Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Incozol 4 did not induce a significant increase of structural or numerical chromosome aberrations in vitro on mammalian cells and was concluded to be non clastogenic in vitro. In addition, Incozol 4 was not mutagenic in gene mutation tests on bacteria (Ames test) and mammalian cells (MLA) both, in the absence and presence of metabolic activation. Incozol 4 is, therefore, considered to be not mutagenic in vitro, although a second Ames test revealed a positive result in the absence of metabolic activation, which is considered to be not relevant for hazard assessment.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-25 to 2012-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

tk+/- (thymidine kinase)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 mediumcontaining 25 mM HEPES and 2 g/L NaHCO3 and 0.3 g/L L-Glutamine
RPMI 5: 5 % (v/v) Horse serum (heat inactivated), 0.01 mL/mL Antibiotic-Antimycotic solution, 0.5 mg/mL Pluronic-F68, 0.2 mg/mL Pyruvic acid
RPMI 10: 10 % (v/v) Horse serum (heat inactivated), 0.01 mL/mL Antibiotic-Antimycotic solution, 0.5 mg/mL Pluronic-F68, 0.2 mg/mL Pyruvic acid
RPMI 20: 20 % (v/v) Horse serum (heat inactivated), 0.01 mL/mL Antibiotic-Antimycotic solution, 0.2 mg/mL Pyruvic acid
- 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 of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver (provided by Trinova Biochem GmbH) and cofactors

Test concentrations with justification for top dose:

Assay 1:
3-hour treatment, -S9: 100; 500; 1000; 1500; 2000; 2500 and 3000 μg/mL
3-hour treatment, +S9: 100; 500; 1000; 1500; 1750; 2000 and 2500 μg/mL
Assay 2:
24-hour treatment, -S9 Mix: 100; 200; 300; 400; 500; 600 and 750 μg/mL
3-hour treatment, +S9 Mix: 500; 1000; 1500; 1750; 2000 and 2500 μg/mL

Vehicle / solvent:

- Vehicle used: Abs. Ethanol
- Justification for choice of vehicle: The test item was solubile in the vehicle up to a concentration of 100 mg/mL. The vehicle has no deleterious or mutagenic effect on the test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Assay 1: 3 hours with and without metabolic activation; Assay 2: 3 hours with and 24 hours without metabolic activation
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 - 14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER: pH and Osmolality

Evaluation criteria:

Assay Acceptance Criteria
The assays are considered valid if all of the following criteria are met:
1. The mutant frequency in the negative (vehicle) control cultures fall within the normal range (above 50 - 170 mutants per 106 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency (PEviability) of the negative controls is within the range of 65 % to 120 % on Day 2 (at the end of the expression period).
4. At least four test concentrations are present, where the highest concentration produces 80 - 90 % toxicity, precipitation, or is 5 mg/mL, or the highest practical concentration.

Mutagenicity Criteria
The test item is considered to be mutagenic in this assay if all the following criteria are met:
1. The assay is valid.
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations.
3. The increases are reproducible between replicate cultures and between tests.
4. There is a significant dose-relationship as indicated by the adequate trend analysis.
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 1E-6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.

Statistics:

For statistical evaluation of mutation frequencies of the test item concentrations were tested on significant differences to the control values by Dunnett’s Test at the positive controls by 2 Sample t-Test by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance for Bartlett’s Test.

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

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
- Precipitation: yes

RANGE-FINDING STUDIES: Yes, the obtained survival data were used to choose the dose levels for the main assays with the aim of covering a concentration range from the maximum possible cytotoxicity to a low effect level.

COMPARISON WITH HISTORICAL CONTROL DATA: In the Assay 1 as well as in the Assay 2 the plating efficiencies of the negative and positive controls in the viability test (PEviability) as well as the mutation frequency of the current negative control were within the acceptable ranges and in accordance with the historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The examined concentrations covered the cytotoxicity range (based on the harmonised relative survival and/or relative total growth) from the 10 - 20 % cytotoxicity to no toxicity. To fulfill the cytotoxicity criterion of the assay, the test item was investigated beyond its limit of solubility under culture conditions.

Conclusions:

Under the conditions of this study, the test item Incozol 4 does not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.

Executive summary:

An in vitro mammalian cell gene mutation assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test item to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix).

The appropriate vehicle, absolute ethanol (Abs. Ethanol) was chosen based on the results of the preliminary solubility test. The test item solutions were prepared in Abs. Ethanol and diluted prior to treatment. Based on the available historical control database, this vehicle was compatible with the survival of the cells and the S9 activity. The test item was tested beyond its limit of solubility under culture conditions.

The concentrations applied in the Assay 1 were chosen according to the solubility and cytotoxicity results of the pre-experiments.

The following concentrations were investigated in the Assay 1:

3-hour treatment (-S9 Mix): 100; 500; 1000; 1500; 2000; 2500 and 3000 μg/mL;

3-hour treatment (+S9 Mix): 100; 500; 1000; 1500; 1750; 2000 and 2500 μg/mL.

Concentrations investigated in the Assay 2:

24-hour treatment (-S9 Mix): 100; 200; 300; 400; 500; 600 and 750 μg/mL;

3-hour treatment (+S9 Mix): 500; 1000; 1500; 1750; 2000 and 2500 μg/mL.

In the performed mutation assays the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x105/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) or diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments.

In the performed Assay 1 and Assay 2 in the absence and also in presence of S9 Mix dose-related toxicity of the test item was observed based on both, the harmonised relative survival (harmonised RS) and the relative total growth (RTG) values. The concentration levels of 2500 and 3000 μg/mL in the Assay 1 and the concentration level of 750 μg/mL in the Assay 2 in absence of exogenous metabolic activation were out of the analysable range (higher than 90 % cytotoxicity occurred at these concentration levels). Apart from this the guideline criteria for the number of analysable concentration levels (at least four) as well as the required cytotoxicity degree (10 - 20 % relative survival or relative total growth) at the maximum concentration (in absence of metabolic activation the maximum analyzable concentration) were fulfilled. In presence of metabolic activation the cytotoxicity caused by the highest examined concentration level was in the acceptable 80 - 90 % range in both assays.

Significant differences to the concurrent control were noted for some test concentrations (causing high toxicity down to the limit described in the guideline) in the first and second experiment (Dunnett’s Test, α= 0.05 or α= 0.01). No clear concentration-response was observed in either of the tests with and without metabolic activation. Apart from these findings the obtained mutation frequencies remained unequivocally well below the GEF criterion for positive response in all cases. The final conclusion was drawn from the results of the statistical and biological evaluation.

Under the conditions of this study, the test item does not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-01-28 to 1988-02-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 test strains were investigated

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium LT2:
TA1535 and TA 100 base pair substitution hisG 46
TA 1537 and TA 98 bear frameshift markers

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 100 and TA98 contain plasmid pKM 101-R factor ampicillin resistance

Metabolic activation:

with and without

Metabolic activation system:

S9 mix induced with Aroclor 1254

Test concentrations with justification for top dose:

The following doses per plate were evaluated in the first test:
With and without S9 mix: 20, 100, 500, 2500, 12500 µg per plate

Due to the substance's toxicity the doses chosen for the repeat tests were as following:
775, 1550, 3100, 6200, 12400 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In the case of DMSO, an "untreated" negative control was not used, because there is sufficient evidence from literature data (i.e. Maron and Ames, 1983) and from own experience (see chapter 9), that this solvent has no influence on the spontaneous mutant counts of the bacterial strains used.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Nitrofurantoin

Remarks:

TA100 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Remarks:

TA1535 and TA98 without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
This study was preformed using the plate incorporation method with and without S9 mix.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: Four plates per strain and dose, both with and without S9 mix.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn

Evaluation criteria:

The following criteria were used for the acceptance of an assay:
a) The negative controls have to be within the expected range, as defined by literature data (i.e. Maron and Ames, 1983) and the laboratories' own historical data.
b) The positive controls have to show sufficient effects as defined by the laboratories' experience.
c) Titer determinations must prove sufficient bacterial density in the suspension.

A reproducible and dose-related increase (ie. > twice the negative control count) in mutant counts for at least one strain is considered positive. Otherwise, the result is evaluated as negative.

Statistics:

No statistical analysis was performed with the test results in this study.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

from 500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

from 500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

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:

from 500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not examined
- Effects of osmolality: Not examined
- Evaporation from medium: No
- Water solubility: Insolube
- Precipitation: Not examined
- Other confounding effects: No

RANGE-FINDING/SCREENING STUDIES
The following doses per plate were evaluated in the first test:
With and without S9 mix: 20, 100, 500, 2500, 12500 µg per plate

COMPARISON WITH HISTORICAL CONTROL DATA
Historical negative and positive controls of experiments performed from July to December 1986 and January to June 1987 were used.
The results were within historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY
There was no indication of a bacteriotoxic effect for the the test item doses of up to and including 100 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Likewise an inhibition of growth was not noted. Higher doses had a bacteriotoxic effect, specific to strain, and could therefore only partly be used for evaluation up to and including 12500 µg per plate.

Additional Result Information :
One of the four strains used revealed without S9 mix a dose-related increase in mutant counts to double those of the negative control. This strain was TA 98.
This increase is to be regarded as a random result due to the low spontaneous rate of the strain. It must also be considered that the negative control with S9 mix was within the range of values for treatment groups without S9 mix, and therefore the increase without S9 mix can be seen as biologically insignificant.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene increased the mutant counts to well over double those of the negative controls, and so demonstrated the system's sensitivity and the activity of the S9 mix.

Conclusions:

The test item Incozol 4 was considered not to have mutagenic effects at evaluable doses up to 12500 μg per plate in any of the Salmonella typhimurium strains used.

Executive summary:

Incozol 4 was investigated in the Salmonella / microsome test for point - mutagenic effects in doses up to 12500 μg per plate on four Salmonella typhimurium LT2 mutants. These were the histidine - auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

Doses up to and including 100 ug per plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. At higher doses, the substance had a strain - specific bacteriotoxic effect. Nevertheless, this range could be used for evaluation purposes.

The positive controls sodium azide, nitrofurantoin, 4-nitro-l,2-phenylene-diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically - relevant increase in mutant colonies compared to the corresponding negative controls .

Evidence of mutagenic activity for Incozol 4 was not found. Neither a dose - related doubling nor a biologically relevant increase of the mutant count, in comparison with the negative controls, was observed.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-28 to 2009-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Testing Methods Concerning New Chemical Substances (Notification No. 1121002, PFSB, MHLW; No. 2 of November 13, 2003, MIB, METI; No. 031121002, EPB, MOE; dated November 21, 2003)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Notice on Standards Issued by Ministry of Labor Based on Provisions of Article 57-3, Paragraph 1 of Industrial Safety and Health Act (Notice No.77, MOL, dated September 1, 1988)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Partial Revision of Notice on Standards Issued by Ministry of Labor Based on Provisions of Article 57-3, Paragraph 1 of Industrial Safety and Health Act (Notice No.67, MOL, dated June 2, 1997)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium (base-pair change: TA100, TA1535) & (frame- shift: TA98, TA1537):All strains are his-(Histidin requirement), uvrB gen deletetd (UV sensitivity) and rfa membrane mutants (Crystal violet sensitivity)
Escherichia coli WP2uvrA-: This strain is uvrA (UV sensitivity) deleted and trp- (base-pair change, Tryptophan requirement).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: TA 100& TA98 ampicillin resistance +pKM101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: With phenobarbital and 5, 6-benzoflavone (Enzym inducing agents) and supplemented with Cofactor-I

Test concentrations with justification for top dose:

Dose finding Test:
With and without S9 mix: 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate

Main Test:
Without S9 mix: 156, 313, 625, 1250, 2500, 5000 µg/plate
With S9 mix: 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: According to information from the sponsor, the test substance is insoluble in water.
In the solvent selection test, the test substance was not dissolved or suspended at 50 mg/mL in sterilized distilled water, and the test substance was dissolved at 50 mg/mL in DMSO and at 100 mg/mL in acetone.
Increases in temperature, discoloration and foaming were not observed when the test substance was mixed with DMSO. According to these results, DMSO was selected as the solvent (negative control) for the test substance in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-AA)

Remarks:

All strains with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

TA100, TA98, WP2uvrA- without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

T1535 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
This study was preformed using the pre-incubation method with and without S9 mix.

DURATION
- Preincubation period: 20 minutes at 37 °C (pre-incubation)
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS:
Dose-finding test: 2 plates/dose
Main test : 2 plates/dose

NUMBER OF CELLS EVALUATED: Revertant colonies were measured using an automatic colony counter (Toyo sokki Co. Ltd., CA-11D) or manual counting.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn
Microbial growth inhibition: Background lawn of the bacterial cells that have amino-acid requirement was observed by a stereoscopic microscope (Nikon, SMZ-10), and microbial growth inhibition was judged by the relationship between the test substance treated plates and the negative (solvent) control plate.

OTHER EXAMINATIONS:
- Sterility Test: In the dose-finding test and main test, bacterial contamination was examined using one plate for each of the highest dose of the test substance solution and S9 mix.
- Other: Observations
Precipitation: Precipitation was judged by observation of the plate surface macroscopically.

Evaluation criteria:

The dose-finding test and the main test were accepted as valid if all the following criteria were satisfied.
(1) The negative (solvent) control values (mean) and the positive control values (mean) are within the proper ranges calculated based on the historical data at our laboratory.
(2) The positive control values (mean) show clear positive responses in the respective tester strains, as evidenced by the number of revertant colonies being greater than 2-fold of the respective negative (solvent) control value (mean).
(3) There are more than 4 doses showing no microbial growth inhibition and more than 5 doses applicable to the evaluation.
(4) The result of the sterility test indicates that there is no bacterial contamination.
(5) There are no plates that became invalid for measurement due to contamination or other unexpected situations.

Test substance was judged to have mutagenicity (positive) when the test substance induced a dose-dependent increase in the number of the revertant colonies (mean) to a level equal to or greater than 2-fold of the negative (solvent) control value (mean value) in any one of the tester strains with or without S9 mix, and when the dose-dependent increase was reproducible.

Statistics:

No statistical analysis was performed with the test results in this study.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

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:

positive

Remarks:

without S9 mix:1250 - 5000 µg/plate; with S9 mix: 5000 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Without S9 mix: 5000 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

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: Insolube
- Precipitation: No
- Other confounding effects: No

RANGE-FINDING/SCREENING STUDIES:
The same result was obtained also in the dose-finding test, and the reproducibility of the test results was confirmed.
Number of revertant colonies: The number of revertant colonies in the test substance-treated groups was more than twice that in the correspondingnegative (solvent) control at the following dose levels.

Without S9 mix; 1250 to 5000 µg/plate (TA100)
5000 µg/plate (WP2uvrA-)
With S9 mix; 5000 µg/plate (TA100)

Microbial growth inhibition: Microbial growth inhibition was observed at the following dose levels.
Without S9 mix; 5000 µg/plate (all strains)

Precipitation: Precipitation of the test substance was not observed in any tester strains with or without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
Acceptable ranges of negative (solvent) and positive control values,background data was calculated using the control values yielded October 07, 2008 to March 25, 2009

Conclusions:

In this Ames test, the number of revertant colonies induced by the test substance was more than twice of that of the corresponding negative (solvent) control for TA100 and WP2uvrA- without S9 mix, and for TA100 with S9 mix. The same result was obtained also in the dose-finding test, and the reproducibility of the test results was confirmed. Furthermore, both tests were performed accurately because the acceptable criteria were satisfied.
Therefore, it is concluded that Incozol 4 is mutagenic under the conditions employed in this study.

Executive summary:

Mutagenicity of the test substance Incozol 4, was assessed in the bacterial reverse mutation test using five strains Salmonella typhimurium TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA-.

The test was conducted by the pre-incubation method with and without S9 mix. A dose-finding test was performed at 5000 μg/plate as the maximum dose using 7 doses with a common ratio of 4.

As the results, the number of revertant colonies treated with the test substance was more than twice that treated with the negative (solvent) control for TA100 and WP2uvrA- without S9 mix, and for TA100 with S9 mix. Microbial growth inhibition of the test substance was observed at 5000 μg/plate in all tester strains without S9 mix. Moreover, precipitation of the test substance was not observed in any tester strain or in any dose with or without S9 mix.

The main test was performed using 5 to 6 doses with a common ratio of 2 based on the results of the dose-finding test. For the strains observed with an increase in the number of revertant colonies more than twice the negative (solvent) control value, the dose range that allows for dose-response relationship analysis was selected. For the strains observed with microbial growth inhibition, the dose that induced clear microbial toxicity was selected as the maximum dose. For the other strains, 5000 μg/plate was selected as the maximum dose.

As the results, the number of revertant colonies treated with the test substance was more than twice that treated with the negative (solvent) control for TA100 and WP2uvrA- without S9 mix, and for TA100 with S9 mix.

Microbial growth inhibition of the test substance was observed at 5000 μg/plate in all tester strains without S9 mix. Moreover, precipitation of the test substance was not observed in any tester strain or in any dose with or without S9 mix.

From the results described above, it was concluded that Incozol 4 was mutagenic under the conditions employed in this study.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-01-16 to 2012-04-02

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:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The test method related new chemical substances (Japanese notification, Yakushokuhatsu 0331 No. 7, Heisei 23 03 29 Seikyoku No. 5, kanpokihatsu No. 110331009, March 31, 2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: CHL/IU (derived form the lungs of female Chinese hamster)

Details on mammalian cell type (if applicable):

- Type and identity of media:Culture medium;
Inactivated (56 °C, 30-min heat treatment) fetal bovine serum (Life Technologies Corp., Lot No. 998811) was added to Minimum Essential Medium (MEM) (1×) liquid (Life Technologies Corp.) to a final concentration of 10 % (v/v).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
Mycoplasma: Negative

Additional strain / cell type characteristics:

other: Number of chromosomes (2n): 25 Doubling time: 14.7/14.04 hour Mycoplasma: negative

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix Aroclor 1254 induced

Test concentrations with justification for top dose:

Cell growth inhibition test:156, 313, 625, 1250, 2500, 5000 μg/mL

According to the result of the cell growth inhibition test, the concentration over the IC50 was set as the highest concentration in each treatment condition.

Chromosome aberration test (short-term treatment test, –S9mix): 163, 325, 650, 1300 μg/mL
Chromosome aberration test (short-term treatment test, +S9mix):125, 250, 500,1000 μg/mL

According to the chromosome aberration test (short-term treatment), the result was negative, so the chromosomal aberration test (continuous treatment test, confirmation test) was continuously conducted.

Chromosome aberration test (continuous treatment –S9mix): 150, 300, 600, 1200 μg/mL
Chromosome aberration test (confimation test +S9mix): 150, 300, 600, 1200 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In the preliminary test for solubility, the test substance at 50 mg/mL did not dissolve in physiological saline (referred to as saline). On the other hand, the test substance was dissolved at 500 mg/mL DMSO. No heat, discoloration or foaming were observed in the formulations. Therefore, DMSO was selected as the vehicle for the test substance (negative control substance) in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: Short term (6h) with and without S9 mix; Continuous (24h) without S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells): After and of exposure period

SPINDLE INHIBITOR:Colcemid

STAIN (for cytogenetic assays): 5 % (v/v) Giemsa solution

NUMBER OF REPLICATIONS: 2 plates/concentration

NUMBER OF CELLS EVALUATED: 100 cells/plate (200 cells/concentration)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
-Observations of precipitation or recrystallization of the test substanc: Yes - Method: Macroscopically
Structural aberration
(1) Chromatid-type breaks
(2) Chromatid-type exchanges
(3) Chromosome-type breaks
(4) Chromosome-type exchanges (dicentric, circular chromosome, etc.)
(5) Fragmentation

Evaluation criteria:

Negative: For any test item treatment group, the frequency of the structural aberration cells and the numerical aberration cells were less than 5 %.

Inconclusive: In some test item treatment group, the frequency of the structural aberration cells and the numerical aberration cells were more than 5 % and less than 10 %.

Positive: In some test treatment group, the frequency of the structural aberration cells and the numerical aberration cells were more than 10 %, and there was a tendency to dose-dependent increase.

The test was accepted when the additional criteria described below were satisfied.
(1) The negative and positive control group and at least 3 concentrations of the test substance are satisfied with the adapted standard of data.
(2) For the negative control group, the frequency of the structural aberration cells and the numerical aberration cells were less than 5 %.
(3) For the positive control group, the frequency of the structural aberration cells was more than 10 %.

Statistics:

No statistical analysis was performed.

Key result

Species / strain:

mammalian cell line, other: CHL/IU (derived form the lungs of female Chinese hamster)

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: Not determined
- Effects of osmolality: Not determined
- Evaporation from medium: No
- Water solubility: Test item insoluble
- Precipitation: Yes in dose-dependent manner
- Other confounding effects: No

RANGE-FINDING/SCREENING STUDIES

Cell growth inhibition test:
In more than 1250 μg/mL, the precipitate was recognized at the start of the treatment.
Rates of cell proliferation in any treatment condition were reduced in a dose-dependent manner.
The cell growth index (50 %) was measured and the concentration was calculated using liner equation:
IC50:
–S9 mix: 1219 μg/mL
+S9 mix: 941 μg/mL
24 hours exposure: 1064 μg/mL

Chromosomal aberration test (Short-term treatment test 6 - 18 h):
In 1300 μg/mL of –S9mix and 1000 μg/mL of +S9mix, the precipitate was recognized at the start of the treatment.
According to the result of the observation, the frequency of the structural anomaly of chromosome in 163, 325, 650 and 1300 μg/mL of -S9mix was 0.5, 0.0, 1.0 and 1.5 %, respectively and the frequency of the numerical abnormality was 1.0, 1.5, 0.5, and 0.5 %, respectively. On the other hand, the frequency of the structural anomaly of chromosome in 125, 250, 500 and 1000 μg/mL of +S9mix was 0.5, 0.5, 0.5 and 1.0 %, and the frequency of the numerical abnormality was 0.0, 0.0, 0.5 and 0.0 % respectively.
For the negative and positive control group of each treatment condition, the frequency of the structural anomaly of chromosome was less than 5 % and more than 10 % respectively.

Chromosomal aberration test (Continuous treatment test 24 h):
In 1200 μg/mL, the precipitate was recognized at the start of the treatment.
According to the result of the observation, the frequency of the structural anomaly of chromosome in 150, 300 and 600 μg/mL was 0.5, 0.5 and 0.5 %, respectively and the frequency of the numerical abnormality was 0.0, 0.5 and 0.5 % respectively. On the other hand, in 1200 μg/mL, due to the cytotoxicity, the observable number of cells did not meet the criteria, so it was not used as data.
For the negative control group, the frequency of the structural anomaly of chromosome and the numerical abnormality were less than 5 %. On the other hand, for the positive control group, the frequency of the structural anomaly of chromosome was more than 10 %.

Chromosomal aberration test (confirmation test 6 - 18 h):
In 1200 μg/mL, the precipitate was recognized at the start of the treatment.
According to the result of the observation, the frequency of the structural anomaly of chromosome in 150, 300, 600 and 1200 μg/mL was 0.5, 1.0, 0.5 and 1.0 %, respectively and the frequency of the numerical abnormality was 0.5, 0.5, 0.0, and 1.0 %, respectively.
For the negative control group, the frequency of the structural anomaly of chromosome and the numerical abnormality were less than 5 %. On the other hand, for the positive control group, the frequency of the structural anomaly of chromosome was more than 10 %.

ADDITIONAL INFORMATION ON CYTOTOXICITY

The preliminary cell growth inhibition test showed that the test material induced toxicity in all exposure groups. Therefore the Inhibition Concentration (IC50) was calculated and set as the highest concentration in each treatment condition.In the chromosomal aberration test (Continuous treatment test) (1200 μg/mL) cytotoxicity occur, so the observable number of cells did not meet the criteria, so it was not used as data.

Conclusions:

Incozol 4 was not considered to have the ability to induce the chromosomal aberrations in CHL/IU cells under the present experimental conditions.

Executive summary:

The chromosomal aberration study by Incozol 4 was conducted in cultured mammalian cells due to the OECD Guidelines for the Testing of Chemicals (No. 473, 1997) and the test method related new chemical substances (Japanese notification, Yakushokuhatsu 0331 No. 7, Heisei 23 03 29 Seikyoku No. 5, kanpokihatsu No. 110331009, March 31, 2011).

Incozol 4 was evaluated for in vitro chromosomal aberration study using CHL/IU cells derived from the lungs of female Chinese Hamsters.

The cell growth inhibition test was performed on cell cultures using a short-term treatment assay in the absence of S9 mix (referred to as –S9 mix) and in the presence of S9 mix (referred to as +S9 mix) and continuous treatment test (referred to as 24 hour exposure). The dose range used was 156, 313, 625, 1250, 2500 and 5000 μg/mL.

As a result, the half maximal Inhibitory Concentration (IC50) was 1219 μg/mL at -S9 mix, 941 μg/mL at +S9 mix and 1064 μg/mL at 24-hour exposure. Based on the result of the cell growth inhibition test, 163, 325, 650 and 1300 μg/mL were set in the absence of S9 mix and 125, 250, 500 and 1000 μg/mL were set in the presence of S9 mix respectively.

Afterwards, chromosomal aberration test (short-term treatment method) was conducted. Specimens of all exposure groups were observed. According to the results of all specimens observed, both in -S9 mix and +S9 mix, the frequencies of aberration cells with structural aberration and numerical aberrations of chromosome were less than 5 %.

Since all results were negative under both conditions of short-term treatments, continuous treatment (-S9 mix) and confirmation test (+S9 mix) were conducted. 24-hour treatment (continuous treatment assay) was conducted at 150, 300, 600 and 1200 μg/mL, and the specimens of 150, 300 and 600 μg/mL were observed. In the confirmation test, 150, 300, 600 and 1200 μg/mL were set up to make observations of all specimens in all qualities.

According to the results of all specimens observed, even in continuous treatment and confirmation test, the frequencies of aberration cells with structural aberration of chromosome and numerical aberrations cells were less than 5%.

Therefore, the test item, Incozol 4 was considered to be non-clastogenic to CHL/IU cells under the present experimental condition.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Chromosomal Aberration Test:

The chromosomal aberration study by Incozol 4 was conducted in cultured mammalian cells according to OECD Guideline No. 473 and the test method related to new chemical substances. Incozol 4 was evaluated for in vitro chromosomal aberration study using CHL/IU cells derived from the lungs of female Chinese Hamsters. The cell growth inhibition test was performed on cell cultures using a short-term treatment assay in the absence of S9 mix (referred to as –S9 mix) and in the presence of S9 mix (referred to as +S9 mix) and continuous treatment test (referred to as 24 hour exposure). The dose range used was 156, 313, 625, 1250, 2500 and 5000 μg/mL.

As a result, the half maximal Inhibitory Concentration (IC50) was 1219 μg/mL at -S9 mix, 941 μg/mL at +S9 mix and 1064 μg/mL at 24-hour exposure. Based on the result of the cell growth inhibition test, 163, 325, 650 and 1300 μg/mL were set in the absence of S9 mix and 125, 250, 500 and 1000 μg/mL were set in the presence of S9 mix respectively. Afterwards, chromosomal aberration test (short-term treatment method) was conducted. Specimens of all exposure groups were observed. According to the results of all specimens observed, both in -S9 mix and +S9 mix, the frequencies of aberration cells with structural aberration and numerical aberrations of chromosome were less than 5 %. Since all results were negative under both conditions of short-term treatments, continuous treatment (-S9 mix) and confirmation test (+S9 mix) were conducted. 24-hour treatment (continuous treatment assay) was conducted at 150, 300, 600 and 1200 μg/mL, and the specimens of 150, 300 and 600 μg/mL were observed.

In the confirmation test, 150, 300, 600 and 1200 μg/mL were set up to make observations of all specimens in all qualities. According to the results of all specimens observed, even in continuous treatment and confirmation test, the frequencies of aberration cells with structural aberration of chromosome and numerical aberrations cells were less than 5 %. Therefore, the test item was considered to be non-clastogenic to CHL/IU cells under the present experimental condition.

Ames Test 1:

Mutagenicity of the test substance was assessed in the bacterial reverse mutation test using five strains Salmonella typhimurium TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA-. The test was conducted by the pre-incubation method with and without S9 mix. A dose-finding test was performed at 5000 μg/plate as the maximum dose using 7 doses with a common ratio of 4. As the result, the number of revertant colonies treated with the test substance was more than twice that treated with the negative (solvent) control for TA100 and WP2uvrA- without S9 mix, and for TA100 with S9 mix.

Microbial growth inhibition of the test substance was observed at 5000 μg/plate in all tester strains without S9 mix. Moreover, precipitation of the test substance was not observed in any tester strain or in any dose with or without S9 mix. The main test was performed using 5 to 6 doses with a common ratio of 2 based on the results of the dose-finding test. For the strains observed with an increase in the number of revertant colonies more than twice the negative (solvent) control value, the dose range that allows for dose-response relationship analysis was selected. For the strains observed with microbial growth inhibition, the dose that induced clear microbial toxicity was selected as the maximum dose. For the other strains, 5000 μg/plate was selected as the maximum dose. As the results, the number of revertant colonies treated with the test substance was more than twice that treated with the negative (solvent) control for TA100 and WP2uvrA- without S9 mix, and for TA100 with S9 mix.

Microbial growth inhibition of the test substance was observed at 5000 μg/plate in all tester strains without S9 mix. Moreover, precipitation of the test substance was not observed in any tester strain or in any dose with or without S9 mix. From the results described above, it was concluded that the test substance was mutagenic under the conditions employed in this study.

Ames Test 2:

The test substance was investigated in the Salmonella / microsome test for point - mutagenic effects at doses up to 12500 μg per plate on four Salmonella typhimurium LT2 mutants. These were the histidine - auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

Doses up to and including 100 μg per plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. At higher doses, the substance had a strain - specific bacteriotoxic effect. Nevertheless, this range could be used for evaluation purposes. The positive controls sodium azide, nitrofurantoin, 4-nitro-l,2-phenylene-diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically - relevant increase in mutant colonies compared to the corresponding negative controls.

Evidence of mutagenic activity for the test substance was not found. Neither a dose - related doubling nor a biologically relevant increase of the mutant count, in comparison with the negative controls, was observed.

L5178Y/TK+/- Mouse Lymphoma Assay:

An in vitro mammalian cell gene mutation assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test item to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix).

The appropriate vehicle, absolute ethanol (Abs. Ethanol) was chosen based on the results of the preliminary solubility test. The test item solutions were prepared in Abs. Ethanol and diluted prior to treatment. Based on the available historical control database, this vehicle was compatible with the survival of the cells and the S9 activity. The test item was tested beyond its limit of solubility under culture conditions.

The concentrations applied in the Assay 1 were chosen according to the solubility and cytotoxicity results of the pre-experiments.

The following concentrations were investigated in the Assay 1:

3-hour treatment (-S9 Mix): 100; 500; 1000; 1500; 2000; 2500 and 3000 μg/mL;

3-hour treatment (+S9 Mix): 100; 500; 1000; 1500; 1750; 2000 and 2500 μg/mL.

Concentrations investigated in the Assay 2:

24-hour treatment (-S9 Mix): 100; 200; 300; 400; 500; 600 and 750 μg/mL;

3-hour treatment (+S9 Mix): 500; 1000; 1500; 1750; 2000 and 2500 μg/mL.

In the performed mutation assays the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x105/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) or diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments.

In the performed Assay 1 and Assay 2 in the absence and also in presence of S9 Mix dose-related toxicity of the test item was observed based on both, the harmonised relative survival (harmonised RS) and the relative total growth (RTG) values. The concentration levels of 2500 and 3000 μg/mL in the Assay 1 and the concentration level of 750 μg/mL in the Assay 2 in absence of exogenous metabolic activation were out of the analysable range (higher than 90 % cytotoxicity occurred at these concentration levels). Apart from this the guideline criteria for the number of analysable concentration levels (at least four) as well as the required cytotoxicity degree (10 - 20 % relative survival or relative total growth) at the maximum concentration (in absence of metabolic activation the maximum analyzable concentration) were fulfilled. In presence of metabolic activation the cytotoxicity caused by the highest examined concentration level was in the acceptable 80 - 90 % range in both assays.

Significant differences to the concurrent control were noted for some test concentrations (causing high toxicity down to the limit described in the guideline) in the first and second experiment (Dunnett’s Test, α= 0.05 or α= 0.01). No clear concentration-response was observed in either of the tests with and without metabolic activation. Apart from these findings the obtained mutation frequencies remained unequivocally well below the GEF criterion for positive response in all cases. The final conclusion was drawn from the results of the statistical and biological evaluation.

Under the conditions of this study, the test item does not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.

 

Conclusion:

Incozol 4 did not induce a significant increase of structural or numerical chromosome aberrations in vitro on mammalian cells and was concluded to be non clastogenic in vitro. In addition, Incozol 4 was not mutagenic in gene mutation tests on bacteria (Ames test) and mammalian cells (MLA) both, in the absence and presence of metabolic activation. Therefore, Incozol 4 is considered to be not mutagenic in vitro, although a second Ames test revealed a positive result in the absence of metabolic activation, which is considered to be not relevant for hazard assessment.

Justification for classification or non-classification

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

Based on the experimental results available the test item is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.