Oxazolidine, 3-butyl-2-(1-ethylpentyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Incozol 2 did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic (negative for genetic toxicity) to human lymphocytes in vitro.

It was concluded that Incozol 2 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed in this study, which included treatment at concentrations up to either 5000 µg/plate or the lower limit of the toxic range, both in the absence and in the presence of a rat liver metabolic activation system (S-9).

According to REACH Regulation 1907/2006/EEC Article 22 information on in vitro gene mutation study in mammalian cells is not required. The submitted registration dossier is an update following a decision made by the MSCA according to Article 16(1) of Directive 67/548/EEC. Information on this endpoint was not required at the time of notification.

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:

1995-11-10 to 1995-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1993

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base-pair substitution (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537).

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA102)
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254 induced)

Test concentrations with justification for top dose:

Experiment 1
with and without S9 mix: 8, 40, 200, 1000, 5000 µg/plate

Experiment 2:
without S9 mix: 1000, 2000, 3000, 4000, 5000 µg/plate
with S9 mix (strains TA98, TA1537, TA102): 6.25, 12.5, 25, 50, 100 µg/plate
with S9 mix (TA100): 3.125, 6.25, 12.5, 50 µg/plate
with S9 mix (TA1535): 12.5, 25, 50, 100, 200 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: analytical grade Dimethylformamide (DMF)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Glutaraldehyde (GLU)

Remarks:

TA102 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (AAN)

Remarks:

TA98, TA100, TA1535 with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
For the range-finding study and Experiment 1 a plate incorporation was used. Experiment 2 employed a pre-incubation step in addition to the plate incorporation.

DURATION
- Preincubation period: 1h
- Exposure duration: 3 days
- Expression time (cells in growth medium): 10 h

AMES-TEST
Incozol 2 was tested for mutation in Five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) using triplicate plates without and with S9. Negative (solvent) controls were included in both assays, in quintuplicate without and with S9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S9. The activity of the S9 mix used in each experiment was confirmed by AAN treatments (again in triplicate) of at least one strain in the presence of S9. Platings were achieved as described for the range-finding below.

As the results of the first experiment were negative, treatments in the presence of S9 in Experiment 2 included a pre-incubation step, where the quantities of test article or control solution, were mixed together and incubated for 1 hour at 37°C, before the addition of 2.5 mL molten agar at 46°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Following initial Experiment 2 treatments of all the test strains in the presence of S9, a high degree of toxicity was observed, extending over all the test doses. As all test doses were affected by toxicity, these data were not considered acceptable for mutagenicity assessment. These treatments in the presence of S9 were therefore repeated using revised test doses, along with concurrent diagnostic control treatments in the absence of S9. Evidence of toxicity was again observed following these repeat treatments, extending over several test doses in each test strain. Although this toxicity was less severe than that observed following the initial treatments, sufficient treatment doses were again affected to consider the data unsuitable for mutagenicity assessment. Subsequent repeat treatments and diagnostic controls were therefore performed, with maximum test doses further reduced, and the results of these treatments are those presented as the mutagenicity data for Experiment 2 in the presence of S9.

DETERMINATION OF CYTOTOXICITY AND RANGE-FINDING
- Method: Incozol 2 was tested for toxicity in strain TA100, at the concentrations 8, 40, 200, 1000, 5000 µg/plate. Triplicate plates without and with S9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate respectively without and with S9 mix. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46°C:

0.1 mL bacterial culture
0.1 mL test article solution or control
0.5 mL 10% S9 mix or buffer solution

followed by rapid mixing and pouring on to Minimal Davis agar plates. When set, the plates were inverted and incubated at 37°C in the dark for 3 day. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

Evaluation criteria:

The test article was considered to be mutagenic if:

1) the assay was valid

2) Dunnett's test gave a significant response (p < 0.01), and the data set showed a significant dose-correlation

3) the positive responses described in 2) were reproducible.

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

This was observed in Experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

This was observed in Experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

This was observed in Experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

This was observed in Experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

This was observed in Experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of Incozol 2 at 8-5000 µg/plate, plus negative and positive controls. Following these treatments, no evidence of toxicity was observed, as would normally be indicated by a thinning of the background bacterial lawn, and these results were therefore considered acceptable for use as the TA100 mutagenicity data for Experiment 1. The remaining test strains were treated in Experiment 1 using the same dose range as in the range-finder experiment. As with the range-finder experiment, no evidence of toxicity was observed following any of these test treatments.

COMPARISON WITH HISTORICAL CONTROL DATA: This was done with the solvent controls, which fell within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The increased toxicity that was observed following Experiment 2 treatments in the presence of S9, compared to the other test treatments, was attributed to the conditions occurring during the pre-incubation step (all other treatments employed a plate-incorporation methodology). As volumes of test agent and bacterial cells are pre-incubated prior to the addition of soft agar, and plating out onto minimal agar plates, the effective concentration of test agent that the bacterial cells are exposed to over the pre-incubation period is increased. In this case, this appears to have resulted in toxic effects which were not manifest when using the same treatment doses under plate-incorporation conditions.

Conclusions:

It was concluded that Incozol 2 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed in this study, which included treatment at concentrations up to either 5000 µg/plate or the lower limit of the toxic range, both in the absence and in the presence of a rat liver metabolic activation system (S9).

Executive summary:

Incozol 2 was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9), in two separate experiments. An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of Incozol 2 at 8-5000 µg/plate, plus solvent and positive controls. Following these treatments, and those of Experiment 1 (which were performed using the same test doses), no evidence of toxicity was observed, as would normally be indicated by a thinning of the background bacterial lawn. Experiment 2 treatments in the absence of S9 were performed up to 5000 µg/plate, and following toxicity resulting in repeat treatments, up to estimates of the lower limit of toxicity in the presence of S9. Experiment 2 treatments were all performed using narrowed dose ranges, in order to more closely investigate those doses of Incozol 2 considered most likely to induce any mutagenic activity. In addition, Experiment 2 treatments in the presence of S9 also employed a pre-incubation step, in order to increase the range of mutagenic chemicals that can be detected using this assay system. No evidence of toxicity was observed following Experiment 2 treatments in the absence of S9. The initial and first repeat treatments of Experiment 2 in the presence of S9 resulted in toxicity at multiple test doses, rendering the data unsuitable for valid mutagenicity assessment. The second repeat treatments produced evidence of toxicity at only the highest one or two test doses, and these results were therefore used as the mutagenicity data for these treatments. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. Incozol 2 treatments of all the test strains, either in the absence or in the presence of S9, produced no increases in revertant numbers that were sufficient to be considered as evidence of mutagenic activity. It was concluded that Incozol 2 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed in this study, which included treatment at concentrations up to either 5000 µg/plate or the lower limit of the toxic range, both in the absence and in the presence of a rat liver metabolic activation system (S9) (Corning Hazleton, 1996).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-04-18 to 2005-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 21st 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

May 19th 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM)
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix ( phenobarbitone and ß-naphthoflavone induced)

Test concentrations with justification for top dose:

Experiment 1
without S9; 4 h treatment, 20 h in treatment free media
0*, 35.47, 70.94*, 141.88*, 283.75*, 567.5, 851.25 µg/mL
with S9; 4 h treatment, 20 h in treatment free media
0*, 17.74, 35.47, 70.94, 141.88*, 212.82*, 283.75* µg/mL

Experiment 2
without S9; 24 h continous exposure
0*, 17.74, 35.47, 70.94*, 106.41*, 141.88, 212.82* µg/mL
with S9; 4 h treatment, 20 h in treatment free media
0*, 35.47, 70.94*, 141.88*, 283.75*, 567.5*, 1135 µg/mL

* Dose levels seleceted for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time: 20 h
- Fixation time: 28 h

SPINDLE INHIBITOR: Colcemid
STAIN: 5% Gurrs Giemsa

NUMBER OF REPLICATIONS: Cultures were prepared in duplicates

NUMBER OF CELLS EVALUATED: 2000 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: no

CHROMOSOME ABERRATION ASSAY
Experiment 1: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were scorable metaphases present up to 283.75 µg/mL in both the absence and presence of metabolic activation (S9). There was no precipitate expected at the dose levels tested, based on observations in the Preliminary Toxicity Test. The mitotic index data confirms the qualitative observations in that a plateau of mitotic index inhibition and an approximate 35% inhibition was observed at and above 35.47 µg/mL in the absence of S9. In the presence of S9 there was no reduction in mitotic index up to the maximum dose level tested, 283.75 µg/mL demonstrating that the test material was not as toxic as observed in the Preliminary Toxicity Test. However scoring proceeded as it was considered that there was a steep toxicity curve and the maximum dose level was close to the toxic limit as there were increases in mitotic index at the upper dose levels that may have been toxicity induced cell cycle delay. Increased concentrations were used for Experiment 2. The maximum dose level selected for metaphase analysis for the without-S9 group was based on toxicity and for the with-S9 group was based on the maximum dose level tested. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. Further, the test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Experiment 2: The qualitative assessment of the slides determined that there were scorable metaphases present at the maximum test material dose level of 212.82 µg/mL in the 24 hours continuous exposure in the absence of S9. In the presence of S9 the maximum dose level with scorable metaphases was 567.5 µg/mL. Precipitate observations were considered to be as those observed in the Preliminary Toxicity Test and therefore a precipitate would be expected at 1135 µg/mL only. The mitotic index data confirms the qualitative observations in that there were metaphases at 212.82 µg/mL and a mitotic index of 43% in the absence of S9. In the presence of S9 there was a mitotic index of 25% at 567.5 µg/mL but a plateau of toxicity observed at 70.94 µg/mL to 283.75 µg/mL, demonstrated by a mitotic index of 58%. The maximum dose level selected for metaphase analysis was based on toxicity and was 212.82 µg/mL for the without-S9 exposure group and 567.5 µg/mL for the with-S9 exposure group. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. Further, the test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test material used was 8.87 to 2270 µg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main study.

COMPARISON WITH HISTORICAL CONTROL DATA: This was done for the vehicle and positive controls using in-house historical aberration ranges. Both vehicle and positive controls were within the historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The preliminary range-finding test showed that the test material induced toxicity in all exposure groups. The selection of the maximum dose level for analysis for all exposure groups was therefore based on toxicity.

Conclusions:

Incozol 2 did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD TG 473 and EU Method B.10. The study design also meets the requirements of the UK Department of Health Guidelines for the Testing of Chemicals for Mutagenicity. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included dose levels that induced mitotic inhibition. Incozol 2 was considered to be non-clastogenic to human lymphocytes in vitro (Safepharm, 2006).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro Mammalian Chromosome Aberration test

An in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells was conducted according OECD TG 473 and EU Method B.10. The study design also meets the requirements of the UK Department of Health Guidelines for the Testing of Chemicals for Mutagenicity. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material was toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included dose levels that induced mitotic inhibition. Incozol 2 was considered to be non-clastogenic to human lymphocytes in vitro (Safepharm, 2006).

Reverse Mutation Assay using Bacteria ( Ames test)

Incozol 2 was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9), in two separate experiments. An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of Incozol 2 at 8-5000 µg/plate, plus solvent and positive controls. Following these treatments, and those of Experiment 1 (which were performed using the same test doses), no evidence of toxicity was observed, as would normally be indicated by a thinning of the background bacterial lawn. Experiment 2 treatments in the absence of S9 were performed up to 5000 µg/plate, and following toxicity resulting in repeat treatments, up to estimates of the lower limit of toxicity in the presence of S9. Experiment 2 treatments were all performed using narrowed dose ranges, in order to more closely investigate those doses of Incozol 2 considered most likely to induce any mutagenic activity. In addition, Experiment 2 treatments in the presence of S9 also employed a pre-incubation step, in order to increase the range of mutagenic chemicals that can be detected using this assay system. No evidence of toxicity was observed following Experiment 2 treatments in the absence of S9. The initial and first repeat treatments of Experiment 2 in the presence of S9 resulted in toxicity at multiple test doses, rendering the data unsuitable for valid mutagenicity assessment. The second repeat treatments produced evidence of toxicity at only the highest one or two test doses, and these results were therefore used as the mutagenicity data for these treatments. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. Incozol 2 treatments of all the test strains, either in the absence or in the presence of S9, produced no increases in revertant numbers that were sufficient to be considered as evidence of mutagenic activity. It was concluded that Incozol 2 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed in this study, which included treatment at concentrations up to either 5000 µg/plate or the lower limit of the toxic range, both in the absence and in the presence of a rat liver metabolic activation system (S9) (Corning Hazleton, 1996).

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 (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.