Butanedioic acid, sulfo-, 4-[2-[(2-hydroxyethyl)amino]ethyl] ester, N-C18-unsatd. acyl derivs., disodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite

IUPAC Name:

Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Reaction product of (R)-12-hydroxy-N-(2-hydroxyethyl)oleamide and maleic anhydride and sodium hydrogensulfite; Dinatrium-4-[2-[(1 2-hydroxy-1-oxooctadec-9-eny)amino]ethyl]-2-sulfosuccinat
- Physical state: Light yellow clear up to opaque liquid
- Analytical purity: 40.20% (of the solid material), correction factor: 2.49
- Impurities (identity and concentrations): See confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: April 27, 2012 (CoA)
- Lot/batch No.: ST01890824
- Expiration date of the lot/batch: February 30, 2013
- Stability under test conditions: Stable
- Storage condition of test material: At + 10° to +25°C
- Other: Manufacturer/Supplier: Evonik Industries AG, Werk Goldschmidt Rewo, Max-Wolf-Strasse 7, 36396 Steinau a. d. Str., Germany

Method

Target gene:

hprt locus at the X-chromosome

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
*V79 cells were maintained in Dulbecco's modified Eagle-Medium supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS. Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2.
* For subculturing, a trypsin (0.05%)-EDTA (ethylenediamine-tetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.
* Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, by using the HOECHST stain 33258
- The spontaneous mutation rate was continuously monitored.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary test: 10, 25, 100, 250, 1000, 2500 µg solid matter/mL medium and the undiluted test item (ca. 4600 µg/mL medium)
Main test: 62.5, 125, 250, 500 and 1000 µg solid matter/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was diluted with aqua ad iniectabilia. The test item was not soluble in any of the other solvents recommended.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

aqua ad iniectabilia

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate dissolved in DMSO

Remarks:

600 and 700 µg EMS/mL, without S9-mix

Controls 2

Positive controls:

yes

Positive control substance:

other: 9,10-dimethyl-1 ,2-benzanthracene dissolved in DMSO

Remarks:

20 and 30 µg DMBA/mL, with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Without S9-mix:4 hours (1st experiment) or 24 hours (2nd experiment); With S9-mix: 4 hours
- Expression time (cells in growth medium): until day 8 with one subcultivation on day 5
- Selection time (if incubation with a selection agent): about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).

SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL)

NUMBER OF REPLICATIONS:
cytotoxicity: triplicate
mutagenicity: for selection of mutants 5 replicate plates; for the estimation of plating efficiencies (PE) 3 replicate plates.


NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: other: relative plating efficiency was determined for each dose to obtain an accurate measure of the toxic effect of the chemical.

Evaluation criteria:

lf in both independent experiments solvent and positive controls show results within the norm and if the test compound does not increase the mutation, or if the mutation frequency is always lower than 40 x 10-6 and if at least 1 000 000 cells per condition have been evaluated, the test item is considered as negative in the test.
In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold solvent control and at least 40 x 10-6 both in the presence and/or absence of S9 mix, the compound is considered as positive in the test.

Statistics:

So far no satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments such as those performed here (see UKEMS guidelines for discussion).

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the main study cytotoxicity in form of decreased plating efficiency (PE1 and PE2) was noted in the first and second experiments at the top concentration of 1000 µg/mL in the absence and presence of metabolic activation, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality:
The pH value of the test item supplied was measured to 5.62.
The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
-pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
-Osmolality: with a semi-micro osmometer .
No relevant changes in the pH or osmolality values of the formulations were noted.
- Water solubility:
The test item was diluted with aqua ad iniectabilia. The test item was not soluble in any of the other solvents recommended.

RANGE-FINDING/SCREENING STUDIES:
The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 10, 25, 100, 250, 1000, 2500 µg solid matter/mL medium and the undiluted test item (ca 4600 µg/mL medium) were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 1000 µg/mL medium in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 1000 µg solid matter/mL were employed as the top concentration for the mutagenicity tests in the absence and in the presence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10 6 survivors in non-activation solvent controls and 6 to 46 per 10 6 survivors in S9 activation solvent controls by Bradley et al. (Mutagenesis by chemical agents in V79 Chinese hamster cells: a report and analysis of the literature. A report of the Gene-Tox Program. Mutation Research 87, 81 - 142 (1981)).
The mutation frequencies of the solvent controls and the positive controls without and with metabolic activation for the last 58 experiments at LPT (most recent background data, not audited by the QAU-department) are given as follows:

Mutation frequency per 106 clonable cells
Without metabolic activation (24-h exposure)
Solvent control (n = 58)
Mean: 14.11
SD: 7.42
Range: 1.30 – 34.80
Positive control (µg/mL) (n = 58) EMS (600)
Mean: 449.1
SD: 444.2
Range: 112.10 - 1708.4
Positive control (µg/mL) (n = 58) EMS (700)
Mean: 468.4
SD: 268.6
Range: 152.0 – 976.9

With metabolic activation(4-h exposure)
Solvent control (n = 58) Mean: 14.88
SD: 8.20
Range: 2.18-38.36
Positive control (µg/mL) (n = 58) DMBA (20)
Mean: 347.1
SD: 241.8
Range: 130.0 – 844.8
Positive control (µg/mL) (n = 58) DMBA (30)
Mean: 563.8
SD: 700.1
Range: 151.3 – 2693.3


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study cytotoxicity in form of decreased plating efficiency (PE1 and PE2) was noted in the first and second experiments at the top concentration of 1000 µg/mL in the absence and presence of metabolic activation, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

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

Under the present test conditions, the active ingredient tested up to cytotoxic concentrations in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

The test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix.

The test item was diluted with aqua ad iniectabilia. A correction factor of 2.49 was used as the supplied test item contains only 40.20% solid matter. Aqua ad iniectabilia served as the vehicle control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 10, 25, 100, 250, 1000, 2500 µg solid matter/mL medium and the undiluted test item (ca. 4600 µg/mL medium) were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 1000 µg/mL medium in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 1000 µg solid matter/mL were employed as the top concentration for the mutagenicity tests in the absence and in the presence of metabolic activation.

Main study

Concentrations of 62.5, 125, 250, 500 or 1000 µg solid matter/mL were selected for the experiments without and with metabolic activation, respectively.

Cytotoxicity

In the main study cytotoxicity in form of decreased plating efficiency (PE1 and PE2) was noted in the first and second experiments at the top concentration of 1000 µg/mL in the absence and presence of metabolic activation, respectively.

Experiments without metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 10.79 and 9.84 x 10^-6clonable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250, 500 or 1000 µg solid matter/mL culture medium ranged from 5.44 to 10.34 x 10^-6 clonable cells. These results are within the normal range of the vehicle controls.

Experiments with metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 10.93 and 11.61 x 10^-6clonable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250, 500 or 1000 µg solid matter/mL culture medium ranged from 4.91 to 12.97 x 10^-6 clonable cells. These results are within the normal range of the vehicle controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 478.40 to 682.86 x 10^-6clonable cells in the case of EMS and ranging from 586.36 to 850.00 x 10^-6clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10^-6clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10^-6clonable cells for EMS and 130.0 to 2693.3 x 10^-6clonable cells for DMBA.

Under the present test conditions, the active ingredient tested up to cytotoxic concentrations in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.