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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in two in vitro bacterial reverse mutation assays (5 and 4 tester stains, respectively) except that in one but not in the second test the tester strain (TA 100) showed an ambiguous positive response at concentrations that were already cytotoxic. In vitro mammalian cell gene mutation assays with the supporting substances phenol, styrenated (Novares LS 500) and Hydrocarbons, C9-unsatd., polymd. (Novares TL 10) did not produce any positive response. In an in vitro chromosomal aberration test in human lymphocytes with the supporting substance Novares TL 10, no structural chromosomal aberration were induced under the conditions of the test. Overall, in vitro mutagenicity/genotoxicity of OAPP is assessed/demonstrated to be negative.

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Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Jun. - 13 Jul. 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
plate incorporation assay and preincubation test
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares LA 300 (phenol, methylstyrenated)
- Lot/batch No.: 24087
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 102
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Microsomal fraction prepared from induced livers of male Wistar rats, induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) orally (3x)
Test concentrations with justification for top dose:
1st experiment: 10, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (all strains, +/-S9)
2nd experiment: 62.5, 125, 250, 500, 1000, and 1500 µg/plate (all strains, -S9)
62.5, 125, 250, 500, 1000, 2000, 3500, and 5000 µg/plate (all strains except TA 100, +S9)
250, 500, 1000, 2000, 4000, and 5000 µg/plate (TA 100, +S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with survival of bacteria and S9 activity
Untreated negative controls:
yes
Remarks:
dist. water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide (TA100, TA1535); 4-NOPD (Ta98, TA1537); MMS (TA102) // with S9: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (1st and 2nd experiment: plate incorporation)

NUMBER OF REPLICATIONS: 3 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth/colony formation


Evaluation criteria:
Considered as mutagenic
- if a clear and dose-related increase in the number of revertants occurs in at least one tester with or without metabolic activation
and/or
- if a biologically relevant positive response for at least one of the dose groups occurs in at least one tester with or without metabolic activation.

An increase is considered relevant
- if in TA 100 and TA 102 mutation rate is at least twice as high as the rate of the solvent control;
- if in TA 98, TA 1535, and TA 1537 the mutation rate is at least 3x higher than that of the solvent control.


Statistics:
According to the OECD guidelines, the biological relevance is the criterion for the interpretation of the results: a statistical evaluation was not considered necessary under this premise (report p. 21).
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 250 µg/pl., depending on strain and presence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
reproducible in both tests at >= 2000 µg/pl.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reproducible in both tests at >= 1000 µg/pl.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Summary:

No biologically relevant increases in revertant colony numbers were observed in the four tester strains, TA 1535, TA 1537, TA 98 and TA 102, following treatment with Novares LA 300, either in the presence or in the absence of metabolic activation.

An unremarkable dose-related increase in the mutation rate was found in tester strain TA 100 under metabolic activation: a maximum mutation factor of 2.8 was reached at a dose of 4000 µg/plate. The mutagenic doses were cytotoxic as well.

All reference mutagens induced distinct increases of revertant colonies indicating the validity of the experiment.

Conclusions:
In one (TA 100) out of five tester strains (other strains TA 1535, TA 1537, TA 98, TA 102), a weak mutagenic dose-related response was observed with metabolic activation. The maximum mutation factor reached was 2.8 at the test substance concentration of 4000 µg/plate. The mutagenic doses were cytotoxic as well.
Overall, the results is considered to be "ambiguous".
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 4 strains of Salmonella used and no E.coli
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Necires EPX-L (phenol, methylstyrenated)
- Lot/batch No.: 9356059
- Expiration date of the lot/batch: 1995-02-01
- Stability under test conditions: Stable for at least 96 hours in dimethylsulphoxide
- Storage condition of test material: At room temperature in the dark
Target gene:
TA1537: hisC3076
TA98: hisD3052/R - factor
TA1535: hisG46
TA100: hisG46?R - factor
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat S9
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: None.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycin, 2-aminoanthracene
Remarks:
Positive control substances were disolved in saline or DMSO
Details on test system and experimental conditions:
Selection of dose levels
Selection of an adequate range of doses was based on a preliminary test with strain TA100, both with and without S9-mix. Eight concentrations were
tested in triplicate (this preliminary test was reported as a part of the first experiment of the mutagenesis assay). The highest concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate.

Direct plate incorporation assay:

Five different doses (with approximately half-log steps) of the test substance were tested in triplicate in each strain. The test substance was
tested both with and without 59-mix in each strain, in two independent experiments. Top agar in top agar tubes was melted and heated to 45°C. The following solutions were successively added to 3 ml top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml
of a dilution of the test substance in DM50 and either 0.5 ml 59-mix (in case of activation assays) or 0.5 ml 0.1 Mphosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48 h. After this period revertant (histidine independent)
colonies were counted.

( *) Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting
carcinogens and mutagens with the Salmonella/mammalian microsome
mutagenicity test, Mutation Res., 31, 347-364.
*) Maron, D.M. and Ames, B.N., 1983, Revised methods for the Salmonella
mutagenicity test, Mutation Res., 113, 173-215.
Erratum, 1983, Mutation Res., 113, 533.

Colony counting
The revertant colonies (histidine independent) were counted automatically with an Artek model 880 colony counter or manually, if less than 40
colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
ACCEPTABILITY OF ASSAY
An Ames test is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably be within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which also reasonably are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding test with strain TA100 or should extend to 5 mg/plate.
Statistics:
DATA EVALUATION AND STATISTICAL PROCEDURES
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic)in the Ames test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9-mix, the survival of strain TA100 was slightly reduced at 3330 and 5000 µg/plate, but in the presence of S9-mix the survival was not reduced up to and including 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Conclusions:
The test substance did not induce mutagenic activity.
Executive summary:

In a bacterial reverse point mutation Ames test with Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537 conducted according to guideline protocols, 'Oligomerisation and alkylation reaction products of 1-phenylpropene and phenol (previous name phenol, methyl stryrenated), was negative in increasing the number of revertant colonies when tested at 3, 10, 33, 100, 333, 1000, 3330, and 5000 ug/plate with and without metabolic activation system from liver of rats treated with Aroclor 1254. Concurrent positive (N-Ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine, 2 -nitrofluorene, and sodium azide), and negative (solvent) controls were used. A slight deviation from OECD 471 is noted in that only 4 strains of bacteria were used.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Cell Mutation Assay at the Thymidine Kinase Locus (TK +/-) in Mouse Lymphoma L5178Y Cells
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares LS 500 (phenol, styrenated)
- CAS No: 61788-44-1
- Substance type: organic
- Purity test date: 2010-01-15
- Lot/batch No.: 28324
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LS 500_phenol, styrenated
- Storage condition of test material: at room temperature, protected from light
Target gene:
Thymidine Kinase Locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Clone 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 2.5; 5.0; 10.0; 20.0; 30.0; 40.0 µg/mL
with S9 mix: 2.5; 5.0; 10.0; 20.0; 30.0; 40.0 µg/mL
Experiment II:
without S9 mix: 2.5; 5.0; 10.0; 20.0; 30.0; and 40.0 µg/mL
with S9 mix: 5.0; 10.0; 20.0; 25.0; 30.0; and 35.0 µg/mL
Following the expression phase of 48 hours the cultures at the highest concentration of 40 µg/mL in the presence and absence of metabolic activation were not continued due to exceedingly strong toxic effects. In experiment II the cultures at the lowest concentrations of 2.5 µg/mL without and 5.0 µg/mL with metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment
and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1.5 x 10^6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle controls and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
Key result
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
- Effects of pH: not effected
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Water solubility: not indicated by the sponsor
- Precipitation: not observed
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
In the range finding pre-experiments test item concentrations between 0.31 and 40 µg/mL in the presence and absence of metabolic activation (4 hours treatment) and 42.2 and 5400 µg/mL in the absence of metabolic activation (24 hours treatment) were used to evaluate toxicity.
The highest applied concentration of 5400 µg/mL was chosen with respect to the current OECD Guideline 476 regarding the purity of the test item (93.1 %).
Relevant toxic effects were observed after 4 hours treatment at 20 µg/mL and above without metabolic activation and at 40 µg/mL with metabolic activation. After 24 hours treatment a strong toxic effect already occurred at 42.2 µg/mL and above.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation was observed by the unaided eye up to the maximum concentration.
Both pH value and osmolarity were determined in the pre-experiment at the maximum concentration of the test item and in the solvent control without metabolic activation. No relevant change in the osmolarity or pH value was observed.
The dose range of the first experiment was set according to the data generated in the pre-experiment. In both main experiments the individual concentrations were generally spaced by a factor of 2.0 in the lower range. A more narrow dose spacing was used at upper concentrations to cover the recommended toxic range of approximately 10 – 20% of survival or RTG even though the toxic gradient was rather steep.
To overcome problems with possible deviations in toxicity or solubility both main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA:
In this study the range of the solvent controls was from 91 up to 198 mutant colonies per 10 exp.6 cells; the range of the groups treated with the test item was from 86 up to 251 mutant colonies per 10 exp. 6 cells. The highest solvent control value (198 mutant colonies per 10 exp.6 cells) exceeded the recommended 50 – 170 x 10 exp. 6 control range as stated under acceptability criteria of the assay of this report. However, the number of mutant colonies per 10 exp. 6 cells in the parallel culture (167) was acceptable. The viability exceeded the upper limit of 120% in the first culture of the first experiment without metabolic activation and in the second culture of the first experiment with metabolic activation. The data are acceptable however, since the cloning efficiency of the parallel culture remained within the acceptable range. Cloning efficiency values above 100% occasionally occur since even suspension cell cultures do not form an ideal solution in medium. The cells tend to form transient aggregates that are counted as single cells during determination of the cell density. The aggregation does not compromise the validity of the data however, since the absolute values of the cloning efficiency are used to calculate the mutation frequency.
MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls. The positive control in the first culture of the second experiment without metabolic activation exceeded the threshold but was below the range of the historical positive control data. The data are acceptable however, since the positive control of the parallel culture met the acceptance criteria.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed in the first experiment at 20 µg/mL and above with and without metabolic activation. In the second experiment relevant toxic effects were observed at 5.0 µg/mL and above in culture II without metabolic activation following 24 hours treatment. In the presence of metabolic activation strong toxic effects were noted at 35 µg/mL in culture I and at 25 µg/mL and above in culture II. The data generated in the first experiment at 30 µg/mL without metabolic activation (both cultures) are not considered valid since the relative total growth (RTG) of both cultures fell short of the threshold of 10 %. The results generated in the second culture of the first experiment at 30.0 µg/mL with metabolic activation are acceptable even though the RTG was just 4.4 since the parallel culture showed a RTG of 36.2 under identical conditions.
Summary Table
  conc. µg S9 total colonies/   total colonies/  
  per mL mix growth 106cells threshold growth 106cells threshold
Column 1 2 3 4 5 6 7 8
Experiment I / 4 h treatment   culture I culture II
Solv. control with acetone - 100.0 152 278 100.0 170 296
Pos. control with MMS  19.5 -  23.9 380 278  34.1 388 296
Test item   2.5 -  75.5 166 278 155.3 129 296
Test item   5.0 -  60.9 183 278 149.1 122 296
Test item  10.0 -  69.4 152 278 106.8 177 296
Test item  20.0 -  36.2 187 278  4.4 251 296
Test item  30.0 -  7.4 294 278  1.9 562 296
Test item  40.0 - culture was not continued# culture was not continued#
Experiment I / 4 h treatment   culture I culture II
Solv. control with acetone + 100.0 198 324 100.0 167 293
Pos. control with CPA   3.0 +  47.3 307 324  41.3 296 293
Pos. control with CPA   4.5  +   28.9 381 324  16.3 381 293
Test item   2.5  +   86.6 192 324  72.8 136 293
Test item   5.0  +   75.7 207 324  55.3 185 293
Test item  10.0  +   96.9 174 324  48.4 157 293
Test item  20.0  +   38.9 237 324  39.3 168 293
Test item  30.0  +   25.1 221 324  12.0 219 293
Test item  40.0  +  culture was not continued# culture was not continued#
Experiment II / 24 h treatment   culture I culture II
Solv. control with acetone - 100.0 119 245 100.0 155 281
Pos. control with MMS  13.0 -  41.5 259 245  25.4 423 281
Test item   2.5 - culture was not continued## culture was not continued##
Test item   5.0 - 103.2 135 245  46.3 180 281
Test item  10.0 - 151.3  92 245  33.2 131 281
Test item  20.0 -  83.4 139 245  38.7 186 281
Test item  30.0 -  92.7 118 245  23.4 183 281
Test item  40.0 -  60.2 189 245  21.5 171 281
Experiment II / 4 h treatment   culture I culture II
Solv. control with acetone + 100.0  91 217 100.0 148 274
Pos. control with CPA   3.0 +  55.8 165 217  52.8 413 274
Pos. control with CPA   4.5 +  36.3 251 217  31.0 365 274
Test item   5.0 + culture was not continued## culture was not continued##
Test item  10.0 +  53.5 120 217  62.5 162 274
Test item  20.0 +  59.9  94 217  64.3 145 274
Test item  25.0 +  58.2 102 217  43.7 143 274
Test item  30.0 +  80.6  86 217  30.2 109 274
Test item  35.0 +  15.4 122 217  49.9 173 274

threshold = number of mutant colonies per 106cells of each solvent control plus 126

The values printed in bold italic are judged as invalid, since the acceptance criteria are not met (RTG < 10%).

#    culture was not continued due to exceedingly severe cytotoxic effects
##
   culture was not continued since a minimum of only four analysable concentrations is required

 

Conclusions:
In conclusion it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

The study was performed to investigate the potential of Novares LS 500 (CAS No 61788-44-1) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Two independent main experiments were performed, using two parallel cultures each.

The first main experiment was performed with and without liver metabolic activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. Due to technical reasons (insufficient cell growth at all of the concentrations and the controls) the second experiment with metabolic activation was terminated prematurely. This experimental part was repeated using identical concentrations (experiment IIA). The results of experiment IIA are included in the second experiment.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:                               2.5; 5.0; 10.0; 20.0; and 30.0 µg/mL
with S9 mix:                                           2.5; 5.0; 10.0; 20.0; 30.0 µg/mL

Experiment II:

without S9 mix:                             5.0; 10.0; 20.0; 30.0; and 40.0 µg/mL
with S9 mix:                                10.0; 20.0; 25.0; 30.0; and 35.0 µg/mL

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed in the first experiment at 20 µg/mL and above with and without metabolic activation. In the second experiment relevant toxic effects were observed at 5.0 µg/mL and above in culture II without metabolic activation following 24 hours treatment. In the presence of metabolic activation strong toxic effects were noted at 35 µg/mL in culture I and at 25 µg/mL and above in culture II. The data generated in the first experiment at 30 µg/mL without metabolic activation (both cultures) are not considered valid since the relative total growth (RTG) of both cultures fell short of the threshold of 10 %. The results generated in the second culture of the first experiment at 30.0 µg/mL with metabolic activation are acceptable, even though the RTG was just 4.4 since the parallel culture showed a RTG of 36.2 under identical conditions.

The recommended toxic range of approximately 10 – 20% of RTG was covered with and without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments up to the maximum concentration with and without metabolic activation. The threshold of 126 plus each solvent control count was not reached or exceeded.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In this study the range of the solvent controls was from 91 up to 198 mutant colonies per 106cells; the range of the groups treated with the test item was from 86 up to 251 mutant colonies per 106cells. The highest solvent control value (198 mutant colonies per 106cells) exceeded the recommended 50 – 170 x 106 control range as stated under paragraph 8.12, acceptability of the assay of this report. However, the number of mutant colonies per 106cells in the parallel culture (167) was acceptable. The viability exceeded the upper limit of 120% in the first culture of the first experiment without metabolic activation and in the second culture of the first experiment with metabolic activation. The data are acceptable however, since the cloning efficiency of the parallel culture remained within the acceptable range. Cloning efficiency values above 100% occasionally occur since even suspension cell cultures do not form an ideal solution in medium. The cells tend to form transient aggregates that are counted as single cells during determination of the cell density. The aggregation does not compromise the validity of the data however, since the absolute values of the cloning efficiency are used to calculate the mutation frequency.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls. The positive control in the first culture of the second experiment without metabolic activation exceeded the threshold but was below the range of the historical positive control data. The data are acceptable however, since the positive control of the parallel culture met the acceptance criteria.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Cell Mutation Assay at the Thymidine Kinase Locus (TK +/-) in Mouse Lymphoma L5178Y Cells
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares TL 10 (Hydrocarbons, C9-unsaturated, polymerized; CAS No 71302-83-5)
- Physical state: liquid
- Lot/batch No.: 28724
- Expiration date of the lot/batch: 2011-09-30
- Storage condition of test material: at room temperature, protected from light
Target gene:
Thymidine Kinase Locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Clone 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 19.5; 39.1; 78.1; 156.3; 312.5; 625.0 µg/mL
with S9 mix: 19.5; 39.1; 78.1; 156.3; 312.5; 625.0 µg/mL
Experiment II:
without S9 mix: 78.1; 156.3; 312.5; 625.0; 1250.0; 2500.0 µ/mL
with S9 mix: 19.5; 39.1; 78.1; 156.3; 312.5; 626.0 µ/mL
Following the expression phase of 48 hours the cultures at the lowest concentrations of 19.5 or 78.1 µg/mL in both main experiments were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and/or vehicle controls and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects.
If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies
clastogenic effects are indicated.
Statistics:
Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
Key result
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
- Effects of pH: not effected
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Water solubility: --
- Precipitation: phase separation at 312.5, 625; 1250; and 2500 µg/mL
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
In the range-finding pre-experiment the highest applied concentration of 5000 µg/mL was chosen with respect to the current OECD Guideline 476. Test item concentrations between 39.1 and 5000 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h
treatment) of metabolic activation.
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hours of treatment.
Following 24 hours of treatment toxic effects were observed at 312.5 and 625 µg/mL close to the limit of solubility. No relevant cytotoxic effects were noted at higher concentrations after phase separation occurred.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Phase separation was observed by the unaided eye at 312.5 µg/mL and above following 4 hours treatment with and without metabolic activation. After 24 hours of treatment precipitation was determined at 1250 µg/mL and above.
The dose range of the first experiment was set according to the data generated in the pre-experiment. In both main experiments the individual concentrations were spaced by a factor of 2.0.


COMPARISON WITH HISTORICAL CONTROL DATA:
In this study the range of the solvent controls was from 99 up to 200 mutant colonies per 10^6 cells; the range of the groups treated with the test item was from 112 up to 310 mutant colonies per 10^6 cells. The highest solvent control value (200, 172, and 198 mutant colonies per 10^6 cells) exceeded the recommended 50 – 170 x 10^6 control range as stated under paragraph 8.12, acceptability of the assay of this report. The data are acceptable however, since the parallel culture remained within the recommended range. The cloning efficiency exceeded the upper limit of 120 % in the culture I of the second experiment with metabolic activation (170 %) and in culture II of the second experiment with and without S9 metabolic activation (130 %). The data are acceptable however, since cloning efficiency values above 100 % occasionally occur since even suspension cell cultures do not form an ideal solution in medium. The cells tend to form transient aggregates that are counted as single cells during determination of the cell density. As well, these values for the solvent controls remained well within the range of 50-200 colonies per 10^6 cells, originally recommended by the IWGT. Therefore, the aggregation does not compromise the validity of the data however, since the absolute values of the cloning efficiency are used to calculate the mutation frequency.
MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls.


ADDITIONAL INFORMATION ON CYTOTOXICITY: --
Summary Table
      relative mutant   relative mutant  
  conc. µg S9 total colonies/   total colonies/  
  per mL mix growth 106cells threshold growth 106cells threshold
Column 1 2 3 4 5 6 7 8
Experiment I / 4 h treatment   culture I culture II
Solv. control with acetone - 100.0 200 326 100.0 139 265
Pos. control with MMS  19.5 -  63.0 365 326  34.6 454 265
Test item  19.5 - culture was not continued# culture was not continued#
Test item  39.1 - 136.1 154 326  68.2 178 265
Test item  78.1 - 119.6 163 326  71.6 176 265
Test item  156.3 - 149.7 143 326  95.0 171 265
Test item 312.5 (p) -  84.4 189 326  52.7 211 265
Test item 625.0 (p) -  92.6 173 326  48.8 203 265
       
Solv. control with acetone + 100.0 172 298 100.0 168 294
Pos. control with CPA   3.0 +  70.9 256 298 103.0 291 294
Pos. control with CPA   4.5  +   50.3 290 298  67.0 408 294
Test item  19.5  +  culture was not continued# culture was not continued#
Test item  39.1  +  115.0 153 298 118.6 148 294
Test item  78.1  +   87.7 203 298  80.2 179 294
Test item  156.3  +   66.2 187 298  66.3 217 294
Test item 312.5 (p)  +   55.3 170 298  49.1 186 294
Test item 625.0 (p)  +   80.0 138 298  65.5 147 294
Experiment II / 24 h treatment   culture I culture II
Solv. control with acetone - 100.0 198 324 100.0 134 260
Pos. control with MMS  13.0 -  24.8 568 324  22.0 554 260
Test item  78.1 - culture was not continued# culture was not continued#
Test item  156.3 -  54.0 177 324  62.6 193 260
Test item 312.5 (p) -  74.2 114 324  53.1 164 260
Test item 625.0 (p) -  69.5 215 324  57.6 154 260
Test item 1250.0 (p) -  90.5 162 324  69.5 117 260
Test item 2500.0 (p) -  85.9 151 324  74.4 199 260
Experiment II / 4 h treatment   culture I culture II
Solv. control with acetone + 100.0  99 225 100.0 110 236
Pos. control with CPA   3.0 +  11.7 639 225  59.7 193 236
Pos. control with CPA   4.5 +  15.6 492 225  50.1 279 236
Test item  19.5 + culture was not continued# culture was not continued#
Test item  39.1 +  44.1 154 225  79.2 165 236
Test item  78.1 +  16.4 255 225  66.7 127 236
Test item  156.3 +  25.0 159 225  49.3 159 236
Test item 312.5 (p) +  17.1 112 225  46.7 185 236
Test item 625.0 (p) +  9.7 310 225  42.9 197 236

threshold = number of mutant colonies per 106cells of each solvent control plus 126

#    Culture was not continued since a minimum of four concentrations is required by the guidelines.

(p)  phase separation visible to the unaided eye

 

Conclusions:
In conclusion it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

The study was performed to investigate the potential of Novares TL 10 (CAS No 71302-83-5) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:                            39.1; 78.1; 156.3; 312.5; 625.0 µg/mL
with S9 mix:                                 39.1; 78.1; 156.3; 312.5; 625.0 µg/mL

Experiment II:

without S9 mix:                   156.3; 312.5; 625.0; 1250; and 2500 µg/mL
with S9 mix:                                 39.1; 78.1; 156.3; 312.5; 625.0 µg/mL

Phase separation of the test item visible to the naked eye was noted in experiments I and II at 312.5 µg/mL and above with metabolic activation (4 hours treatment) and without metabolic activation (4 and 24 hours treatment).

No toxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation in the first experiment. In the second experiment relevant toxic effects were observed in the presence of metabolic activation at 156.3 µg/mL and above in culture II.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the first experiment up to the maximum concentration with and without metabolic activation. In the second experiment the threshold of 126 plus each solvent control count and the historical range of solvent controls were exceeded in culture I at 78.1 µg/mL and 625 µg/mL. However, no comparable increase was noted in the parallel culture under identical conditions; so this isolated increase was judged as irreproducible artefact. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency using SYSTAT11statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of experiment II with metabolic activation. However, a small increase of the mutation frequency at toxic concentrations is common in this assay system and does not indicate a possible mutagenic potential provided that the mutation frequency does not exceed the threshold of 126 colonies per 106 cells above the corresponding solvent control. Since the mutation frequency neither exceeded the historical range of solvent controls nor the threshold as indicated above, the statistical results were considered as biologically irrelevant.

In this study the range of the solvent controls was from 99 up to 200 mutant colonies per 106cells; the range of the groups treated with the test item was from 112 up to 310 mutant colonies per 106cells. The highest solvent control value (200, 172, and 198 mutant colonies per 106cells) exceeded the recommended 50 – 170 x 106control range as stated under acceptability criteria of the assay of this report. The data are acceptable however, since the parallel culture remained within the recommended range. The cloning efficiency exceeded the upper limit of 120 % in the culture I of the second experiment with metabolic activation (170 %) and in culture II of the second experiment with and without S9 metabolic activation (130 %). The data are acceptable however, since cloning efficiency values above 100 % occasionally occur since even suspension cell cultures do not form an ideal solution in medium. The cells tend to form transient aggregates that are counted as single cells during determination of the cell density. As well, these values for the solvent controls remained well within the range of 50-200 colonies per 106cells, originally recommended by the IWGT. Therefore, the aggregation does not compromise the validity of the data however, since the absolute values of the cloning efficiency are used to calculate the mutation frequency.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Chromosome Aberration Test in Human Lymphocytes in vitro
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares TL 10 (CAS No 71302-83-5)
- Lot/batch No.: 28724
- Expiration date of the lot/batch: September 30, 2011
- Stability under test conditions: Not indicated by the Sponsor
- Storage condition of test material: At room temperature, protected from light
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 3.5, 6.1, 10.6, 18.6, 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mL
Experiment II: 0.4, 0.7, 1.3, 2.3, 4.0, 7.0, 12.2 21.3, 37.3, 65.3, 114.3, 200.0 µg/mL

Without metabolic activation:
Experiment I: 3.5, 6.1, 10.6, 18.6, 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mL
Experiment II: 0.7, 1.1, 2.0, 3.5, 6.1, 10.6, 18.6, 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: about 1.5

NUMBER OF CELLS EVALUATED: 100 per culture, except for the positive control in Experiment II without metabolic activation,
where only 50 metaphases were scored.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were scored.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Species / strain:
lymphocytes: human
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:
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were scored.
1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 5000.0 µg/mL, was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, visible precipitation of the test item in the culture medium was observed at 18.6 µg/mL and above in the absence of S9 mix and at 6.1 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 1.1 µg/mL and above and in the presence of S9 mix at 21.3 µg/mL and above. The occurrence of test item precipitation in the cultures has no detrimental impact on the outcome of the study, since the precipitate did not interfere with the scoring.
No relevant influence in the osmolarity or pH value was observed in Experiment I (solvent control: 363 mOsm, pH 7.5 versus 316 mOsm and pH 7.4 at 5000.0 µg/mL). In Experiment II the osmolarity was markedly decreased at the highest applied concentration only (solvent control: 397 mOsm, pH 7.3 versus 316 mOsm and pH 7.3 at 5000.0 µg/mL). Phase separation was observed in Experiment I at 174.1 µL/mL and above in the absence and presence of S9 mix and in Experiment II at 533.1 µL/mL and above in the absence of S9 mix.
In Experiment I, in the absence and presence of S9 mix and in Experiment II, in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. In Experiment II, in the absence of S9 mix, clear cytotoxic effects were observed after continuous treatment with 32.5 µg/mL (47.1 % of control).
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (770.0 or 825.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Summary of results of the chromosomal aberration study with
Novares TL 10 (CAS No 71302-83-5)

Exp.

Preparation

Test item

Mitotic indices

Aberrant cells

 

 

interval

concentration

in %

in %

 

 

 

in µg/mL

of control

incl. gaps*

excl. gaps*

carrying exchanges

 

 

Exposure period 4 hrs without S9 mix

 

I

22 hrs

Solvent control1

100.0

2.0

1.5

0.0

 

 

 

Positive control2

62.5

10.0

9.5S

2.0

 

 

 

6.1

85.5

0.5

0.0

0.0

 

 

 

10.6

102.3

1.5

0.5

0.0

 

 

 

18.6P

92.2

2.5

1.5

0.0

 

 

Exposure period 22 hrs without S9 mix

 

II

22 hrs

Solvent control1

100.0

3.5

2.5

0.0

 

 

 

Positive control3#

46.8

40.0

39.0S

7.0

 

 

 

0.7

92.7

2.5

2.0

0.0

 

 

 

1.1P

103.2

1.0

0.5

0.0

 

 

 

18.6P

56.7

0.0

0.0

0.0

 

 

 

32.5P

47.1

2.5

2.0

0.0

 

 

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

0.5

0.5

0.0

 

 

 

Positive control4

58.0

9.0

9.0S

1.0

 

 

 

3.5

96.7

2.0

1.5

0.0

 

 

 

6.1P

89.6

1.5

1.5

0.0

 

 

 

10.6P

122.6

1.0

0.5

0.0

 

II

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

 

Positive control4

50.2

21.5

21.5S

4.5

 

 

 

7.0

94.9

1.0

0.5

0.0

 

 

 

12.2

91.3

1.0

1.0

0.0

 

 

 

21.3P

106.1

1.5

1.0

0.5

 

*  Including cells carrying exchanges
#
   Evaluation of 50 metaphases per culture

P  Precipitation occurred at the end of treatment

S  Aberration frequency statistically significantly higher than corresponding control values

1   Acetone 0.5 % (v/v)

2     EMS 825.0 µg/mL

3     EMS  770.0 µg/mL

4   CPA   15.0 µg/mL

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, Novares TL 10 (CAS No 71302-83-5) is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic and/or precipitating concentrations.
Executive summary:

The test item Novares TL 10 (CAS No 71302-83-5), dissolved in acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I & II

Exposure period

 4 hrs

22 hrs

  4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were scored.

The highest applied concentration in the pre-test on toxicity (5000.0 µg/mL of the test item) was chosen with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. The following treatment concentrations were chosen for cytogenetic evaluation:

Experiment I:  without S9 mix:            6.1, 10.6 and 18.6 µg/mL,   
with S9 mix:                  3.5, 6.1 and 10.6 µg/mL,

Experiment II:  without S9 mix:          0.7, 1.1, 18.6 and 32.5 µg/mL,        
with S9 mix:                  7.0, 12.2 and 21.3 µg/mL.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration (47.1 % of control).

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo mammalian somatic cell study (erythrocyte micronucleus test, OECD TG 474), the supporting substance phenol, styrenated did not show any positive response. This result is adopted for the target substance OAPP.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Acclimation period, animals (2nd pre-experiment), relative humidity in the room 45 – 80 % , maximum temperature 20 – 25°C for about 6 hours; Supplier of the animals changed from Harlan Laboratories B.V. to Charles River Lab.(except 1st pre-experiment)
GLP compliance:
yes
Type of assay:
other: Micronucleus Assay in bone marrow of the Mouse
Specific details on test material used for the study:
Identity: Novares LS 500 (phenol, styrenated; CAS No 61788-44-1)
Batch No.: 28324
Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LS 500_phenol, styrenated
Stability in solvent: not indicated by the sponsor
Storage: At room temperature, protected from light
Expiration Date: unknown
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
Strain: NMRI
Source: Charles River Laboratories
Research Models and Services Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number of Animals
used in the pre experiments: 2 males and 2 females (for each tested dose level)
used in the main experiment: 35 males
Initial Age at Start of
Experiment: 8 - 12 weeks
Acclimation: minimum 5 days
Initial Body Weight
at Start of Treatment: 1st application: mean value 40.3 g (SD  2.1 g)
2nd application: mean value 40.6 g (SD  2.1 g)



ENVIRONMENTAL CONDITIONS
Housing: single
Cage Type: Makrolon Type II/III, with wire mesh top
(EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG,
73494 Rosenberg, Germany)
Feed: pelleted standard diet, ad libitum
(Harlan Laboratories B.V.; Postbus 6174;
5960 AD Horst; The Netherlands)
Water: tap water, ad libitum,
(Gemeindewerke, 64380 Rossdorf, Germany)
Environment: temperature 22  3°C
relative humidity 45 - 80 %
artificial light 6.00 a.m. - 6.00 p.m.



IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
Name: 30% dimethylsulfoxide / 70% polyethylene glycol 400
(30% DMSO / 70% PEG 400)
Supplier: VWR-Merck
64295 Darmstadt
Catalogue no.: DMSO: 1.02931.1000; PEG 400: 1.09726.0100
Route and Frequency of Administration: orally, twice
Volume Administered: 10 mL/kg b.w.
Details on exposure:
orally
Duration of treatment / exposure:
--
Frequency of treatment:
twice
Post exposure period:
--
Remarks:
Doses / Concentrations:
250, 500, and 1000 mg/kg b.w..
Basis:

No. of animals per sex per dose:
2 males and 2 females (for each tested dose level) used in the pre experiments
35 males were used in the main experiment
Control animals:
yes
Positive control(s):
Name: CPA; Cyclophosphamide
Supplier: Fisher Scientific GmbH
61130 Nidderau, Germany
Catalogue no.: 203960010 (purity: > 98 %)
Dissolved in: sterile water
Dosing: 40 mg/kg b.w.
Route and frequency of administration: orally, once
Volume administered: 10 mL/kg b.w.
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.
Evaluation criteria:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect, the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
All animals per test group were evaluated as described.

The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the negative control.
Statistics:
nonparametric Mann-Whitney test
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
in range finding studies, the lowest dose not exhibiting toxic effects was evaluated. This is the highest dose applied in the main study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (Clinical Signs on Toxicity):

In the first pre-experiment 4 animals (2 males, 2 females) received once orally a single dose of 2000 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 2000 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
toxic
reactions hours post-treatment
male / female
1 h 2-4 h 6 h 24 h 30 h 48 h
ruffled fur 2/0 2/2 2/2 1/- 1/1 1/1
In order to increase the exposure of the animals to the test item upon sponsor’s request the study design changed from single treatment to double treatment spaced by 24 hours.

In the second pre-experiment 4 animals (2 males, 2 females) received twice orally at 24 h intervals a dose of 2000 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 2000 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
Number of males / females with finding
hours post 1. application hours post 2. application
1 h 2-4 h 6 h 24 h 1 h 2-4 h 6 h 24 h
- reduction
of spontaneous
activity 0/0 0/0 0/0 0/0 0/0 0/0 1/1 0/0
- abdominal
position 0/0 0/0 0/0 0/0 0/0 0/0 1/1 0/0
- ruffled fur 0/0 0/0 0/0 0/0 0/0 1/0 0/0 0/0
death 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/1


In the third pre-experiment 4 animals (2 males, 2 females) received twice orally at 24 h intervals a dose of 1500 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 1500 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
Number of males / females with finding
hours post 1. application hours post 2. application
1 h 2-4 h 6 h 24 h 1 h 2-4 h 6 h* 24 h
- reduction of
spontaneous
activity 0/0 0/0 0/0 0/0 1/2 2/2 2/2 -/-
- abdominal
position 0/0 0/0 0/0 0/0 0/0 0/0 2/2 -/-
- eyelid
closure 0/0 0/0 0/0 0/0 0/2 0/2 2/2 -/-
- ruffled fur 2/0 2/2 0/0 0/0 2/2 2/2 2/2 -/-
- tumble 0/0 0/0 0/0 0/0 0/0 0/0 1/1 -/-
- apathy 0/0 0/0 0/0 0/0 0/0 0/0 2/2 -/-
- convulsion 0/0 0/0 0/0 0/0 0/0 0/0 1/0 -/-
- death 0/0 0/0 0/0 0/0 0/0 0/0 2/2 -/-
*: Due to the severity of the clinical signs observed at that time point it was decided to euthanize all of the animals.
-/-: no observation possible.
In the fourth pre-experiment 4 animals (2 males, 2 females) received twice orally at 24 h intervals a dose of 1000 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 1000 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
Number of males / females with finding
hours post 1. application hours post 2. application
1 h 2-4 h 6 h 24 h 1 h 2-4 h 6 h 24 h
ruffled fur 1/0 0/0 0/0 0/0 2/0 0/0 0/0 0/0


In the fifth pre-experiment 4 animals (2 males, 2 females) received twice orally at 24 h intervals a dose of 1250 mg/kg b.w. Novares LS 500 (CAS No 61788-44-1) formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 1250 mg/kg b.w. showed signs indicative of toxicity as shown in the table:
Number of males / females with finding
hours post 1. application hours post 2. application
1 h 2-4 h 6 h 24 h 1 h 2-4 h 6 h 24 h
- reduction of
spontaneous
activity 0/0 0/0 0/0 0/0 1/2 1/1 1/1 0/-
- abdominal
position 0/0 0/0 0/0 0/0 0/0 0/1 0/1 0/-
- eyelid
closure 0/0 0/0 0/0 0/0 0/0 0/1 0/1 0/-
- ruffled fur 0/0 0/0 0/0 0/0 1/2 0/1 1/1 0/-
- tremor 0/0 0/0 0/0 0/0 0/0 0/0 0/1 0/-
- death 0/0 0/0 0/0 0/0 0/0 1/1 0/0 0/1
-/-: no observation possible.

On the basis of these data 1000 mg/kg b.w. was considered the maximum tolerated dose by using a double application spaced by 24 hours.
No sex specific differences were observed for clinical signs. In agreement with the sponsor the main study was performed using males only.


RESULTS OF DEFINITIVE STUDY:

In the main experiment the treated animals of the high (1000 mg/kg b.w.), mid (500 mg/kg b.w.) and low (250 mg/kg b.w.) dose of the test item, as well as the animals treated with the vehicle control (30% DMSO / 70% PEG 400) did not show any evidence of toxicity.

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes
scoring 48 hours after treatment

Table1: vehicle

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

    1

m

30% DMSO / 70% PEG 400

0

8

        1145

    2

m

 

2

        1276

    3

m

 

 

2

        1048

    4

m

 

 

3

        1150

    5

m

 

 

1

        1185

    6

m

 

 

2

        1063

    7

m

 

 

0

        1257

 

sum

18

        8124

 

mean

2.6

        1161

 

percent cells with micronuclei

0.129

 

Table2: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

8

m

Novares LS 500

(CAS No 61788-44-1)

250

1

        1078

9

m

 

0

        1098

10

m

 

3

        1088

11

m

 

 

2

        1135

12

m

 

 

4

        1269

13

m

 

 

0

        1152

14

m

 

 

1

        1255

 

sum

11

        8075

 

mean

1.6

        1154

 

percent cells with micronuclei

0.079

 

Table3: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  15

m

Novares LS 500

(CAS No 61788-44-1)

500

2

        1082

  16

m

 

4

        1149

  17

m

 

0

        1135

  18

m

 

 

2

        1275

  19

m

 

 

2

        1149

  20

m

 

 

1

        1324

  21

m

 

 

0

        1217

 

sum

11

        8331

 

mean

1.6

        1190

 

percent cells with micronuclei

0.079

 

Table4: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  22

m

Novares LS 500

(CAS No 61788-44-1)

1000

1

        1350

  23

m

 

2

        1210

  24

m

 

2

        1350

  25

m

 

 

0

        1245

  26

m

 

 

0

        1174

  27

m

 

 

2

        1214

  28

m

 

 

4

        1139

 

sum

11

        8682

 

mean

1.6

        1240

 

percent cells with micronuclei

0.079

 

Table5: positive control

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  29

m

Cyclophosphamide

40

56

        1248

  30

m

 

 

45

        1144

  31

m

 

 

39

        1257

  32

m

 

 

57

        1040

  33

m

 

 

51

        1137

  34

m

 

 

36

        1158

  35

m

 

 

34

        1190

 

sum

318

        8174

 

mean

45.4

        1168

 

percent cells with micronuclei

2.271

 

Summary of Micronucleus Test Results

test group

dose mg/kg b.w.

application before preparation (h)

PCEs with micronuclei (%)

range

PCE per 2000 erythocytes

vehicle

          0

    48

0.129

 0 -8

         1161

test item

      250

    48

0.079

 0 -4

         1154

test item

      500

    48

0.079

 0 -4

         1190

test item

    1000

    48

0.079

 0 -4

         1240

positive control

        40

    24

2.271

34 -57

         1168

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle control versus test group

Significance

p

 250 mg Novares LS 500 (CAS No 61788-44-1)/kg b.w.

-

n.t.

 500 mg Novares LS 500 (CAS No 61788-44-1)/kg b.w.

-

n.t.

1000 mg Novares LS 500 (CAS No 61788-44-1)/kg b.w.

-

n.t.

   40 mg CPA/kg b.w.

+

0.0003

       -     =    not significant
        +    =    significant
       n.t. =    not tested

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

This study was performed to investigate the potential of Novares LS 500 (CAS No 61788-44-1) to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

Novares LS 500 was administered orally twice at an interval of 24 hours. The test item was formulated in 30% dimethylsulfoxide / 70% polyethylene glycol 400, which was also used as vehicle control; the dose volume was 10 mL/kg b.w.. A positive control group received cyclophosphamide once at 40 mg/kg b.w.. Forty eight hours after the first administration of the test item (24 h after the last treatment) the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated: 250, 500, and 1000 mg/kg b.w..

The highest dose was estimated by a pre-experiment to be suitable. As observed in pre-experimental assessments, at higher dose levels, animals died.

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that Novares LS 500 (CAS No 61788-44-1) did not have any cytotoxic properties in the bone marrow when given orally in this experiment. It is considered that the clinical signs and deaths expressed at doses higher than 1000 mg/kg bw, used in this experiment indicated that Novares LS 500 was absorbed and that the bone marrow would have been exposed to the test item.

In comparison to the corresponding vehicle controls, there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with Novares LS 500 (CAS No 61788-44-1) were below to the value of the vehicle control group. Additionally all values were within the historical vehicle control data base.

Cyclophosphamide administered once orally at 40 mg/kg b.w. was used as positive control, which showed a statistically significant increase of induced micronucleus frequency.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay

Two bacterial reverse mutation assays (OECD 471) were conducted according to GLP standards using Salmonella typhimurium strains with and without metabolic activation at concentrations up to 5000 µg/plate of the test substance (oligomerisation and alkylation reaction products of 2-phenylpropene and phenol - phenol, methylstyrenated) (EC no. 700-960-7), the target compound. Inhibitory/cytotoxic concentrations of Novares LA 300 produced a weak mutagenic response in just one tester strain, likewise did LA 1200 (the latter not shown). These results are considered "ambiguous". A third product carrying this EC number showed a negative result. Overall, no biologically relevant mutagenicity was noted.

Cell gene mutation test

Read-across is performed from two similar substances (resins) used as supporting/source substances: phenol, styrenated (Novares LS 500) (CAS no. 61788-44-1) and Hydrocarbons, C9-unsatd.,polymd. (Novares TL 10) (EC no. 615-276-3). An in-vitro Cell gene mutation test (OECD 476) was conducted according to GLP standards with both substances using mouse lymphoma cells with and without the presence of metabolic activation. Neither test substance induced gene mutations at any concentration.

Mammalian Chromosome Aberration test

Read-across is performed from the supporting/source substance Hydrocarbons, C9-unsatd.,polymd. (Novares TL 10) (EC no. 615-276-3). An in-vitro Mammalian Chromosome Aberration test (OECD 473) was conducted according to GLP standards using human lymphocytes cells with and without the presence of metabolic activation. The test substance did not induce chromosomal aberration at any concentration.

In-vivo Mammalian erythrocyte micronucleus test

Read-across is performed from the supporting/source substance phenol, styrenated (CAS no. 61788-44-1). An in-vivo mammalian erythrocyte micronucleus test (OECD 474) was conducted according to GLP standards in male mice. The test substance did not induce micronucleus formation at any concentration.

Justification for read-across is given in the respective study records. The results obtained with the source substances are adopted for the target substance oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP).

Justification for classification or non-classification

The substance was not mutagenic or only marginally positive when tested in vitro in a bacterial reverse mutation assay. Read-across from similar resins indicates that the substance oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (phenol, methylstyrenated) is very likely to be negative in the following tests: mammalian chromosome aberration assay, a cell gene mutation test, and in vivo mammalian erythrocyte micronucleus test. Thus, classification according to Regulation (EC) No 1272/2008 is not required.