehicle control), 7.8, 31.3, and 125.0 μg/ml were analyzed.

There were no significant increases in the frequency of cells with aberrations administered octenylsuccinic acid in either the absence or presence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of aberrant cells. Based upon these results, octenylsuccinic acid was considered to be negative in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: (CHO/HGPRT) forward gene mutation assay

Specific details on test material used for the study:

Test Material Name: Octenylsuccinic acid
Chemical Name: 2-(Octen-1-yl) butanedioic acid
Synonyms: DF-20 Acid, OSAC
Lot/Reference/Batch Number: 8515025
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area by high performance liquid chromatography with identification by liquid chromatography mass spectrometry and nuclear magnetic resonance (Megregian, 2016).
Test Material Stability Under Storage Conditions: Octenylsuccinic acid, lot 8515025, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6313. (Megregian and Crispin, 2016).

Target gene:

The gene for Hgprt is located on the mammalian X-chromosome.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Indicator Cells and Justification of Their Use:
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, Tennessee, was used in this study. The CHO-K1-BH4 cell line was selected as the test system for this study because it is sensitive to mutagens, has a low background mutant frequency, and is readily available. Stock cultures were stored at approximately -80°C or below. The cultures were periodically checked for mycoplasma contamination (American Type Culture Collection, Manassas, Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture lab-ware under standard conditions of approximately 5% CO2 in air at 37°C in a humidified incubator.

Media:
The cells were routinely maintained in Ham's F-12 nutrient mix supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum, 25 mM HEPES, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 0.25 μg/ml), and an additional 2 mM L-glutamine. Treatment medium was the above-mentioned medium without serum. The selection medium used for the detection of Hgprt- mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 μM 6-thioguanine, 5% serum, 25 mM HEPES, 2 mM L-glutamine, and the above-mentioned antibiotics.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 50 to 2000 μg/ml in the absence and in the presence of S9. The highest concentration was based on the 2000 μg/ml assay limit.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO, CAS No. 67-68-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Controls:
Dimethyl sulfoxide (DMSO, CAS No. 67-68-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive control for the non-activation system (without S9 factor) at a final concentration of 621 μg/ml. The positive control for assays performed with S9 (activation system) was 3-methylcholanthrene (3-MCA, CAS No. 56-49-5) at concentrations of 4 and 8 μg/ml. The dose levels of EMS and 3-MCA were based upon our unpublished findings.

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be soluble in DMSO up to 228.5 mg/ml. All test material solutions were prepared fresh on the day of treatment and used within one hour of preparation. The test material was first dissolved in DMSO and further diluted (1: 100) in medium to obtain the desired concentrations as recommended in the test guidelines. This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al., 1981). EMS was dissolved in treatment medium. 3-MCA was dissolved first in DMSO and further diluted in the treatment medium. All dosing units were expressed in μg/ml. The pH of the medium was adjusted back to approximately 6.8-7.8 as the pH was appreciably altered following the addition of the test compound provided that the osmolality of the medium was not unduly affected in doing such an adjustment.

Treatment Procedure:
Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 10^6 cells/T-75 flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with treatment medium, S9 mix (when applicable), the test chemical, the vehicle control, or the positive control chemical. The cells were treated for approximately 4 hours at 37°C and the exposure was terminated by washing the cells with phosphate buffered saline (Ca++ and Mg++ free).

Identification of the Test System:
All test cultures were identified using self-adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay B1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by high performance liquid chromatography with mass spectrometry detection (HPLC/MS). Analytical method validation was performed concurrently with sample analysis. Homogeneity analysis was not conducted as dosing solutions were not administered as suspensions.

In Vitro Metabolic Activation:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats were purchased from a commercial source and stored at approximately -80°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a “mix” (O’Neill et al., 1982). The S9 mix consisted of the following co-factors: 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the treatment medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the medium is 1/5 of the concentrations stated above.

Evaluation criteria:

Evaluation Criteria:
For an assay to be acceptable, the mutant frequency in positive controls should be significantly higher than the vehicle controls. The mutation frequency in the vehicle and positive controls should be within the control limits of the laboratory historical control values as calculated using previous laboratory values. The test chemical was considered positive if it induced a statistically significant, dose-related increase in mutant frequency, and the mutant frequency was outside the control limit of the laboratory historical vehicle control range. The test chemical was considered negative if it did not induce a statistically significant, dose-related increase in mutant frequency, and the mutant frequency was not outside the control limits of the laboratory historical vehicle control range. If a test chemical did not meet either of the above criteria it may have been considered equivocal. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the vehicle and positive controls.

Statistics:

Statistical Analysis:
The frequency of mutants per 10^6 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutant frequency variance. The actual plate counts were assumed to follow a Poisson distribution; therefore, the mean plate count was used as an estimate of variance (Kirkland, 1989).
If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

RS values ranging from 101.1 to 132.6%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

RS values ranging from 92.5 to 121.8%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

other:

Remarks:

There was little to no toxicity observed as indicated by the RS values ranging from 78.9 to 112.5%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

other:

Remarks:

RS values showed moderate toxicity at the highest concentration (i.e., 2000 μg/ml) with RS values ranging from 25.0 to 106.9%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

other:

Remarks:

Moderate toxicity was observed with RS values ranging from 37.2 to 104.5%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 2285 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays (Thilagar et al., 1984; Galloway et al., 1985; Cifone, 1985). There was no appreciable change in the osmolality at this concentration as compared to the treatment medium with solvent alone. An appreciable change in pH was observed, therefore, the pH of the test material treated cultures was adjusted back to 6.8 – 7.8 during treatment (treatment medium with the test material, pH = 6.71, osmolality = 412 mOsm/kgH2O; treatment medium with 1% DMSO, pH = 7.52, osmolality = 446 mOsm/kgH2O).

Remarks on result:

other: Assay B2 Results

Assay A1 – Preliminary Toxicity Assay:

In the preliminary toxicity assay, the test material was tested at concentrations of 0 (vehicle control), 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest concentration tested was based upon the limitations imposed by 2000 μg/ml assay limit. The treated cultures without S9 activation showed little to no toxicity with the relative survival (RS) values ranging from 74.1 to 118.8%. In the presence of S9 activation, excessive toxicity was seen at concentrations of 1000 and 2000 μg/ml as exhibited by limited or no observable colonies. The remaining concentrations had RS values ranging from 49.2 to 101.3%. Based upon the results of this assay, concentration levels of 125, 250, 500, 1000, and 2000 μg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and 50, 250, 550, 700, 850, and 1000 μg/ml in the presence of S9.

Assay B1 – Initial Mutagenicity Assay:

Due to a technical error in the initial mutagenicity assay, this portion of the assay was discontinued and repeated in a separate assay (Assay B2).

Assay B2 – Repeat Initial Mutagenicity Assay:

In the repeat initial mutagenicity assay (Assay B2), in the absence of S9, no toxicity was observed with RS values ranging from 101.1 to 132.6%. The mutant frequencies observed in cultures treated with the test material in the absence were not significantly different from the concurrent vehicle control values. All average mutant frequencies, between replicates, were within the control limits of the laboratory historical vehicle control range.

In the presence of S9, no toxicity was observed with RS values ranging from 92.5 to 121.8%. Based on these results the desired limit of toxicity was not achieved nor was the maximum concentration based on the 2000 μg/ml assay limit for cultures treated in the presence of S9. Therefore, this portion of the assay was required to be repeated in a separate assay. The mutant frequencies observed in cultures treated with the test material in the presence of S9 were not significantly different from the concurrent vehicle control values. All average mutant frequencies, between replicates, were within the control limits of the laboratory historical vehicle control range.

Assay C1 – Confirmatory Mutagenicity Assay in the Absence of S9 and Repeat Mutagenicity Assay in the Presence of S9:

In the confirmatory assay (Assay C1), the concentrations ranged from 125 to 2000 μg/ml in the absence of S9 and 50 to 2000 μg/ml, in the presence of S9. In the absence of S9 there was little to no toxicity observed as indicated by the RS values ranging from 78.9 to 112.5%. In the presence of S9, RS values showed moderate toxicity at the highest concentration (i.e., 2000 μg/ml) with RS values ranging from 25.0 to 106.9%. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values. All average mutant frequencies, between replicates, were within the control limits of the laboratory historical vehicle control range, with the exception of the 800 and 1100 μg/ml concentrations in the presence of S9. Despite the discrepancy the average mutant frequencies of the 800 and 1100 μg/ml concentration were within a reasonable range of the control limits of the laboratory historical vehicle control range. There was no apparent dose dependent increase amongst the test material treated cultures verifying there was no treatment-related increase in mutant frequency amongst the test material treated cultures when compared to the vehicle control cultures.

Assay D1 – Repeat Confirmatory Mutagenicity Assay:

In the repeat confirmatory assay (Assay D1), the concentrations ranged from 125 to 2000 μg/ml in the presence of S9 and moderate toxicity was observed with RS values ranging from 37.2 to 104.5%. The mutant frequencies observed in cultures treated with the test material in the presence of S9 were not significantly different from the concurrent vehicle control values and were within the control limits of the laboratory historical vehicle control range.

In all mutagenicity assays, the positive control chemicals induced significant increases in mutation frequencies and these data confirmed the adequacy of the experimental conditions for detecting induced mutations. The mutant frequencies exhibited by the positive control chemicals were either within or exceeded the control limits of the laboratory historical positive control range, with the exception of the 4 and 8 μg/ml concentrations of MCA in Assay C1 (presence of S9). Although the average mutant frequencies of the 4 and 8 μg/ml MCA were slightly below the control limits of the laboratory historical positive control range, the average mutant frequencies were statistically significant when compared to the vehicle control verifying the validity of this assay.

The analytically observed concentrations of the test material in the stock dosing solutions in Assay B1 ranged from 85.4 to 101.0% of target and verified that concentrations used for treatment were within acceptable range.

Conclusions:

It was concluded that under the experimental conditions used, octenylsuccinic acid was negative in this in vitro CHO/HGPRT forward gene mutation assay.

Executive summary:

Octenylsuccinic acid (2-(Octen-1-yl) butanedioic acid) was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 50 to 2000 μg/ml in the absence and in the presence of S9. The highest concentration was based on the 2000 μg/ml assay limit. The analytically determined concentrations of octenylsuccinic acid in the dose preparations ranged from 85.4 to 101.0%. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Vehicle control cultures were treated with the solvent used to dissolve the test material (i.e., dimethyl sulfoxide).

There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. Cultures treated with the positive control chemicals had significantly higher mutant frequencies. Based upon these results, octenylsuccinic acid was considered to be negative in this in vitro CHO/HGPRT forward gene mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

As the registered substance was negative in the in vitro tests, no in vivo follow up testing for the registered substance was necessary.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the negative outcomes in the key in vitro genetic toxicity assays, the registered substance is not self-classified for the genotoxicity endpoint.