Octadecanedioic acid, 1,18-dimethyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria:

An in vitro study was conducted to investigate the potential of test item to induce gene mutations in Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strains, according to OECD Guideline 471, in compliance with GLP. The strains were tested at concentrations up to 5000 µg/plate. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item was therefore considered to be non-mutagenic (Harlan Laboratories Ltd, 2013).

 

in vitro cytogenicity / micronucleus study: Micronucleus in vitro, human lymphocytes (OECD 487):

An in vitro study was conducted to investigate the potential of test item to induce micronuclei in isolated cultured human peripheral blood lymphocytes, according to OECD Guideline 487, in compliance with GLP. Human peripheral blood lymphocytes, cultured in vitro, were exposed to Octadecanedioic acid, 1,18-dimethyl ester at different concentrations, in the absence and presence of metabolic activation (2% v/v S9 mix). Under the test conditions Octadecanedioic acid, 1,18-dimethyl ester did not show any potential to induce micronuclei or clastogenic or aneugenic potential in isolated cultured human peripheral blood lymphocytes, in the absence or presence of metabolic activation (JRF, 2019).

 

in vitro gene mutation study in mammalian cells: Mouse Lymphoma Assay (OECD 476)

The mutagenic potential of the test item 9-dodecenoic acid, methyl ester (9DDAME) has been read-across from the substance 9-decenoic acid, methyl ester from the following study.

An in vitro study was conducted to investigate the potential of test item to induce gene mutations at the HPRT locus in mouse lymphoma L5178Y cells, according to OECD Guideline 476, in compliance with GLP. The assay was performed in the presence and absence of metabolic activation (S9). The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. 9-decenoic acid, methyl ester (9-DAME) was therefore considered to be non mutagenic to L5178Y cells under the conditions of the test (Harlan Laboratories Ltd, 2012). Based on the results of the read across study, a similar result can be expected for the test item.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

05 December 2011 to 24 January 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Read-across justification: A comparison target substance (9DDAME) and the read-across substance (9DAME) shows that the two substances share structural similarities, increasing from a chain length of C10 to C12 with similar functional groups and also have ‘mechanistic action’ similarities.

Reason / purpose for cross-reference:

read-across source

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 2011-07-19 to 2011-07-21; Date of signature 2011-08-31

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal enzyme fraction

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 7.19, 14.38, 28.75, 57.5, 115, 230, 460, 920 and 1840 µg/mL

Experiment 1 (without S-9 mix): 0, 1.25, 2.5, 5, 10, 20, 40, 50 and 60 µg/mL
Experiment 1 (with S-9 mix): 0, 5, 10, 20, 40, 60, 80, 100 and 120 µg/mL

Experiment 2 (without S-9 mix): 0, 2.5, 5, 10, 20, 40, 50, 60 and 80 µg/mL
Experiment 2 (with S-9 mix): 0, 10, 20, 40, 60, 80, 100, 120 and 140 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Experiment 1: 400 µg/mL; Experiment 2: 150 µg/mL; without metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

2 µg/mL; with metabolic activation

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10E-06 for the TK+/- locus in L5178Y cells. Vehicle control results should ideally be within this range.
Positive control chemicals should induce at least 3 to 5 fold increases in mutant frequency greater than the corresponding vehicle control.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The test item, 9-decenoic acid, methyl ester (9DAME) did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

The mutagenic potential of the test item 9-dodecenoic acid, methyl ester (9DDAME) has been read-across from the substance 9-decenoic acid, methyl ester from the following study.

An in vitro study was conducted to investigate the potential of test item to induce gene mutations at the HPRT locus in mouse lymphoma L5178Y cells, according to OECD Guideline 476, in compliance with GLP. The assay was performed in the presence and absence of metabolic activation (S9). The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. 9-decenoic acid, methyl ester (9-DAME) was therefore considered to be non mutagenic to L5178Y cells under the conditions of the test (Harlan Laboratories Ltd, 2012). Based on the results of the read across study, a similar result can be expected for the test item.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus for Salmonella strains, tryptophan for E. coli.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal enzyme fraction

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Mutation test - Experiment 1: 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation test - Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

The test item was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully soluble in acetone (100 mg/ml), dimethyl formamide (50 mg/ml) and tetrahydrofuran (200 mg/ml) in solubility checks performed in-house. Acetone was selected as the vehicle.

Untreated negative controls:

yes

Remarks:

spontaneous reversion rate

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535 without metabolic activation

Untreated negative controls:

yes

Remarks:

spontaneous reversion rate

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537 without metabolic activation

Untreated negative controls:

yes

Remarks:

spontaneous reversion rate

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA98 without metabolic activation

Untreated negative controls:

yes

Remarks:

spontaneous reversion rate

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

10 µg/plate for WP2uvrA, 1 µg/plate for TA100, 2 µg/plate for TA1535 and 2 µg/plate for TA1537 with metabolic activation

Untreated negative controls:

yes

Remarks:

spontaneous reversion rate

Negative solvent / vehicle controls:

no

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA98 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and preincubation for Experiment 2.

PRELIMINARY TOXICITY TEST
Ten concentrations of the test item formulation and a vehicle control (acetone) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 μg/plate because of excessive test item precipitation.

Mutation Test - Experiment 1:
Six concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Weakened TA100 bacterial background lawns were noted in the preliminary toxicity test, therefore an additional dose level and an expanded dose range were selected in order to achieve four non-toxic dose levels and the potential toxic limit of the test item.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the vehicle, test item formulation or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter. Manual counts were performed at 5000 μg/plate because of excessive test item precipitation.

Mutation Test - Experiment 2:
The test item dose range was the same as Experiment 1. An additional dose level and an expanded dose range were again selected in order to achieve four non-toxic dose levels and the potential toxic limit of the test item.

As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were therefore dosed as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method described in Section 3.5.2.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter. Manual counts were again performed at 5000 μg/plate because of excessive test item precipitation.

NUMBER OF CELLS EVALUATED: All tester strain cultures should be in the range of 0.9 to 9 x 10^09 bacteria per mL.

Evaluation criteria:

There were several criteria for determining a positive result:

1). A dose-related increase in mutant frequency over the dose range tested.
2). A reproducible increase at one or more concentrations.
3). Biological relevance against in-house historical control ranges.
4). Statistical analysis of data as determined by UKEMS.
5). Fold increase greater than 2 times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item was considered non-mutagenic (negative) in the test system if the above criteria were not met.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preliminary test only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preliminary test only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preliminary test only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preliminary test only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preliminary test only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Weakened bacterial background lawns were noted to TA100 at 5000 μg/plate (absence and presence of S9-mix) in the preliminary toxicity test. However, in both Experiments 1 and 2, the test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. The response noted in the preliminary assay was considered spurious and of no biological relevance. The test item was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 50 μg/plate in Experiment 2. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.1 times the concurrent vehicle control.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

The test item, octadecanedioic acid, 1,18-dimethyl ester (ODDAME) was considered to be non-mutagenic under the conditions of this test.

Executive summary:

An in vitro study was conducted to investigate the potential of test item to induce gene mutations in Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strains, according to OECD Guideline 471, in compliance with GLP. The strains were tested at concentrations up to 5000 µg/plate. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item was therefore considered to be non-mutagenic (Harlan Laboratories Ltd, 2013).

 

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29th July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Elevance Renewable Sciences, Inc.; Batch/Lot No. 11631837A
- Expiration date of the lot/batch: Indefinitely if stored appropriately
- Purity: 93%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At less than 40 deg C in original containers. Under nitrogen when not in use.

Species / strain / cell type:

other: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy, male volunteer (of 28 years old)

For lymphocytes:
- Whether whole blood or separated lymphocytes were used: whole blood

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cultures were prepared separately in a centrifuge tube containing heparin sodium. Each contained 0.5 mL of whole blood in 9.5 mL of complete medium [containing 20% heat-inactivated (56°C; 30 min.) fetal bovine serum and 2% Phytohaemagglutinin (PHA-M)] in centrifuge tubes and incubated at 37 ± 1°C and 5% CO2 in an incubator for approximately 48 hours. Same procedure was used for preparation of cultures for all the phases of the experiments.

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: rat liver treated with Aroclor 1254
- source of S9 : Dr. G. P. Meshram
- method of preparation of S9 mix : The S9 fraction was buffered and supplemented with the essential co-factors beta-NADP, KCl and D-Glucose-6-phosphate to form the “S9 mix”.
- concentration or volume of S9 mix and S9 in the final culture medium : 2% in the final culture medium

Test concentrations with justification for top dose:

Cytotoxicity Test: 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL

Main study:
Phase I (4 h exposure): 31.25, 62.5, 125, 250 and 500 µg/mL, with and without metabolic activation
Phase II (24 h exposure): 31.25, 62.5, 125, 250 and 500 µg/mL, without metabolic activation

Concentrations were selected based on results from a cytotoxicity test.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

vinblastine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : two
- Number of independent experiments : two

HARVEST:
-Each culture was harvested and processed separately for micronuclei frequency evaluation. Cells were hypotonically treated with cold (2-8°C) KCl solution and centrifuged for 8 minutes. The supernatant was removed and replaced with chilled Caronoy's fixative and centrifuged for 8 minutes. Cells were given a further two changes of chilled Carnoy's fixative washing.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Slides were prepared from each culture tube by pouring approximately 0.5 mL of fixed cell suspension, drop by drop on two, pre-cleaned, ice-chilled slides. The slides were dried over a slide warmer and labelled with the study number, treatment code, absence/presence of metabolic activation and slide number. Two slides were prepared per replicate culture per test concentration (i.e. four slides per test concentration). Out of these two slides, one was used for scoring and the other served as a reserve or used for additional scoring, if required. The dried slides were stained with 5% Giemsa in phosphate buffer for 35 minutes. The slides were made permanent by mounting a cover slip with DPX mountant. To prevent bias in the scoring procedure for micronuclei, the slide numbers were masked with coded numbers. All slides were coded before microscopic analysis and decoded after scoring was completed.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): minimum of 1000 binucleated cells per replicate were screened per concentration to evaluate the incidence of micronuclei.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cytostasis - reduction in replicative index

ASSAY ACCEPTANCE CRITERIA:
The following acceptance criteria were used to determine a valid assay:
- The negative (untreated) control and vehicle (DMSO) control is considered acceptable for addition to the laboratory historic negative control database.
- The positive controls should induce responses that are compatible with the historical positive control data base and produce a statistically significant increase when compared to the concurrrent vehicle control.
- Cell proliferation (Replicative Index) in the negative and vehicle control should be used to demonstrate that a sufficient number of treated cells have reached mitosis during the test and that the treatments are conducted at appropriate levels of cytotoxicity.
- All experimental conditions should be tested unless one resulted in a positive result.
- An adequate number of cells and concentrations should be analysable.
- The criteria for the selection of the highest concentration should be met i.e. 55 ± 5% reduction in RI compared to the vehicle control, and if cytotoxicity is not observed, 2000 µg/mL or 2 µL/mL, 10 mM or limit of solubility, whichever is lower, should be tested as the maximum concentration.

Rationale for test conditions:

The assay measures the ability of test item to induce micronucleus formation in binucleated cells of cultured human lymphocytes, known for their reliability and reproducibility in mutagenicity assays and also recommended by the OECD.

Evaluation criteria:

The test item was considered clearly negative in the study if the following criteria were met:
- None of the test concentrations exhibits a statistically significant increase compared with the vehicle control.
- There is no concentration-related increase when evaluated with an appropriate trend test
- All results were inside the distribution of the historical control data

Statistics:

Micronuclei data containing binucleated cells was statistically analysed for normality using Shapiro-Wilk’s test, and for homogeneity of variance using the Bartlett test before conducting appropriate parametric test (ANOVA, t-test).

Species / strain:

other: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Cytostasis (reduction in replicative index of 55%) was not observed at 500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Precipitation, pH and Osmolality Tests:
No significant change in pH (±1 unit) or osmolality (≥ 50 mOsm/kg H2O) was observed at any tested concentration (62.5 - 2000 µg/mL in culture medium) at 0 h and 4 h. Concentration dependent visible precipitate in the culture media was observed from 500 µg/mL to 2000 µg/mL, at the beginning and after 4 hour incubation. Due to limitations in solubility and presence of precipitation in culture media, 500 µg/mL was selected as the highest applicable concentration for cytotoxicity test.

CYTOTOXICITY TEST:
No significant change in the pH or osmolality was observed up to 500 µg/mL, in the absence and presence of the metabolic activation even after incubation.
Precipitation was observed at the concentration of 500 µg/mL, in the beginning and end of 4 hour treatment, in the absence and presence of metabolic activation. Precipitations were not observed at lower concentrations up to 250 µg/mL.

The cytostsis obsrberved in the absence of metabolic activation was 8.34, -0.11, 3.43, 5.22, 5,75 and 15.32%, and in presence of metabolic activation was 4.15, 0.27, 1.97, 3.39, 4.71 and 10.86% at 15.63, 31.25, 62.5, 125, 250 and 500 µg test item/mL.

No adverse impact of the vehicle control (DMSO) was observed in cultures when compared with the concurrent negative control.

MAIN STUDY:
PHASE I (Absence and presence of metabolic activation (2% v/v S9 mix) and 4 h exposure:
The observed cytostasis was -083, 5.37, 4.26, 10.05 and 16.36% and 0.04, 2.26, 9.33, 14.39 and 12.27% at 31.25, 62.5, 125, 250 and 500 µg test item/mL, respectively in the absence and presence of metabolic activation.
The observed cytostasis for the positive control in the presence of metabolic activation was 16.15%.

Cytostasis (reduction in replicative index of 55%) was not observed at 500 µg/mL. However, due to the presence of visible precipitates at 500 µg/mL, the limit concentration for the main study was selected as 500 µg/mL. Therefore, test concentrations selected for scoring of binucleated cells containing micronuclei were: 125, 250 and 500 µg/mL.

In the absence and presence of metabolic activation, the test item did not induce any statistically significant or biologically relevant increase in number of binucleated cells with micronuclei.

PHASE II (Absence of metabolic activation and 24 h exposure):
The observed cytostasis was 6.14, 9.50, 23.29, 23.33 and 39.45% at 31.25, 62.5, 125, 250 and 500 µg of test item.
The observed cytostasis was 10.54% in cultures treated with the postive control.

Cytostasis (reduction in replicative index of 55%) was not observed at 500 µg/mL. However, due to the presence of visible precipitates at 500 µg/mL, the limit concentration for the main study was selected as 500 µg/mL. Therefore, test concentrations selected for scoring of binucleated cells containing micronuclei were: 125, 250 and 500 µg/mL.

In the absence of metabolic activation, the test item did not induce any statistically significant or biologically relevant increase in number of binucleated cells with micronuclei.

The bacgground replicative index and micronucleus frequency results for the negative and vehicle control were comparable, and there was no adverse effect of DMSO observed on cultures when compared with that of the concurrent negative control during the main study.

ACCEPTANCE CRITERIA:
The number of binucleated cells with micronculei within the negative and vehicle control cultures were within the published range and acceptable for addition into the laboratories historical data base. The positive control, vinlastine, produced a statistically significant increase in the frequency of micronuclei containing binucleated cells in phase II. Cyclophosphamide produced a statistically significant increase in the frequency of micronuclei containing binucleated cells in Phase I in the presence of metabolic activation.
The response of the positive controls demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Conclusions:

Under these guideline test conditions, Octadecanedioic acid, 1,18-dimethyl ester did not show any potential to induce micronuclei or clastogenic or aneugenic potential in isolated cultured human peripheral blood lymphocytes, in the absence or presence (2% v/v S9 mix) of metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of test item to induce micronuclei in isolated cultured human peripheral blood lymphocytes, according to OECD Guideline 487, in compliance with GLP. Human peripheral blood lymphocytes, cultured in vitro, were exposed to Octadecanedioic acid, 1,18-dimethyl ester at different concentrations, in the absence and presence of metabolic activation (2% v/v S9 mix). Under the test conditions Octadecanedioic acid, 1,18-dimethyl ester did not show any potential to induce micronuclei or clastogenic or aneugenic potential in isolated cultured human peripheral blood lymphocytes, in the absence or presence of metabolic activation (JRF, 2019).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results of an in-vitro gene mutation study in bacterial, an in vitro micronucleus study and an in vitro gene mutation study in mammalian cells (read-across from a structural analogue substance) were all negative. Therefore, it is concluded that Octadecanedioic acid, 1,18-dimethyl ester is not genotoxic and does not warrant classification for mutagenicity according to CLP regulation EC 1272/2008.