Phenol, mono- + distyrenated (MSP+DSP)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Besides an ambiguous result in a bacterial reverse mutation assay (Ames test, OECD TG 471) (four tester strains negative, one strain weakly positive), phenol styrenated did not show a mutagenic/genotoxic response in an in vitro mammalian cell gene mutation study (OECD TG 476). Overall, in vitro mutagenicity/genotoxicity of phenol, styrenated is assessed/demonstrated to be negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jun. - 07 Aug. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

plate incorporation assay

GLP compliance:

yes (incl. QA statement)

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 LS 500
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 24430
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LS 500_phenol, styrenated
- Substance type: organic
- Purity test date: 2010/08/05
- 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:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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: 3.16, 10, 31.6, 100, 316, and 1000 µg/plate (TA 98 and TA 100, +/-S9)
1.0, 3.16, 10, 31.6, 100, and 316 µg/plate (TA 1535 and TA 1537, +/-S9)
1.0, 3.16, 10, 31.6, 100, 316, and 1000 µg/plate (TA 102, +/-S9)
2nd experiment: 15.63, 31.25, 62.5, 125, 250, and 500 µg/plate (TA 98 and TA 100, +/-S9)
3.91, 7.81, 15.63, 31.25, 62.5, 125, and 250 µg/plate (TA 1535 and TA 1537, +/-S9)
7.81, 15.63, 31.25, 62.5, 125, 250, and 500 µg/plate (TA 102, +/-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:

2-acetylaminofluorene

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (1st and 2nd experiment: plate incorporation test)

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:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

reproducible in both tests

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reproducible, >=31 µg/pl. (-S9); >= 125 µg/pl. (+S9), depending on tester strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

reproducible in both tests

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reproducible, >=31 µg/pl. (-S9); >= 125 µg/pl. (+S9), depending on tester strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

reproducible in both tests

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reproducible, >=31 µg/pl. (-S9); >= 125 µg/pl. (+S9), depending on tester strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

reproducible in both tests

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reproducible, >=31 µg/pl. (-S9); >= 125 µg/pl. (+S9), depending on tester strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

not specified

Remarks:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reproducible: >=31 µg/pl. (-S9); >= 125 µg/pl. (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

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 LS 500, either in the presence or in the absence of metabolic activation.

Biologically relevant, dose-related increases were found in tester strain TA 100 at 100 µg/pl. without S9 and at 316 µg/pl. under metabolic activation (1st exp.) and at 62.5 and 125 µg/pl. without S9 and at 250 µg/pl. with S9 (2nd exp.): a maximum mutation factor of 3 was reached in the presence of S9. The mutagenic doses in the absence or presence of metabolic activation were cytotoxic as well.

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

Conclusions:

The test substance is not considered to be positive in the Ames test as tester strains TA 98, TA 102, TA 1535, and TA 1537 did not show any cytotoxic effects with and without metabolic activation even at cytotoxic concentrations. Only strain TA 100 responded slightly positve at cytotoxic concentrations without clear dose-response relationship.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source substance LA 300 (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol, previous name phenol, methylstyrenated) is produced by the same process as LS 500 (phenol, mono- & distyrenated, target substance). In both cases, one reactant is phenol. The second reactant is different being 2-phenylpropene and just styrene for LA 300 and LS 500 respectively.
During the manufacturing process (acid catalysed alkylation reaction), the starting monomeric reactants form di- or higher substituted/condensed products. Reaction products are on the one hand the alkylation products mono- and di-substituted phenol (LS 500 1-phenylethyl and LA 300 1-methyl-1-phenylethyl substituents, respectively). On the other hand, manufacture of LA 300 results, in addition to the 1-methyl-1-phenylethyl substituted phenols, also in di-/oligomerisation reaction products of the olefinic reaction component. Substituted phenol components of LA 300 and LS 500 differ only in one methyl group that in case of LA 300 is additionally attached to the bridging carbon atom, the substituents being 1-methyl-1-phenylethyl- (LA 300) and 1-phenylethyl- (LS 500), respectively.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Main difference between LA 300 and LS 500 is, that the production of LA 300 results in dimers and oligomers of 2-phenylpropene (olefinic reaction component) in addition to substituted phenols. These di/oligomers are purely alkylaromatic substances with no phenol moiety present. They are composed of an alkyl chain (C5 or longer) that carries two or more phenyl-rings (benzene moieties). Ratio of non-phenolic to phenolic substances in LA 300 is > 0.5.

3. ANALOGUE APPROACH JUSTIFICATION
Use of LA 300 as supporting substance for LS 500 is justified because both substances contain very similar components (substituted phenols), which differ only in one methyl substituent at the bridging carbon chain. In addition, components with the same degree of condensation (equal number of aromatic rings) (either alkylated or di-/oligomerised products) are quite similar in size and have similar spatial arrangements of structural elements. Structural elements are basically the same for both substances, as components are comprised of aromatic rings (benzene and phenol or purely benzene) and of a smaller aliphatic chain, to which the aromatic moieties are attached. Due to the similarity of the structural elements, metabolism and biotransformation will proceed in a similar way. There will be attack at the aliphatic chain resulting in hydroxylated products that subsequently are transformed to aldehydes and carboxylic acids. Metabolism of the aromatic rings will also result in hydroxylated products. Thus, similar metabolites are formed for LA 300 and LS 500. Based on these grounds, it is justified to adopt results of toxicity tests obtained with the source substance LA 300 (phenol, methylstyrenated) for the target substance LS 500 (phenol, mono- & distyrenated).

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Read-across to the preceding entry:
Source substance: Phenol, methylstyrenated - LA 300 (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol)
Reference: Kraft M, Lutterbach A 2007

Species / strain:

S. typhimurium TA 1535

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

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium 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

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

not specified

Remarks:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reproducible in both tests at >= 1000 µg/pl.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

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

True negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The test substance is not considered to be positive in the Ames test as tester strains TA 98, TA 102, TA 1535, and TA 1537 did not show any genotoxic effects with and without metabolic activation even at cytotoxic concentrations. Only strain TA 100 responded slightly positive (with metabolic activation only) at high concentrations (>= 2000 µg/pl) that were already cytotoxic (>= 1000 µg/pl).
The genotoxic effects observed in the Ames test with the source substance LA 300 (phenol, methylstyrenated) are similar to the effects found in the Ames test of the target substance phenol, mono- & distyrenated (LS 500). Thus, the low mutagenic potency of the target substance against bacteria is confirmed by a test with the source substance.

Reference 3

Endpoint:

genetic toxicity in vitro, other

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source substance Necires EPX-L (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol, previous name phenol, methylstyrenated) is produced by the same process as LS 500 (phenol, mono- & distyrenated, target substance). In both cases, one reactant is phenol. The second reactant is different being 2-phenylpropene and just styrene for Necires EPX-L and LS 500 respectively.
During the manufacturing process (acid catalysed alkylation reaction), the starting monomeric reactants form di- or higher substituted/condensed products. Reaction products are on the one hand the alkylation products mono- and disubstituted phenol (LS 500 1-phenylethyl and Necires EPX-L 1-methyl-1-phenylethyl substituents, respectively). On the other hand, manufacture of Necires EPX-L results, in addition to the 1-methyl-1-phenylethyl substituted phenols, also in di-/oligomerisation reaction products of the olefinic reaction component. Substituted phenol components of Necires EPX-L and LS 500 differ only in one methyl group that in case of Necires EPX-L is additionally attached to the bridging carbon atom, the substituents being 1-methyl-1-phenylethyl- (Necires EPX-L) and 1-phenylethyl- (LS 500), respectively.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Main difference between Necires EPX-L and LS 500 is, that the production of Necires EPX-L results in dimers and oligomers of 2-phenylpropene (olefinic reaction component) in addition to substituted phenols. These di/oligomers are purely alkylaromatic substances with no phenol moiety present. They are composed of an alkyl chain (C5 or longer) that carries two or more phenyl-rings (benzene moieties). Ratio of non-phenolic to phenolic substances in Necires EPX-L is > 0.5.

3. ANALOGUE APPROACH JUSTIFICATION
Use of Necires EPX-L as supporting substance for LS 500 is justified because both substances contain very similar components (substituted phenols), which differ only in one methyl substituent at the bridging carbon chain. In addition, components with the same degree of condensation (equal number of aromatic rings) (either alkylated or di-/oligomerised products) are quite similar in size and have similar spatial arrangements of structural elements. Structural elements are basically the same for both substances, as components are comprised of aromatic rings (benzene and phenol or purely benzene) and of a smaller aliphatic chain, to which the aromatic moieties are attached. Due to the similarity of the structural elements, metabolism and biotransformation will proceed in a similar way. There will be attack at the aliphatic chain resulting in hydroxylated products that subsequently are transformed to aldehydes and carboxylic acids. Metabolism of the aromatic rings will also result in hydroxylated products. Thus, similar metabolites are formed for Necires EPX-L and LS 500. Based on these grounds, it is justified to adopt results of toxicity tests obtained with the source substance Necires EPX-L (phenol, methylstyrenated) for the target substance LS 500 (phenol, mono- & distyrenated).

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Read-across to the preceding entry:
Source substance: Phenol, methylstyrenated (Necires EPX-L)
Reference: van de Waart EJ 1994

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: In the absence of S9-mix the survival of strain TA100 was slightly reduced at 3330 and 5000 µg/plate. In the presence of S9-mix the survival of strain TA100 was not reduced up to and including 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The test substance (Necires EPX-L; phenol, methylstyrenated) did not induce mutagenic activity in the four strains tested neither with nor without metabolic activation. This is generally in line with results obtained with Ames tests performed with the target substance phenol, mono- & distyrenated.

Reference 4

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:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

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. QA statement)

Type of assay:

mammalian cell gene mutation assay

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)
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 28324
- Composition of test material: composition is reported in the document (Certificate of Analysis) attached under IUCLID Sect. 13, Certificate of Analysis_Novares LS 500_phenol, styrenated
- Substance type: organic
- Purity test date: 2010/01/15
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- 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-Naphthoflavone 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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

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 10E6 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 con¬trols 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)

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	10^6cells	threshold	growth	10^6cells	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 (phenol, mono- & distyrenated; CAS No --/ EC No 701-443-9) 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.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

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), phenol, styrenated (LS 500) did not show any positive response. No mutagenic effect in vivo was observed with the target substance.

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:

other: GLP Guideline study

Qualifier:

according to guideline

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 (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Novares LS 500 (CAS No 61788-44-1)
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, 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
- Substance type: organic
- Purity test date: 2010-01-15
- Stability in solvent: not indicated by the sponsor
- Storage: at room temperature, protected from light
- Expiration date of the lot/batch:: unknown

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system 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.

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, time intervall between treatments was 24 hours

Post exposure period:

bone marrow cells were collected 24 hours after the second treatment (48 hours after the start of the experiment)

Dose / conc.:

250 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

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 (7 animals per group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
Supplier: Fisher Scientific GmbH, 61130 Nidderau, Germany
Catalogue no.: 203960010 (purity: > 98 %)
Dissolved in: sterile water
Route and frequency of administration:orally, once
Dosing: 40 mg/kg b.w.
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

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: in all three test groups, there was no incidence of an enhanced frequency of micronucleus formation in polychromatic erythrocytes after test substance treatment compared to controls

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 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

Phenol, styrenated (LS 500) has been tested for genetic toxicity in two in vitro and one in vivo mutagenicity tests (in vitro bacterial reverse mutation test (Ames), in vitro mammalian cell gene mutation test and in vivo mammalian somatic cell erythrocyte micronucleus test). In addition, there are two in vitro bacterial reverse mutation tests with the supporting substance phenol, methylstyrenated (LA 300, Necires EPX-L). All tests were performed under GLP according to EU/OECD test guidelines. Reliability is 1.

Bacterial reverse mutation assay (Ames test)

Phenol, styrenated (LS 500) was tested in a bacterial reverse mutation assay according to OECD TG 471 at concentrations between ≥ 1.0 and ≤ 1000 µg/plate in two independent experiments with tester strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, and TA 1537 with and without metabolic activation. Cytotoxicity was observed at concentrations ≥ 31 µg/plate without metabolic activation and ≥ 125 µg/plate with metabolic activation, depending on tester strain. Biologically relevant, dose-related increases in revertant colonies were only found in tester strain TA 100 at 100 µg/plate without S9 mix and at 316 µg/plate under metabolic activation (1st exp.) and at 62.5 and 125 µg/plate without S9 and at 250 µg/plate with S9 (2nd exp.). A maximum mutation factor of 3 was reached in the presence of S9. The mutagenic doses in the absence or presence of metabolic activation were cytotoxic as well (Kraft/BSL 2007).

Phenol, methylstyrenated (LA 300 and Necires EPX-L) was tested in similar assays (OECD TG 471). LA 300 was tested with the same five tester strains as LS 500 at concentrations between ≥ 10 and ≤ 5000 µg/plate with and without metabolic activation. At concentrations ≥ 2000 µg/plate with metabolic activation, LA 300 induced a dose-related increase in revertant colonies in tester strain TA 100 (maximum mutation factor 2.8 at 4000 µg/plate). Cytotoxicity was observed at concentrations ≥ 1000 µg/plate (LA 300_Kraft/BSL 2007).

Necires EPX-L was tested with only 4 strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, and TA 1537) at concentrations between ≥ 3 and ≤ 5000 µg/plate with and without metabolic activation. No genotoxicity was observed at any test concentration with and without metabolic activation. Slight cytotoxicity was noted in tester strain TA 100 at 3330 and 5000 µg/plate with metabolic activation (Nec EPX_van de Waart/NOTOX 1994).

Data indicate, that the mutagenic potential as well as the cytotoxicity of the supporting substance phenol, methylstyrenated is somewhat lower compared to phenol, styrenated.

Phenol, styrenated (LS 500) was demonstrated to be weakly mutagenic in one strain (TA 100) out of the five strains tested in the in vitro bacterial reverse mutation assay (Kraft/BSL 2007). Overall, results of the bacterial reverse mutation assays are assessed to be not clearly positive but ambiguous.

In vitro mammalian cell gene mutation test

In an in vitro mammalian cell gene mutation test with mouse lymphoma cells according to OECD TG 476, phenol styrenated (LS 500) was tested in parallel cultures at concentrations between 2.5 and 40.0 µg/mL with and without metabolic activation (S9 mix) in two independent experiments. Relevant cytotoxic effects were observed at concentrations ≥ 20 µg/mL after 4 h treatment with and without metabolic activation and at concentrations ≥ 5 mg/mL after 24 h treatment without metabolic activation. No reproducible dose dependent increase of mutation frequencies was observed in any of the experiments conducted up to the highest dose with and without metabolic activation. Phenol, styrenated (LS 500) was demonstrated not to induce mutations in mouse lymphoma cells under the conditions of the test (Wollny/Harlan; 2010).

In vivo mammalian erythrocyte micronucleus test

In an in vivo micronucleus test with polychromatic erythrocytes from mouse bone marrow according to OECD TG 474, phenol, styrenated (LS 500) was tested using three concentrations (250, 500, and 1000 mg/kg bw). 1000 mg/kg bw was the highest dose which could be applied in a range finding test without mortality. In the main test, no apparent signs of toxicity were observed. In addition, the ratio between polychromatic and normochromatic erythrocytes was not altered in the treatment groups compared to controls indicating that the test substance had no cytotoxic effects on bone marrow. There was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei in comparison to the corresponding vehicle controls at any dose level and preparation interval after administration of the test item. The mean values of micronuclei observed after treatment with test substance were below the value of the vehicle control group. Additionally, all values were within the historical vehicle control database. Under the conditions of the tests conducted, phenol styrenated /LS 500) did not induce cytogenetic damage to bone marrow erythroblasts (Merker/Harlan; 2010).

Conclusion

In bacterial reverse mutation tests, phenol, styrenated (LS 500) showed a weakly mutagenic response. A slight mutagenic effect was demonstrated only for one of five tester strains (TA 100) with and without metabolic activation at concentrations being already notably cytotoxic. Maximum mutation factor was 3. The result has been graded as ambiguous.

Genotoxicity of phenol, styrenated (LS 500) was further investigated with an in vitro gene mutation test in mammalian cells and with an in vivo micronucleus test with mouse bone marrow. In both mammalian cell tests, genotoxicity was tested negative. With these two tests, information requirement on structural and numerical chromosome aberrations and gene mutations according to REACH Annex VIII, sections 8.4.2 and 8.4.3 are covered. In accordance with Column 2 of Annex VIII, section 8.4.2, the in vitro micronucleus study is substituted by an available in vivo micronucleus test. The data available are sufficient to evaluate the mutagenic potential of phenol, styrenated (LS 500).

As in both tests with mammalian cells results are negative, phenol, styrenated is assessed to be not mutagenic in mammalian somatic cells.

Justification for classification or non-classification

Even having a weakly mutagenic response in one strain (TA 100) out of five in a bacterial reverse mutation assay, phenol, styrenated did not show any mutagenic/genotoxic activity in mammalian cell systems (in vitro gene mutation study and in vivo mammalian somatic cell erythrocyte micronucleus study) (see under discussion). According to Regulation (EC) No 1272/2008 (CLP regulation), classification of phenol, styrenated as germ cell mutagenic is not warranted.