Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro: Ames: negative, Evonik, 1571/0020, 2009, OECD 471
HPRT: negative, BASF AG, 1290609, 2010, OECD 476
MNT: negative, BASF SE, 33M0015/08M018, 2014, OECD487

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 21st July 1997
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinwiesen 19, Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I: without S9-mix: 0.16 - 2.5 µg/mL; with S9-mix: 39.1 - 1250 µg/mL
Experiment II: without S9-mix: 1.3 - 20 µg/mL; with S9-mix: 40 - 640 µg/mL
Experiment III: without S9.mxi: 2.5 - 30 µg/mL; with S9-mix: 40 -640 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 15 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: two parallel cultures

NUMBER OF CELLS EVALUATED: at least 50 colonies

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6 – 31.7 mutants per 1000000 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cloning efficiancy of less than 50 % in both parallel cultures occurred in experiment I at 2.5 μg/mL without metabolic activation (4 hours treatment). In experiment II relevant toxic effects as described above occurred at 20 μg/mL in the absence of metabo
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Phase separation: found at 10µg/mL in 4 hour Experiement without S9-mix and at 80 µg/mL in 4 hour experiment with S9-mix

RANGE-FINDING/SCREENING STUDIES: Relevant toxic effect occurred at 10 μg/mL and above without metabolic activation after 4 hours of treatment. In the presence of metabolic activation (4 h treatment) the cell growth was completely inhibited at 1084.8 μg/mL and above. Following 24 hours treatment a clear toxic effect was determined at the maximum concentration of 40 μg/mL.
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 2169.5 and 4339 μg/mL in the presence of metabolic activation (4 hours treatment).


ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects as indicated by a relative cloning efficiency I (CE I) of less than 50 % in both parallel cultures occurred in experiment I at 2.5 μg/mL without metabolic activation (4 hours treatment). In experiment II relevant toxic effects as described above occurred at 20 μg/mL in the absence of metabolic activation and at 320 μg/mL and above with metabolic activation. In experiment III cytotoxic effects were determined at 30 μg/mL and above without and 320 μg/mL and above with metabolic activation. The substantial variability of the cytotoxicity with metabolic activation is based on phase separation phenomena. If a clear separation in two layers occurs the cells adhering to a wall of the culturing flasks do not get into contact with the upper layer at all. However if an emulsion is formed the cells get into contact with both phases and toxic effects are likely to occur. The protein and lipid content of the S9 fraction used in the experimental parts with metabolic activation tends to stabilize emulsions acting like a detergent.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

No relevant and reproducible increase in mutant colony numbers/1000000 cells was observed in the main experiments up to the maximal concentration. The induction factor of three times the corresponding solvent control was exceeded in experiment III, culture II with metabolic activation at almost all of the concentrations. However, this effect was not considered biologically relevant since it was not reproduced in the parallel culture performed under identical experimental conditions and was not dose dependent as indicated by the lacking statistical significance. In fact, this increase is based on the low corresponding solvent control of just 5.0 colonies per 1000000 cells. All of the mutation frequency values remained within the historical range of solvent controls. An isolated but substantial increase of the mutation frequency was noted in the second culture of the second experiment at 20 μg/mL. However, no comparable increase occurred in the parallel culture under identical conditions. Still, the increase was statistically relevant so, the experiment was repeated to verify this increase. No increase of the mutation frequency was observed in the repeat experiment though the cytotoxic range of approximately 10-20% relative cloning efficiency I was covered.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in the second culture of the first experiment with metabolic activation. Since the mutation frequency did not exceed the threshold of three times the corresponding solvent control the statistical result was considered as biologically irrelevant. Another significant trend was detected in the second culture of the second experiment without metabolic activation. This trend was relevant since the mutation frequency exceeded the threshold as well and resulted in an additional verifying experiment. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was also detected in the first culture of the first experiment with metabolic activation and in the second culture of the second experiment with metabolic activation. These trends however, were judged as irrelevant since both were reciprocal, going down versus increasing concentrations.

In the main experiments (with and without S9 mix) the range of the solvent controls was from 5.0 up to 31.0 mutants per 1000000 cells; the range of the groups treated with the test item was from 0.0 up to 453.1 mutants per 1000000 cells.

EMS (0.15 mg/mL in experiment I and II, and 0.075 mg/mL in experiment III) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD487 (MNT)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The V79 cell line (a permanent cell line derived from the Chinese hamster) was used in the experiment
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL), 1% (v/v) amphotericine B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (rats)
Test concentrations with justification for top dose:
1st Experiment:
4 hours exposure, 24 hours harvest time, without S9 mix:
0; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix:
0; 50.00; 100.00; 200.00; 1 250.00; 2 500.00; 5 000.00 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix
0; 25.00; 50.00; 100.00; 200.00; 400.00; 800.00 μg/mL
3rd Experiment
4 hours exposure, 24 hours harvest time, with S9 mix:
0; 25.00; 50.00; 100.00; 200.00; 400.00; 800.00 μg/mL
Vehicle / solvent:
Due to the insolubility of the test substance in water, acetone was selected as vehicle, which had been demonstrated to be suitable in the in vitro mircronucleus test and for which historical control data are available.
The final concentration of the vehicle acetone in culture medium was 1% (v/v)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: ethyl methanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Experiment 1 and 3: 4 h exposure, 20 h recovery
- Experiment 2: 24 h exposure, no recovery without S9 or 4 h exposure, 40 h recovery with S9 mix
In Experiment I and III, CytB was added for the recovery period; in experiment II without S9, the the actin polymerisation inhibitor CytB was added immediately to the treatment medium and in Experiment II with S9 it was added 24 h before harvest

Just before preparation the culture medium was completely removed. Single cell suspensions were prepared from each test group by trypsination. Then, the cell numbers per flask of each single cell suspension were determined using a cell counter. Subsequently, 5x104 cells per slide were centrifuged at 1.400 rpm for 7 minutes onto labelled slides using a Cytospin centrifuge. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.

DNA was stained using DAPI, cytoplasm was stained with PI


NUMBER OF CELLS EVALUATED: 1000 cells per culture using duplicates for each condition -> 2000 cells

DETERMINATION OF CYTOTOXICITY
- Method: cells were counted after trypsination before they were spun on slides; equal numbers were then put on slides


OTHER:
- At the end of the treatment period, the cultures of all test groups were examined microscopically with regard to cell morphology, which is a further indication for cytotoxicity
- The cytokinesis-block proliferation index (CBPI; a direct measure of the proliferative activity of the cells) was determined in at least 1 000 cells per culture (at least 2 000 cells per test group)
- The pH value was measured at least for the vehicle and Top Dose with and without S9
- Osmolarity was measured at least for the top dose and for the vehicle control with and without S9 mix
Evaluation criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data
• The positive control substances both with and without S9 mix induced a distinct increase in the number of micronucleated cells .

A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the test groups is not distinctly increased above the concurrent vehicle control and is within our laboratory’s historical negative control data range.
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
Species / strain:
Chinese hamster lung fibroblasts (V79)
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 influenced
- Effects of osmolality: not influenced
- Precipitation: observed from 100 µg/ml (Experiment I and II) or 200 µg/ml (Experiment III)
- Cell morphology: In the absence of metabolic activation cell attachment/morphology was adversely influenced (grade > 2) at 200 μg/mL in the 1st Experiment and at 25 μg/mL and above in the 2nd Experiment. In all experiments in the presence of metabolic activation test substance treatment did not lead to adverse effects on cell attachment/morphology.
- Other confounding effects: no clearly reduced proliferative activity of above 50% cytostasis was observed after 4 and 24 h exposure


COMPARISON WITH HISTORICAL CONTROL DATA: The positive control data are within the historical control data; dIn all experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.2 – 0.8% micronucleated cells) were close to the concurrent vehicle control values (0.2 – 0.6% micronucleated cells) and clearly within our historical negative control data range (0.1 - 1.8% micronucleated cells)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix clearly reduced cell numbers were obtained from 50 μg/mL onward (38.9% of control) after 4 hours exposure in the 1st Experiment and from 25 μg/mL onward (32.3% of control) after 24 hours exposure in the 2nd Experiment. However, in the 2nd Experiment the concentration of 25 μg/mL was not scorable for the occurrence of micronucleated cells due to strong cytotoxicity. Besides, in the presence of S9 mix the cell numbers were clearly reduced after 4 hours exposure from 1 250 μg/mL onward (33.5% of control) at 24 hours preparation interval in the 1st Experiment, from 400 μg/mL onward (15.0% of control) at 44 hours preparation interval in the 2nd Experiment and at 800 μg/mL (41.0% of control) at 24 hours preparation interval in the 3rd Experiment, respectively. However, these test groups at 24 hours preparation interval were not scorable for the occurrence of micronucleated cells due to strong cytotoxicity.

PROLIFERATION INDEX:
In this study, clearly reduced proliferative activity of above 50% cytostasis was only observed in the 2nd Experiment with metabolic activation at 400.0 μg/mL.
However, in the absence of S9 mix slides could not be evaluated due to strongly reduced proliferation in the 1st Experiment at 200 μg/mL and in the 2nd Experiment at 25 μg/mL. In addition, in the presence of S9 mix non-scorable slides were obtained in the 1st Experiment at 1 250 μg/mL and in the 3rd Experiment at 800 μg/mL.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The exact numbers of micronucleated cells as well as on cytotoxicity are summarized in the attached documents

Conclusions:
Interpretation of results (migrated information):
negative

The ability of the test substance to induce mircronuclei in V79 cells in vitro with and without the addition of liver S9 mix was determined according to OECD Test guideline 487 and GLP. At least 1000 cells per condition were analyzed for micronuclei.
The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.
Cytotoxicity indicated by clearly reduced cell count was observed at least at the highest applied test substance concentration in all experimental parts of this study. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.
Thus, under the experimental conditions described, Oxooel 9N is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his-/trp-gene
Species / strain / cell type:
other: Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/ß-naphthoflavone induced rat liver microsomes
Test concentrations with justification for top dose:
50 - 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see: Details on test system and conditions
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

NUMBER OF REPLICATIONS: in triplicate

Positive controls:

without S9-mix:
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA;
9-Aminoacridine (9AA):80 µg/plate for TA1537;
4-Nitroquinoline-1-oxide (4NQO): 0.2 µg/plate for TA98

with S9-mix:
2-Aminoanthracene (2AA): 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA;
Benzo(a)pyrene (BP): 5 µg/plate for TA98;

Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 10^9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolie activation. Biological relevance of the results will be considered first, statistical methods, can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive er negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Species / strain:
other: Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(precipitate was observed at and above 1500 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A fine, particulate precipitate was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

 1st experiment, Standard plate test (50 - 5000 µg/plate)
Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 22 1 (1500) no  negative 9.5 (4NQO)
  yes 28 1.1 (150) no  negative 8.8 (BP)
TA 100 no 102 0.97 (150) no  negative 6.1 (ENNG)
  yes 106 1.1 (150/1500) no  negative 28.7 (2AA)
TA 1537 no 13 0.85 (150) no  negative 75.6 (9AA)
  yes 10 1.2 (1500) no  negative 47.3 (2AA)
TA 1535 no 22 1.1 (5000) no  negative 49.2 (ENNG)
  yes 10 1.2 (5000) no  negative 17 (2AA)
WP2uvrA no 30 1 (150/1500) no  negative 55.1 (ENNG)
  yes 44 1 (50/500/5000) no  negative 13.4 (2AA)
2nd experiment, Standard plate test (50 - 5000 µg/plate)
Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 23 0.96 (22) no negative 9.5 (4NQO)
  yes 26 1.1 (150) no negative 7.5 (BP)
TA 100 no 106 1 (150/5000) no negative 11.6 (ENNG)
  yes 101 0.99 (50) no negative 31.5 (2AA)
TA 1537 no 13 1.1 (500) no negative 94.1 (9AA)
  yes 14 1 (500) no negative 34.9 (2AA)
TA 1535 no 25 1.1 (500) no negative 66.5 (ENNG)
  yes 11 1.1 (150/500) no negative 21.3 (2AA)
WP2uvrA no 27 1 (150/5000) no negative 79.6 (ENNG)
  yes 38 1 (50) no negative 14.4 (2AA)

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine; 9AA: 9-Aminoacridine; 4NQO: 4-Nitroquinoline-1-oxide; 2AA: 2-Aminoanthracene; BPBenzo(a)pyrene;
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Valid experimental data were available to assess the genetic toxicity of Octene (hydroformylation products, high-boiling) in vitro.

in vitro:

Gene mutation in bacteria:

The substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci in Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98, and E. coli WP2uvrA- at concentrations ranging from 50 to 5000 µg/plate in the Ames test according to OECD Guideline 471 (Evonik, 1571/0020, 2009). The assay was performed using the Standard plate test with and without metabolic activation (phenobarbitone/ß-naphthoflavone induced rat liver microsomes), respectively. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A fine, particulate precipitate was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. This result is supported by another study similar to OECD Guideline 407 which shows similar results.

Gene mutation in mammalian cells:

A valid study according to OECD Guideline 476 was done by BASF in 2010. In this study, three independent experiments were performed, using two parallel cultures each. The first main experiment was performed with and without liver microsomal 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. To verify the results of the second experiment a third experiment was performed under identical general conditions with and without metabolic activation and adjusted concentrations. The tested concentrations were between 0.16 and 640 µg/mL. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In vitro Micronucleus test:

The ability of the test substance to induce mircronuclei in V79 cells in vitro (clastogenic or aneugenic activity) was determined according to OECD Test guideline 487 and GLP (BASF SE, 2014). Three independent experiments with duplicates for each condition were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). At least 1000 cells per condition were analyzed for micronuclei.

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

Cytotoxicity indicated by clearly reduced cell count was observed at least at the highest applied test substance concentration in all experimental parts of this study.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, Oxooel 9N is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008.