Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-07 to 2017-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

EC Number:

700-064-6

Cas Number:

2105830-60-0

Molecular formula:

Not applicable as this is a UVCB substance

IUPAC Name:

Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

Test material form:

liquid: viscous

Details on test material:

- Name: Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters
- Product Name: Multiester P97-463
- CAS No.: 2105830-60-0
- Batch No.: X24Jan17
- Physical State: viscous liquid
- Colour: amber
- Density: 1.06 g/cm³
- Active Components: 94%
- Expiry Date: 31 December 2018
- Storage Conditions: Room temperature, protected from light and kept in closed container to avoid air contact
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Target gene:

HPRT gene

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

0.01, 0.02, 0.05, 0.10, 0.15, 0.20, 0.25 and 0.5 mg/mL

Vehicle / solvent:

DMSO (1%; v/v)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure)
Expression time (cells in growth medium): 7 - 9 days
Selection time (if incubation with selection agent): approximately 9 - 11 days

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: one experiments with single exposure; cells were seeded in 5 cell culture petri dishes and evaluated
NUMBER OF CELLS SEEDED: 4 x 10^5 cells per petri dish
DETERMINATION OF CYTOTOXICITY: relative survival (RS)

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative control. Additionally, a dose-response realationship was determined in the X² test for trend.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Precipitation

Precipitation of the test item was noted at concentrations of 0.25 mg/mL and higher without metabolic activation and with metabolic activation at concentrations of 0.2 mg/mL and higher.

Toxicity

A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment I with and without metabolic activation.

In experiment I, the relative survival was 66% (without metabolic activation) and 8% (with metabolic activation) for the highest tested concentration of 0.50 mg/mL.

Mutagenicity

In the experiment without and with metabolic activation all validity criteria were met. The mutant values of the negative controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%.

The positive controls, DMBA (1.0 μg/mL) and EMS (300 μg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.

In the experiment without metabolic activation the mutant values of the negative controls, the solvent controls and all mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 7.8 - 39.7 mutants per 10E6 cells). The positive control EMS induced a distinct increase in mutant frequency with 240.5 mutants/10E6 cells.

The mutant frequencies of the negative controls were 21.5 and 20.1 mutants per 10E6 cells, of the solvent controls 30.4 and 15.6 mutants per 10E6 cells, respectively, and in the range of 9.9 to 31.4 mutants per 10E6 cells with the test item.

The highest mutant frequency was observed at a concentration of 0.25 mg/mL (31.4 mutants per 10E6 cells) with a relative survival of 42%.

The mutant frequencies induced by the test item did not show a biologically relevant increase. None of the observed mutant frequencies were statistically significantly increased over those of the solvent controls.

In the experiment with metabolic activation the mutant values of the negative controls, the solvent controls and all mutant values of the test item concentrations found were within or even lower than the historical control data of the test facility Eurofins Munich (about 9.2 – 38.8 mutants per 10E6 cells). The positive control DMBA induced a distinct increase in mutant frequency with 272.7 mutants/10E6 cells.

The mutant frequencies of the negative controls were 13.4 and 23.1 mutants per 10E6 cells, of the solvent controls 14.6 and 14.0 mutants per 10E6 cells, respectively, and in the range of 4.0 to 21.6 mutants per 10E6 cells with the test item, respectively.

The highest mutant frequency was observed at a concentration of 0.05 mg/mL (21.6 mutants per 10E6 cells) with a relative survival of 72%.

A statistical analysis displayed that one of the mutant frequencies was significantly increased over those of the solvent controls, but there was no evidence for a dose-response relationship. Therefore, this effect was considered as not biologically relevant.

Applicant's summary and conclusion

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

The selection of the concentrations was based on data from the pre-experiment. With and without metabolic activation were performed as a 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation: 0.05, 0.10, 0.15, 0.20 and 0.25 mg/mL

with metabolic activation: 0.01, 0.05, 0.10, 0.15, 0.20 and 0.25 mg/mL

Precipitation of the test item was noted at concentrations of 0.2 mg/mL (with metabolic activation) and 0.25 mg/mL (without metabolic activation).

Biologically relevant growth inhibition (relative survival < 70%) was observed in the experiment with and without metabolic activation. Without metabolic activation the relative survival was 66% and with metabolic activation 8% for the highest concentration (0.5 mg/mL).

In the experiments no biologically relevant increase of mutants was found after treatment with the test item without and with metabolic activation. All mutant values were within the historical data base of the test facility.

A statistical analysis displayed that one of the mutant frequencies was significantly increased over those of the solvent controls, but there was no evidence for a dose-response relationship.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusion

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.