2,3-Epoxypropyl neodecanoate, oligomeric reaction products with toluene-4-sulfonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In conclusion, the majority of the available mutagenicity studies with the test item showed no mutagenic effect: Under the present test conditions, the test item, tested up to concentrations of 5 mg/plate in the in vitro bacterial mutagenicity assay (Salmonella reverse mutation Assay) was mutagenic with metabolic activation only. Both mammalian in vitro genotoxicity assays, for the test substance, namely the in vitro mammalian mutagenicity assay (HPRT Assay, OECD 476; LPT, 2012) and the in vitro mammalian cytogenetic assay (Micronucleus Assay, OECD 487; LPT, 2012), carried out in the presence and absence of metabolic activation, revealed no genotoxic activation.

The starting component 2,3-epoxypropyl neodecanoate, which is mutagenic in vitro, and in vivo, as has been shown in bacterial mutagenicity tests and in an in vivo somatic cell test, is classified as mutagenic Cat. 2. Therefore, the test item Copolymer of 2,3-epoxypropyl neodecanoate and 4-methylbenzenesulfonic acid itself has to be classified as mutagenic Cat 2, because it contains > 1% of the mutagenic substance 2,3-epoxypropyl neodecanoate.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-01-29 to 1986-02-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions: TA 102 or E.coli WP2 were not tested,

Principles of method if other than guideline:

Ames BN et al. (1975). Mutat. Res. 31, 347-364

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Arochlor induced rat S9 fraction

Test concentrations with justification for top dose:

10, 25, 50, 100, 200, 250, 1000, 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

other: except simultaneous test of several substances  including one positive result

Details on test system and experimental conditions:

Type: Ames test
ADMINISTRATION: 
- Number of replicates: 2
- Application: Solvent dimethyl sulfoxide (CAS No. 67-68-5)
- Positive and negative control groups and treatment:    
Negative: Blank   
Positive: None (except simultaneous test of several substances  including one positive result)
- Incubation: 4 days

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:    Mutagenic effects (i.e  ratio of revertant rates treated/control >= 2) 

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

with metabolic activation, depending on strain, see results for details

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

no further information

Remarks on result:

other: other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Remarks:

Migrated from field 'Test system'.

CYTOTOXIC CONCENTRATION: none

GENOTOXIC EFFECTS: 

without metabolic activation: none with metabolic activation:   TA 100: >= 50 µg/plate
  TA 1535: >= 25 µg/plate TA 98: none TA 1537: none TA 1538: none

Conclusions:

The mutagenic effect in the in vitro bacterial mutagenicity test is induced by not reacted 2,3-epoxypropyl neodecanoate, which is mutagenic in vitro.

Executive summary:

Under the conditions employed in the Salmonella reverse mutation assay,

2,3-Epoxypropyl neodecanoate, oligomeric reaction products with toluene-4-sulfonic acid

was mutagenic with metabolic activation in strain TA1535 at doses higher 25 ug/plate and in strain TA100 at doses higher than 50 ug/plate.

However, the mutagenic effect in the in vitro bacterial mutagenicity test is induced by not reacted 2,3-epoxypropyl neodecanoate, which is mutagenic in vitro.

Reference 2

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:

2011-10-05 to 2012-02-09

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin
(100 µg/mL) called DMEM-FCS; Cells were periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258;
Spontaneous mutation rate was continuously monitored

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial supernatant fraction derived from livers of Aroclor 1254-treated rats (S9 mix)

Test concentrations with justification for top dose:

Five concentrations: 6.25; 12.5; 25; 50; 100µg/mL without metabolic activation
62.5, 125, 250, 500, 1000 µg/mL with metabolic activation

Vehicle / solvent:

Test item was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethyl methanesulphonate (EMS) in direct mutagenicity experiment; 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay; both EMS and DMBA were dissolved in DMSO. The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL

Details on test system and experimental conditions:

CELLS AND TISSUE CULTURE MEDIA
- V79 cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin 3 (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS
- Incubation of cultures: at 37°C in a humidified atmosphere (90%) containing 10% CO2
- For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.

METHOD OF APPLICATION:
- 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).


DURATION (see tables below)
- Preincubation period:
* 1 day (in 30 mL DMEM-FCS)
- Exposure duration:
* 4 hours (1st experiment) and 24 hours (2nd experiment) without S9 mix, respectively;
* in the experiments with S9 mix, the medium was replaced by 18 mL S9 mix and the exposure limited to 4 hours.
* the negative control was treated with acetone (the vehicle) in the same way
* After removal of the test item and washing of the plates with PBS cells were trypsinised and a relative plating efficiency was determined for each
dose to obtain an accurate measure of the toxic effect of the chemical
- Expression time (cells in growth medium):
* Three replicate plates (60 mm diameter) were used with a known number of cells.
* Remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS with one subcultivation on day 5.
* Afterwards cells were harvested by trypsinisation and replated at a density of 1 000 000 per 150mm diameter dish in DMEM-FCS containing
6-thioguanine (10 µg/mL) for selection of mutants (5 replicate plates), or at approx. 100 to 150 cells (exact number known) per 60 mm diameter
dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE 2), (3 replicate plates).
- Fixation time:
* Plates were fixed and stained after about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).
- Positive control:
* ethyl methanesulphonate (EMS) in direct mutagenicity experiment;
* 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay
both EMS and DMBA were dissolved in DMSO.
* The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL


NUMBER OF REPLICATIONS: three
NUMBER OF CELLS EVALUATED: 1 500 000

DETERMINATION OF CYTOTOXICITY (same procedure was used as employed for the mutagenicity experiments, except that no mutant selection was carried out)
- Method: survival
- A concentration of the test item which produces a low level of survival (10 to 20%) would be used as highest concentration and the survival in the
lowest concentration being approximately the same as that in the negative control.
- Five adequately spaced concentrations are employed
- In this preliminary experiment without and with metabolic activation test item precipitation was noted at the top concentration of 5000 µg
test item/mL. Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg test item/mL in the
experiment without and with metabolic activation, respectively. Hence, 100 µg test item/mL were employed as the top concentration for the
mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

Rationale for test conditions:

The study was carried out according to the following guidelines: Council Regulation (EC) No 440/2008 method B.17. “Mutagenicity – In vitro mammalian cell gene mutation test” (published in the Official Journal of the European Union L 142, dated May 31, 2008); and OECD Guideline for Testing of chemicals No. 476: In vitro mammalian cell gene mutation test (adopted July 21, 1997).

Evaluation criteria:

The following predetermined descriptive criteria are used for interpretation of the results:
- If in both independent experiments solvent and positive controls show results within the norm and if the test item does not increase the mutation
frequency 2-fold above the mean of the solvent controls under any condition, 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 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 item is considered as positive in the test.

Statistics:

No satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments. See evaluation criteria

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitation were noted at concentrations of 5000 µg test item/mL. Cytotoxicity in form of decreased plating efficiency: 100 µg/mL without S9mix, 1000 µg /mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (Preliminary cytotoxicity test):
In the preliminary experiment without and with metabolic activation test item precipitation was noted at the top concentration of 5000 µg test
item/mL. Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg/mL in the experiment without and with metabolic activation, respectively. Hence, 100 µg test item/mL were employed as the top concentration for the mutagenicity tests in the
absence and 1000 µg/mL 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 106 survivors in non-activation solvent
controls and 6 to 46 per 106 survivors in S9 activation solvent controls [1]. The background data obtained at LPT are given at the end of this
chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls EMS (600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation
frequency.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10 6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA
(see table below).
.


TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Criteria for assay acceptance

Solvent control: As the total number of colonies is normally low and as a single mutation may cause several colonies due to cell division during the expression period, a relatively large variation of the mutation frequency may result. This is especially true, if a low spontaneous mutation frequency is forced by cloning (in order to achieve a high sensitivity of the test).

The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10⁶survivors in non-activation solvent controls and 6 to 46 per 10⁶survivors in S9 activation solvent controls [1]. The background data obtained atLPTare given at the end of this chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls(600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation frequency.

The background mutation frequency atLPTranges from 1.30 to 38.36 x 10^-6clonable cells for the negative controls. The mutation frequency of the positive controls atLPTranges from 112.1 to 1708.4 x 10^-6clonable cells forand 130.0 to 2693.3 x 10^-6clonable cells for DMBA (see table below).

The mutation frequencies of the solvent controls andthe positive controls without and with metabolic activation for the last 58 experiments (most recent background data, not audited by the QAU-department) are given as follows:

Mutation frequency per 10^6 clonable cells
	Without metabolic activation (24-h exposure)		With metabolic activation (4-h exposure)
Solvent control (n = 58)
mean	14.11		14.88
SD	7.42		8.20
range	1.30 - 34.80		2.18 - 38.36
Positive control (µg/mL) (n = 58)
	EMS (600)	EMS (700)	DMBA (20)	DMBA (30)
mean	449.1	468.4	347.1	563.8444.2
SD	444.2	268.6	241.8	700.1
range	112.1 – 1708.4	152.0 – 976.9	130.0 – 844.8	151.3 – 2693.3

SD     = Standard deviation

EMS   = ethyl methanesulfonate

DMBA = 9,10-dimethyl-1,2-benzanthracene

Conclusions:

Under the present test conditions, the test item was 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 completely dissolved in acetone.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item precipitation was noted at the top concentration of 5000 µg test item/mL. Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 100 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

 

Main study

Five concentrations ranging from 6.25 to 100 or 62.5 to 1000 µg test item/mL were selected for the experiments without and with metabolic activation.

 

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 concentrations 100 or 1000 µg/mL in the absence and presence of metabolic activation, respectively.

 

Experiments without metabolic activation

The mutation frequency of the negative control acetone was 8.25 and 9.09 x 10 -6 clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 6.25, 12.5, 25, 50 or 100 µg test item/mL culture medium ranged from 6.28 to 13.87 x 10 -6 clonable cells. These results are within the normal range of the negative controls.

 

Experiments with metabolic activation

The mutation frequency of the negative control acetone was 9.52 and 11.50 x 10 -6 clonable cells. Hence, the negative 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 test item/mL culture medium ranged from 7.59 to 13.73 x 10 -6 clonable cells. These results are within the normal range of the negative 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 221.62 to 288.38 x 10-6clonable cells in the case of EMS and ranging from 155.43 to 486.69 x 10 -6 clonable 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 -6 clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10 -6 clonable cells for and 130.0 to 2693.3 x 106 clonable cells for[S214501] DMBA.

 

Conclusion

Under the present test conditions, test item 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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-26 to 2012-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit), No. 487, Guideline July 22, 2010

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

mammalian cell system( Chinese hamster Ovary cells)

Species / strain / cell type:

other: Chinese hamster ovary (CHO-K1) cells

Details on mammalian cell type (if applicable):

Species/cell type: CHO cells as originally derived from the ovary of Chinese hamster, obtained from ATCC
CHO-K1, modal chromosome number of 20

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix based on liver homogenate fraction  from male rats, induced with Aroclor 1254 (i.p.)

Test concentrations with justification for top dose:

31.5, 62.5, 125, 250 µg/mL with and without metabolic activation, 4 h exposure
7.8, 15.6, 31.3, 62.5 µg/ml without metabolic activation, 20 h exposure

Vehicle / solvent:

Vehicle: test item was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle: acetone

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

cyclophosphamide

Remarks:

+S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle: acetone

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

mitomycin C

Remarks:

-S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehivle acetone

True negative controls:

no

Positive controls:

yes

Remarks:

aneugen

Positive control substance:

other: colchicine

Remarks:

-S9-mix

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Species/cell type: CHO-K1 cell line, cell cycle length 12 hours
- Metabolic activation system: male rat liver S9 from  Aroclor 1254 induced animals
ADMINISTRATION: 
- Solubility: test item was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.
- Preliminary experiment: cytotoxicity was noted starting at concentrations of 250 or 100 µg test item/mL in the experiments with and without
metabolic activation, (4 h and 20-h exposure, respectively). Hence, 250 µg test item 1203/mL were employed as the top concentration for the
mutagenicity genotoxicity tests without and with metabolic activation (4 h exposure). 62.5 µg/mL were employed as the top concentration for the experiment without metabolic activation with an extended exposure of 20 hours.
- Dosing:  31.5, 62.5, 125, 250 µg/mL with and without metabolic activation, 4 h exposure
7.8, 15.6, 31.3, 62.5 µg/ml without metabolic activation, 20 h exposure
- Positive and negative control groups and treatment:    
negative: acetone
positive (+S9): cyclophosphamide 
positive (-S9): mitomycin C 
positive (-S9): colchicine 

DURATION
- most aneugens and clastogens are detected by a short term treatment period of 4 hours in the presence and absence of S9, followed by removal of
the test item and a growth period of 1.5 – 2.0 cell cycles. Cells were sampled at a time equivalent to about 1.5 – 2.0 times the normal (i.e. untreated)
cell cycle length either after the beginning or at the end of treatment. Because of the potential cytotoxicity of S9 preparations for cultured
mammalian
cells, an extended exposure treatment of 1.5 – 2.0 normal cell cycles was used only in the absence of S9.
Cell treatment and harvest times for the used CHO cell line see table below.
As both initial tests of the short 4-h treatment are negative or equivocal, a subsequent, extended exposure treatment without S9 was used.
- Harvesting time: harvesting time was 20 hours after the end of exposure

STAIN (for cytogenetic assays): Each culture was harvested and processed separately. High-quality cell preparations for scoring were obtained. Cell
cytoplasm were retained to allow the detection of micronuclei and (in the cytokinesis-block method) reliable identification of binucleate cells. The
slides were stained using Giemsa.

NUMBER OF REPLICATIONS: 2, duplicate cultures were used for each test item concentration and for the solvent control cultures.

NUMBER OF CELLS EVALUATED: The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000
binucleated cells per culture; two cultures per concentration).

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
The CBPI indicates the average number of cell cycles per cell during the period of exposure to cytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis:

OTHER EXAMINATIONS: 1000 binucleated cells per duplicate cell culture were scored to assess the frequency of cells with one, two, or more than
two micronuclei. Additionally, the cells were classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a
measure of toxicity.

Rationale for test conditions:

The study was carried out in compliance with: OECD Guidelines for Testing of Chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit) (No. 487, Guideline July 22, 2010).

Evaluation criteria:

The assay demonstrates its ability to reliably and accurately detect substances of known aneugenic and clastogenic activity, with and without
metabolic activation.
Solvent/vehicle control and untreated cultures give reproducibly low and consistent micronuclei frequencies, typically 5 – 25 micronuclei per 1000
cells according to OECD 487. Data from negative and positive controls are used to establish historical control ranges. These values are used in
deciding the adequacy of the concurrent negative/positive controls for an experiment .

Statistics:

The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for
continuity according to YATES.
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing
micronuclei, it is classified as a positive result.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

250 or 100 µg test item/mL in the experiments with and without metabolic activation, (4‑h and 20-h exposure, respectively). 62.5 µg/mL for the experiment without metabolic activation with an extended exposure of 20 h

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The pH and osmolality of the negative control and 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 pH or osmolality of the formulations were noted, see table below.

Test item was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.

Data from negative and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the
concurrent negative/positive controls for an experiment i.e. the negative/positive control data must be within the historical ranges

Remarks on result:

other: other: Chinese hamster Ovary (CHO)

Remarks:

Migrated from field 'Test system'.

Concentrationof Test item [µg/mL medium]	pH value	osmolality [mosmol/kg]
Negative control: exposure medium, containing 0.5% acetone	7.9	431
15.6	8.1	450
31.3	8.1	448
62.5	8.0	451
125	8.0	457
250	7.9	470

Conclusions:

Under the present test conditions, the test item tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of genotoxic properties in the in vitro micronucleus test.

Executive summary:

The in vitro micronucleus assay is a genotoxicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e. micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes which are unable to migrate with the rest of the chromosomes during the anaphase of cell division.

The purpose of the micronucleus assay is to detect those agents which modify chromosome structure and segregation in such a way as to lead to induction ofmicronuclei in interphase cells.

Test item was assayed in an in vitro micronucleus test using CHO cell cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.

The test item was completely dissolved in acetone.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation cytotoxicity was noted starting at concentrations of 250 or 100 µg test item/mL in the experiments with and without metabolic activation, (4‑h and 20-h exposure, respectively). Hence, 250 µg test item/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation (4‑h exposure). 62.5 µg/mL were employed as the top concentration for the experiment without metabolic activation with an extended exposure of 20 hours.

In the main study cytotoxicity was noted at the top concentrations of 250 or 100 µg test item/mL in the experiments without and with metabolic activation (4‑h and 20-h exposure, respectively).

Mitomycin C, colchicine and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated with the test item at concentrations from 31.3 to 250 or 7.8 to 62.5 µg/mL medium (4‑h and 20-h exposure, respectively) in the absence of metabolic activation ranged from 4.0 to 6.0 micronuclei per 1000 binucleated cells. The results obtained are considered to be within the normal range of the vehicle acetone where a mean incidence of micronucleus frequencies of 5.0 or 4.5 micronuclei per 1000 binucleated cells was observed after a 4-hour and 20-hour exposure, respectively. The micronucleus frequency of the untreated controls was 5.0 or 4.0 micronuclei per 1000 binucleated cells after a 4-hour and 20-hour exposure, respectively.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the test item at concentrations from 31.3 to 250 µg/mL medium in the presence of metabolic activation ranged from 6.5 to 9.5 micronuclei per 1000 binucleated cells. The results obtained are considered to be within the normal range of the vehicle acetone where a mean incidence of micronucleus frequencies of 10.5 or 7.5 micronuclei per 1000 binucleated cells was observed in the first and second experiment, respectively. The micronucleus frequencies of the untreated controls were 5.0 or 8.0 micronuclei per 1000 binucleated cells in the first and second experiment, respectively.

Conclusion

Under the present test conditions, test utem tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of genotoxic properties in the in vitro micronucleus test.

In the same test, Mitomycin C, colchicine and cyclophosphamide induced significant damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No data available. A data waiver was claimed.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test item has to be classified as mutagen Cat. 2 according to CLP regulation (1272/2008), because it contains > 1% of the mutagenic substance 2,3-epoxypropyl neodecanoate.