Reaction mass of 1,1'-oxybis(ethylbenzene) and Styrene, oligomers

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:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD and GLP guidelines, and was considered to be relevant, adequate and reliable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see textbox below

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see textbox below

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

Deviations:

yes

Remarks:

see textbox below

Principles of method if other than guideline:

Protocol deviations:
1. In the first experiment in the absence and presence of S9-mix, only seven dose levels could be analysed for expression of the TK mutation.
Evaluation: Too many dose levels showed a toxic response to the treatment. Both in the absence and presence of S9-mix, dose levels selected gave a cell survival of approximately 10%, approximately 50% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Furthermore the dose ranges used were narrow and clear negative results were obtained. Therefore, the testing of an extra lower dose level would have given no additional information and the use of only seven dose levels had no effect on the integrity of the study.
2. In the first experiment in the presence of S9-mix, for determination of the mutation frequency a total number of 475 wells was used at the dose level of 40 µg/mL instead of 480 as specified in the protocol.
Evaluation: Five wells were infected. Since the fall out was only 1%, observed at a low dose level and the mutation frequency was well within the range, this deviation had no effect on the results of the study.
The study integrity was not adversely affected by the deviations.

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 mass of (1,1’ oxybis(ethylbenzene) and styrene, oligomers

IUPAC Name:

Reaction mass of (1,1’ oxybis(ethylbenzene) and styrene, oligomers

Constituent 2

Reference substance name:

SMPO Heavy Ends

IUPAC Name:

SMPO Heavy Ends

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): SMPO Heavy Ends; Reaction mass of (1,1’ oxybis(ethylbenzene) and styrene, oligomers
- Substance type: UVCB
- Physical state: Brown liquid
- Analytical purity: 100% (UVCB)
- Purity test date: No data
- Lot/batch No.: BC604 17-10-2014
- Expiration date of the lot/batch: No data
- Stability under test conditions: Not indicated
- Storage condition of test material: At room temperature protected from light

Method

Target gene:

Thymidine kinase (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

-Type and identity of media:
Horse serum
Horse serum (Invitrogen Corporation) was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing
penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) (Invitrogen), 1 mM sodium pyruvate
(Sigma) and 2 mM L-glutamin (Invitrogen Corporation).
Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium
-For 3 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
-For 24 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse
serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
-Properly maintained: yes
-Periodically checked for Mycoplasma contamination: yes. The cultures were checked for mycoplasma contamination.
-Periodically checked for karyotype stability: no data
-Periodically”cleansed” against high spontaneous background: yes. Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Merck) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 -mix

Test concentrations with justification for top dose:

Dose range finding test:
concentration range of 5.4 to 512 µg/mL
First mutagenicity test:
Without S9-mix: 0.1, 1, 10, 25, 40, 52, 65, 80, 100 and 125 µg/mL exposure medium.
With S9-mix: 1, 10, 25, 40, 65, 100, 164, 200, 250, 325 and 400 µg/mL exposure medium.
Second mutagenicity test:
Without S9-mix: 0.5, 1, 5, 10, 20, 30, 40, 50, 55, 60, 65 and 70 µg/mL exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No
- Exposure duration:
First mutagenicity test : 3 hours
Second mutagenicity test : 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 or 12 days (+2 hours for staining for TFT-selection)
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
5 (selection plate)
2 (cloning plate)

NUMBER OF CELLS EVALUATED:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium.
For determination of the MF a total number of 9.6 x 105 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). In the treatment group of 40 µg/mL in the first experiment a total number of 475 wells was used for determination of the mutation frequency.


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth;

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is = 50 per 106 survivors and = 170 per 106 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent/control (a small colony IMF of 150 x 10-6).
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Results and discussion

Test results

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: SMPO Heavy Ends precipitated in the exposure medium at concentrations of 164 µg/mL and above.
SMPO Heavy Ends was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 512 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/mL for 3 hours treatment) or 5 x 106 cells (1.25 x 105 cells/mL for 24 hours treatment) with a number of test substance concentrations increasing with approximately half log steps. The cell cultures for the 3 hours treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm. The cell cultures for the 24 hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. SMPO Heavy Ends was tested in the absence and presence of S9-mix.
Since SMPO Heavy Ends was poorly soluble in the exposure medium, the highest tested concentration was 512 µg/mL exposure medium.
Cell cultures were exposed to SMPO Heavy Ends in exposure medium for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 5 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.
For determination of the cytotoxicity, the surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted (day 1) and subcultured again for another 24 hours, after which the cells were counted (day 2). The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 105 cells/mL were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 5.4 to 512 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 48% at the test substance concentration of 52 µg/mL compared to the relative suspension growth of the solvent control. At greater than 164 µg/mL, less than 1% relative suspension growth was observed.
In the presence of S9-mix, the relative suspension growth was 33% at the test substance concentration 164 µg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 512 µg/mL.
In the absence of S9-mix, the relative suspension growth was 19% at the test substance concentration of 52 µg/mL compared to the relative suspension growth of the solvent control. At greater than 164 µg/mL, less than 1% relative suspension growth was observed.


COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The growth rate over the two-day expression period for cultures treated with DMSO was between 16 and 20 (3 hours treatment) and 101 and 103 (24 hours treatment).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Solubility

SMPO Heavy Ends precipitated in the exposure medium at concentrations of 164 µg/mL and above.

SMPO Heavy Ends was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 512 µg/mL.

Dose range finding test

In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 5.4 to 512 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.

In the absence of S9-mix, the relative suspension growth was 48% at the test substance concentration of 52 µg/mL compared to the relative suspension growth of the solvent control. At greater than 164 µg/mL, less than 1% relative suspension growth was observed.

In the presence of S9-mix, the relative suspension growth was 33% at the test substance concentration 164 µg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 512 µg/mL.

In the absence of S9-mix, the relative suspension growth was 19% at the test substance concentration of 52 µg/mL compared to the relative suspension growth of the solvent control. At greater than 164 µg/mL, less than 1% relative suspension growth was observed.

First mutagenicity test

Based on the results of the dose range finding test, the following dose range was selected for the first

mutagenicity test:

Without S9-mix: 0.1, 1, 10, 25, 40, 52, 65, 80, 100 and 125 µg/mL exposure medium.

With S9-mix: 1, 10, 25, 40, 65, 100, 164, 200, 250, 325 and 400 µg/mL exposure medium.

-Evaluation of toxicity

In the absence of S9-mix, the dose levels of 80 to 125 µg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 200 to 400 µg/mL were not used for mutation frequency

measurement, since these dose levels were too toxic for further testing.

Only seven dose levels were used for the mutagenicity determination.

The dose levels selected to measure mutation frequencies at the TK-locus were:

Without S9-mix: 0.1, 1, 10, 25, 40, 52 and 65 µg/mL exposure medium.

With S9-mix: 1, 10, 25, 40, 65, 100 and 164 µg/mL exposure medium.

In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 91% compared to the total growth of the solvent controls.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 94% compared to the total growth of the solvent controls.

-Evaluation of the mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with SMPO Heavy Ends either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the SMPO Heavy Ends treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test

To obtain more information about the possible mutagenicity of SMPO Heavy Ends, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.

Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 0.5, 1, 5, 10, 20, 30, 40, 50, 55, 60, 65 and 70 µg/mL exposure medium.

-Evaluation of toxicity

The dose levels of 0.5 to 20 µg/mL showed no cytotoxicity and 50 to 60 µg/mL showed similar cytotoxicity. Therefore, the dose levels of 1 and 55 µg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 65 and 70 µg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were: 0.5, 5, 10, 20, 30, 40, 50 and 60 µg/mL exposure medium.

The relative total growth of the highest test substance was reduced by 87% compared to the total growth of the solvent controls.

-Evaluation of mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with SMPO Heavy Ends. The numbers of small and large colonies in the SMPO Heavy Ends treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

DISCUSSION AND CONCLUSION

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

The growth rate over the two-day expression period for cultures treated with DMSO was between 16 and 20 (3 hours treatment) and 101 and 103 (24 hours treatment).

Mutation frequencies in cultures treated with positive control chemicals were increased by 10- and 11- fold for MMS in the absence of S9-mix, and by 10-fold for CP in the presence of S9-mix. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this as.

It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, SMPO Heavy Ends did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, SMPO Heavy Ends did not induce a significant increase in the mutation frequency.

In conclusion, SMPO Heavy Ends is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

SMPO Heavy Ends is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

SMPO Heavy Ends was tested in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells to study the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

Batch BC604 17-10-2014 of SMPO Heavy Ends was a brown liquid with a purity of 100% (UVCB). The test substance was dissolved in dimethyl sulfoxide. In the first experiment, SMPO Heavy Ends was tested up to concentrations of 65 µg/mL and 164 µg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 9% and 6% in the absence and presence of S9-mix, respectively. SMPO Heavy Ends precipitated in the culture medium at the dose level of 164 µg/mL.

In the second experiment, SMPO Heavy Ends was tested up to concentrations of 60 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 13%. The spontaneous mutation frequencies in the solvent-treated control cultures were within the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 10- and 11-fold for MMS in the absence of S9-mix, and by 10-fold for CP in the presence of S9-mix. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, SMPO Heavy Ends did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the duration of treatment time.

In the presence of S9-mix, SMPO Heavy Ends did not induce a significant increase in the mutation frequency.

It is concluded that SMPO Heavy Ends is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.