Hexanoic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was examined for mutagenic activity in a study according to OECD TG 471 (Ames test, direct plate incorporation procedure) with the five histidine-dependent Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98 and one tryptophan-dependent Escherichia coli WP2 strain (WP2uvrA), all with and without metabolic activation. The test substance concentration ranged from 0.005 to 5.0 µL/plate

None of the six tester strains (S. typhimurium: TA1535, TA100, TA1537, TA1538, TA98 and E. coli: WP2uvrA) showed increased reversion to prototrophy, when tested up to the maximum recommended dose level of the substance (5 ¿L/plate) in the absence and presence of a metabolic activation system (S9 mix), whereas the respective positive controls produced the expected distinct increase in the number of revertant controls. Thus, no genotoxicity is to be concluded based on the available data.

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cellsin vitroin three independent experiments.The highest applied concentration in this study (2000 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487.In all experimental parts in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration, which showed phase separation. Under the experimental conditions reported, the test item did not induce micronuclei as determined by thein vitromicronucleus test in Chinese hamsterV79cells.Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

The test substance was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The cells were exposed to the test item for 4 hours in concentrations up to and including 500 µg/mL (with S9 mix) and 375 µg/mL (without metabolic activation). The maximum test item concentration was limited by the solubility of the test item in aqueous media. No substantial and reproducible dose dependent increase of the mutation frequency above was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions described, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance was considered to be non-mutagenic in this HPRT assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Oct - Nov 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according 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:

Histidine gene locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

0.005 to 5.0 ¿L/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

phosphate buffer

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

cyclophosphamide

ethylmethanesulphonate

other: 4-Nitro-o-phenylenediamine, Anthracene-2-amine, 2-Nitro-9H-fluorene, N-Methyl-N¿-nitro-N-nitrosoguanidine

Remarks:

concentrations of positive controls: 4-NPDA 10 µg/plate, 2-NF 10 µg/plate, NaN3 5 µg/plate, MNNG 5 µg/plate, EMS 5 µL/plate, 2-AA 2.5 and 10 µg/plate, BP 2.5 µg/plate, CP 400 µg/plate

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

Migrated from field 'Test system'.

None of the six tester strains (S. typhimurium: TA1535, TA100, TA1537, TA1538, TA98 and E. coli: WP2uvrA) showed increased reversion to prototrophy with ZK 56404 at the doses tested (0.005 to 5.0 ¿L/plate) either in the absence or presence of S9 mix.

Precipitates in the agar were not found.

Growth inhibition of the background lawn could be observed with all tester strains. In the experiments without S9 mix growth inhibition of the background lawn was observed with strains TA98, TA100, TA1535 and TA1537 from 0.05 ¿L/plate, with strain TA1538 from

0.1 ¿L/plate and with strain WP2uvrA from 0.25 ¿L/plate onwards.

In the experiments with metabolic activation (S9 mix) growth inhibition of the background lawn started at 0.5 ¿L/plate (strain TA1535), at 1.0 ¿L/plate (strains TA1537, WP2uvrA) or at 2.5 ¿L/plate (strains TA98, TA100, TA1538).

The colony counts recorded on appropriate negative control plates confirmed the characteristic spontaneous reversion rates of the tester strains.

Furthermore, appropriate positive controls with known mutagens (anthracene-2-amine; benzo[a]pyrene; cyclophosphamide; ethyl methanesulfonate; N-Methyl-N'-nitro-Nnitrosoguanidine; 2-nitro-9H-fluorene; 4-nitro-o-phenylenediamine; sodium azide) produced the expected distinct increase in the number of revertant colonies.

Executive summary:

The substance was examined for mutagenic activity in a study according to OECD TG 471 (Ames test, direct plate incorporation procedure) with the five histidine-dependent Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98 and one tryptophan-dependent Escherichia coli WP2 strain (WP2uvrA), all with and without metabolic activation. The test substance concentration ranged from 0.005 to 5.0 µL/plate

None of the six tester strains (S. typhimurium: TA1535, TA100, TA1537, TA1538, TA98 and E. coli: WP2uvrA) showed increased reversion to prototrophy, when tested up to the maximum recommended dose level of the substance of 5 ¿L/plate in the absence and presence of a metabolic activation system (S9 mix), whereas the respective positive controls produced the expected distinct increase in the number of revertant controls.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: over 4 hours at room temperature in the light (Watson P, Study Number FP 57HY Envigo Report 13 April 2017)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: All formulations were prepared freshly before treatment and used within two hours of preparation.

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany
- Suitability of cells: used successfully for many years in in vitro experiments; high proliferation rate, and a reasonable plating effeiciency
- Cell cycle length, doubling time or proliferation index: doubling time approximately 13 hours
- Methods for maintenance in cell culture if applicable: stocks are stored in liquid nitrogen; before freezing each batch is screened for mycoplasm contamination and
checked for karyotype stability
- Modal number of chromosomes: 22 +/- 1

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal essential medium (MEM) with different supplements; incubations were done at 37°C in a humidified atmosphere with 1.5% carbon dioxide.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Exp. I, (4 hrs): 3.9, 7.8, 15.5, 31.3, 62.5, 125, 250, 500,1000 and 2000μg/mL (without and
with S9 mix)
Exp. IIA, (24 hrs): 62.5, 125, 250, 500,1000 and 2000 μg/mL (without S9 mix)
Exp. IIA, (4 hrs): 62.5, 125, 250, 500,1000 and 2000 μg/mL (with S9 mix)
Exp. IIB (4 hrs): 62.5, 125, 250, 500,1000 and 2000 μg/mL (with S9 mix)

The highest applied concentration was chosen with respect to the current OECD TG 487.

The following concentrations were selected for reading in all experiments: 500, 1000, 2000 µg/mL

Vehicle / solvent:

DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin

Details on test system and experimental conditions:

- Culture conditions: Thawed stock cultures were propagated at 37 °C in 80 cm^2 plastic flasks. About 5 x 10^5 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week.
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes containing microscopic slides. Into each chamber 1.0 x 10^5 – 1.5 x 10^5 cells were seeded. For RICC (relative increase in cell count) determination, 16600 cells per well (Exp. I), 16720 cells per well (exp. IIA) and 15600 cells per well (Exp. IIB) were seeded in duplicates in a 24-well-plate.
All incubations were done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

- TREATMENT:

Pre-experiment:
A preliminary cell growth inhibition test (determination of proliferation index) was performed to determine the concentrations to be used in the main experiment. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure.

Pulse exposure: The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.

Continuous exposure (without S9 mix) : The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item. The medium was not changed until preparation of the cells.

Preparation of micronuclei: The cells were treated on the slides in the chambers with deionised water for 1 – 1.5 min at 37 °C. Afterwards the cells were fixed twice with a mixture of ethanol and glacial acetic acid (3+1 parts, respectively) containing 1.25 % formaldehyde. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. Per culture at least 1000 cells from clones with 2 - 8 cells were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells.

Cytotoxicity was assessed by the determination of the relative increase in cell counts (RICC).

RICC = (increase in number of cells in treated cultures (final - starting)) diveded by (increase in number of cells in control cultures (final - starting)) multiplied by 100

Cytotoxicity [%] = 100 – RICC

In addition, cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), and 5 - 8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represent the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated (see formula below). Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee a sufficient cell proliferation during treatment and recovery.

PI = [(c1 x 1) + (c2 x 2) + (c4 x 3) + (c8 x 4)] / [c1 + c2 + c4 + c8]
PI = Proliferation index
cx = Number of clones with x cells (with x: 1, 2, 4, or 8)

Evaluation criteria:

Acceptability criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range.
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described in details on test system and conditions were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
− The criteria for the selection of top concentration are consistent with those described above.

Evaluation Criteria:
Clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant influence
- Effects of osmolality: no relevant influence
- Precipitation: phase separation was observed in all experimental parts at 2000 µg/mL at the end of treatment

In all experimental parts in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration, which showed phase separation.

In Experiment I and IIA, in the absence of S9 mix, no relevant increases in micronucleated cells were observed after treatment with the test item.     
In Experiment IIA in the presence of S9 mix, two statistically significant increases in micronucleated cells (1.45 and 2.05 %) were observed after treatment with 500 and 1000 µg/mL, respectively. The second value exceeded the range of the laboratory historical solvent control (0.20 – 1.72 % micronucleated cells).      
In the confirmatory experiment IIB, the values of all evaluated concentrations were within the range of the laboratory historical solvent control data (0.20 – 1.72 % micronucleated cells) and no statistical significance was observed after treatment with the test item.

Table            Summary of results

Exp.	Preparation	Test item	Proliferation	RICC	Cytotoxicity	Micronucleated
	interval	concentration	Index	in %	in %	Cells*
		in µg/mL				in %
Exposure period 4 hrs without S9 mix
I	24 hrs	Solvent control^1	2.66	100.00	0.00	0.90
		Positive control^2	2.31	74.06	25.94	4.05^S
		500	2.65	97.63	2.38	0.60
		1000	2.60	95.56	4.44	0.70
		2000^PS	2.61	109.34	n.c.	0.70
Exposure period 24 hrs without S9 mix
IIA	24 hrs	Solvent control^1	3.06	100.00	0.00	0.45
		Positive control^3	2.54	78.20	21.80	8.40^S
		500	2.80	71.93	28.07	0.45
		1000	2.47	73.18	26.82	0.60
		2000^PS	2.93	72.55	27.45	0.60
Exposure period 4 hrs with S9 mix
IIA	24 hrs	Solvent control^1	2.02	100.00	0	0.75
		Positive control^4	1.33	36.25	63.75	14.80^S
		500	1.79	72.18	27.82	1.45^S
		1000^#	1.93	82.44	17.56	2.05^S
		2000^PS	1.84	79.68	20.32	0.95
IIB	24 hrs	Solvent control^1	2.40	100.00	0.00	1.25
		Positive control^5	1.40	45.21	54.79	7.65^S
		500	2.24	84.67	15.33	1.70
		1000	2.38	104.03	n.c.	1.40
		2000^PS	2.52	106.69	n.c.	1.65

*      The number of micronucleated cells was determined in a sample of 2000 cells

^#      The number of micronucleated cells was determined in a sample of 4000 cells

n.c. Not calculated as the RICC is equal or higher than the solvent control value

^S       Number of micronucleated cells statistically significantly higher than corresponding control values

^PS     Phase separation was observed at the end of treatment

¹         DMSO                    0.5 % (v/v)

²         Mitomycin C         0.1 µg/mL

³         Griseofulvin         8.0 µg/mL

⁴         CPA                      10.0 µg/mL

⁵         CPA                      15.0 µg/mL

 

* The number of micronucleated cells was determined in a sample of 2000 cells

# The number of micronucleated cells was determined in a sample of 4000 cells

S Number of micronucleated cells statistically significantly higher than corresponding control values

PS Phase separation was observed at the end of treatment

n.c. Not calculated as the RICC is equal or higher than the solvent control value

1 DMSO 0.5 % (v/v)

2 Mitomycin C 0.1 μg/mL

3 Griseofulvin 8.0 µg/mL

4 CPA 10.0 μg/mL

5 CPA 15.0 µg/mL

Conclusions:

negative

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cellsin vitroin three independent experiments. The highest applied concentration in this study (2000 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487. In all experimental parts in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration, which showed phase separation. Under the experimental conditions reported, the test item did not induce micronuclei as determined by thein vitromicronucleus test in Chinese hamsterV79cells. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Reference 3

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

Study period:

Jan - Feb 2017

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:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: over 4 hours at room temperature in the light (Watson P, Study Number FP 57HY Envigo Report 13 April 2017)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately before treatment), the test item was formulated in the vehicle

Target gene:

HPRT locus

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 supplements; for the selection of mutant cells the medium was supplemented with 11 µg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from the liver of phenobarbital/ß-naphthoflavone induced rats

Test concentrations with justification for top dose:

Pre-experiment: 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0, 2000.0 µg/mL (-/+ S9 mix)
Main experiment: 31.3, 62.5, 125.0, 250.0, 375.0 µg/mL ( without S9 mix); 62.5, 125.0, 250.0, 375.0, 500.0 µg/mL (with S9 mix)

Vehicle / solvent:

Dimethylsulfoxid (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

EMS was used without and DMBA with metabolic activation

Details on test system and experimental conditions:

- DOSE SELECTION: According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye and microscopically. Furthermore, test item induced changes in the osmolarity will influence dose selection. The highest applied concentration in the pre-test on toxicity (2000 μg/mL) was chosen with respect to the OECD duideline 476. A relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 250.0 µg/mL and above without metabolic activation. In the presence of metabolic activation a relevant cytotoxic effect was determined at the highest concentration of 2000.0 µg/mL.The test medium was checked for precipitation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Phase separation occurred at 500.0 μg/mL and above after 4 hours treatment with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the main experiment was set according to data generated in the preexperiment. The individual concentrations were spaced by a factor of 2.0. The spacing was narrower at high concentrations to cover the critical range of possible cytotoxicity and precipitation more closely.To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.The cultures at the lowest concentration with metabolic activation 31.3 µl/mL were not continued as a minimum of only four analysable concentrations is required by the guidelines. The cultures at the highest concentration (500 µl/mL) without metabolic activation were not continued to avoid analysis of too many precipitating concentrations.

Culture Medium:
For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
Seeding:
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approx. 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2x10exp.-7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

Treatment:

After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0106 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

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 within the laboratory´s historical control data range, 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. 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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Phase separation occurred at 250.0 µg/mL and above in the absence of metabolic activation and at 375.0 µg/mL and above in the presence of metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiment up to the maximum concentration. The 95% confidence interval was slightly exceeded at 375.0 µg/mL in the first culture of the first experiment with metabolic activation (39.8 versus an upper limit of 30.2 mutant colonies/106 cells). This isolated increase was judged as irrelevant as it was not reproduced and there was no dose dependent increase as indicated by a lacking statistical significance.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture without metabolic activation. As the trend was not reproduced in the parallel culture and the absolute values of the mutation frequency remained within the 95% confidence interval, it is consequently judged as irrelevant fluctuation.
In the main experiment with and without S9 mix the range of the solvent controls was from 11.6 up to 24.7 mutants per 106 cells; the range of the groups treated with the test item was from 5.2 up to 39.8 mutants per 106 cells.

Conclusions:

negative

Executive summary:

The test substance was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The cells were exposed to the test item for 4 hours in concentrations up to and including 500 µg/mL (with S9 mix) and 375 µg/mL (without metabolic activation). The maximum test item concentration was limited by the solubility of the test item in aqueous media. No substantial and reproducible dose dependent increase of the mutation frequency above was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions described, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance was considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data no classification for genotoxicity is warranted according to Regulation (EC) 1272/2008 (CLP).