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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ (NAF-AO) [EC no. 701-299-7] (see Chapter 1) was not mutagenic when tested in vitro in bacterial reverse mutation assays. Furthermore, data from an in vitro mammalian cell gene mutation test and an in vitro mammalian chromosome aberration assay provided only results indicating that the substance was negative for genotoxicity. Overall, genetic toxicity in vitro was tested negative based on results from in vitro genotoxicity studies mandatory under REACH.

Link to relevant study records

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. - 10 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 L 100; CAS-no. 71302-83-5 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- Lot/batch No.: 24106
- 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 rat livers (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 and 2nd experiment: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (all strains, +/-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
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-p-phenylenediamine: TA 98, TA 1537 without S9; 2-aminoanthracene: all strains with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (1st experiment: plate incorporation); 2nd experiment: preincubation 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. 20).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance 'Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised' (Novares L 100) at any concentration level, neither in the presence nor in the absence of metabolic activation. All reference mutagens induced distinct increases of revertant colonies indicating the validity of the experiment. The test substance was shown to be not mutagenic in this bacterial reverse mutation assay.
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 L 800; CAS-no. 71302-83-5 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- Lot/batch No.: 21633
- 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 rat livers (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 and 2nd experiment: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (all strains, +/-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:
sodium azide
methylmethanesulfonate
other: 4-nitro-p-phenylenediamine: TA 98, TA 1537 without S9; 2-aminoanthracene: all strains with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (1st experiment: plate incorporation); 2nd experiment: preincubation 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. 20).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
except in Exp. 2 at 5000 µg/pl without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
except in Exp. 2 at 5000 µg/pl without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance 'Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised' (Novares L 800) at any concentration level, neither in the presence nor in the absence of metabolic activation. All reference mutagens induced distinct increases of revertant colonies indicating the validity of the experiment. The test substance was shown to be not mutagenic in this bacterial reverse mutation assay.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20/10/2009 to 27/11/2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
plate incorporation 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 TL 10; CAS-no. 71302-83-5 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- Lot/batch No.: 28724
- 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 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
microsomal fraction prepared from rat livers (male Wistar rats) induced with Delor 106 (PBC mixture, 500 mg/kg i.p); 30 µL/plate
Test concentrations with justification for top dose:
1st test series: 50, 150, 500, 1500, and 5000 µg/plate
2nd test series: 5, 15, 50, 150, and 500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: N,N-dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: complete solubility
Untreated negative controls:
yes
Remarks:
no solvent, tested in TA 100 within the series of toxicity testing
Negative solvent / vehicle controls:
yes
Remarks:
N,N-dimethylformamide
Positive controls:
yes
Positive control substance:
other: see field below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 - 72 h at 37 ± 1 °C

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth ≥ bacterial background lawn

POSITIVE CONTROLS
4-Nitro-o-phenylenediamine (20 µg/plate): TA 98 without metabolic activation;
Sodium azide (20 µg/plate): TA 100 and TA 1535 without metabolic activation;
9-Aminoacridine hydrochloride monohydrate (100 µg/plate): TA 1537 without metabolic activation;
N-methyl-N´-nitro-N-nitrosoguanidine (20 µg/plate): E. coli without metabolic activation;
2-Aminofluorene (10 µg/plate): TA 98 and TA 100 with metabolic activation;
2-Aminoanthracene (1.0, 2.5, 25 µg/plate): TA 1535, TA 1537, E.coli with metabolic activation
Evaluation criteria:
The result is considered positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
A test substance is considered as mutagenic if
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

An increase is considered as „biologically relevant“,
- if in the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversions of >10;
- if in the number of reversions is at least three times as high as that of the solvent control for the strains having spontaneous reversions of ≤ 10.

A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response in any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
According to OECD Guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
The test substance 'Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised' (Novares TL 10) did not cause significant increases in revertant colony numbers of any of the five tester strains over controls thus showing that the test substance is not mutagenic in this bacterial reverse mutation assays.
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; CAS-no. 71302-83-5 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- Lot/batch No.: 28724
- Expiration date of the lot/batch: 2011-09-30
- 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-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 µg/mL
with S9 mix: 19.5; 39.1; 78.1; 156.3; 312.5; 626.0 µg/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: experiment 1: 4 hours with and without metabolic activation; experiment 2: 24 hours without metaoblic activation and 4 hours with metabolic activation
- 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: >1,5E+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 10E+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 10E+6 cells; the range of the groups treated with the test item was from 112 up to 310 mutant colonies per 10E+6 cells. The highest solvent control value (200, 172, and 198 mutant colonies per 10E+6 cells) exceeded the recommended 50 - 170 x 10E+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 10E+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 (EC no. 615 -276 -3) 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 SYSTAT11 statistics 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 10E+6 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 10E+6 cells; the range of the groups treated with the test item was from 112 up to 310 mutant colonies per 10E+6 cells. The highest solvent control value (200, 172, and 198 mutant colonies per 10E+6 cells) exceeded the recommended 50 - 170E+6 control 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 10E+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.

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:
in vitro mammalian chromosome aberration test
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 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- 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):
MEDIA USED
- Type and composition 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:
cyclophosphamide
Remarks:
with metabolic activiation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activiation
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).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
except experiment II without S9 at 32.5 µg/L
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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 (EC no. 615 -276 -3)

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 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 (EC no. 701 -299 -7), 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 vivo genetic toxicity testing is not required, because in all tests on in vitro genetic toxicity (see above) negative results were observed.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic and germ cell study: gene mutation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genotoxic potency of NAF-AO was tested in vitro in three bacterial reverse mutation assays (Ames tests), in an in vitro mammalian gene mutation study, and in an in vitro chromosomal aberration study.

Bacterial reverse mutation assays

Three bacterial reverse mutation assays (Ames, OECD 471) were conducted according to GLP standards using S. typhimurium and E. coli strains with and without metabolic activation and concentrations up to 5000 µg/plate. No mutagenicity was noted.

Mammalian cell gene mutation test

An in-vitro mammalian cell gene mutation test (OECD 476) was conducted according to GLP standards using mouse lymphoma cells with and without the presence of metabolic activation. NAF-AO did not induce gene mutations at any concentration.

Mammalian chromosome aberration test

An in-vitro mammalian chromosome aberration test (OECD 473) was conducted according to GLP standards using human lymphocytes with and without the presence of metabolic activation. NAF-AO did not induce chromosomal aberration at any concentration.

All study results from three different in vitro genotoxicity tests did not provide any evidence of a positive genotoxic response. The substance did not produce any adverse effects in in vitro genotoxicity studies mandatory according to REACH regulation Annex VII and VIII.

Therefore, in vivo genotoxicity testing can be waived.

Justification for classification or non-classification

The substance NAF-AO did not provide any positive response when tested in three different types of in vitro mutation/genotoxicity assays (see above). Overall, no adverse effects were observed in all the genotoxicity tests performed. Thus, classification according to Regulation (EC) No 1272/2008 (CLP regulation) is not required.