butanedioic acid, 2-octenyl

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

butanedioic acid, 2-octenyl

EC Number:

820-780-3

Cas Number:

62568-82-5

Molecular formula:

C12 H20 O4

IUPAC Name:

butanedioic acid, 2-octenyl

Test material form:

other: White solid

Details on test material:

Test Item Name : Octenylsuccinic Acid
Appearance/Colour/Odour : White solid
Lot N° : 8515025

Method

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

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).