1,6-Hexandiammonium dihydroxide, N,N'-bis-(2-hydroxypropyl)-N,N,N',N'-tetramethyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not reveal any mutagenic activity in an in vitro gene mutation assay in bacteria (Ames test, OECD 473, RL 1).

In an in vitro clastogenicity test in CHO-V79 cells (OECD 473, RL 1), the test item was not clastogenic under the experimental conditions.

Negative results were olso obtained in an in vitro gene mutation study in mammalian CHO cells according to OECD TG 476 (RL 1).

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:

From 28 JUN 2002 to 23 SEP 2002

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: stock cultures in the bank of "Genetic Toxicology", Aventis Pharma Germany, ProTox, prepared from the original bacterial strains
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: approx. 37 °C in an incubator

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: stock cultures in the bank of "Genetic Toxicology", Aventis Pharma Germany, ProTox, prepared from the original bacterial strains
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: approx. 37 °C in an incubator

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9 mix (induced with Aroclor 1254)

Test concentrations with justification for top dose:

plate incorporation test: 0, 119, 381, 1190, 3810 and 11905 µg/plate; corresponding to 0, 50, 160, 500, 1600 and 5000 µg active ingredient/plate
preincubation test: 0, 38.1, 119, 381, 1190, 3810 and 11905 µg/plate; corresponding to 0, 16, 50, 160, 500, 1600 and 5000 µg active ingredient/plate

Vehicle / solvent:

Deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for strain TA 100 and TA 1535, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for strain TA1537, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

for strain TA 98, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

for strain WP2uvrA, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

for strain WP2uvrA, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1) plate incorporation assay
Experiment 2) preincubation assay

DURATION
- Preincubation period: 37 °C for 1 h
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: 3 replicates per concentration

Rationale for test conditions:

Test conditions as described in the guideline

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Statistics:

Arithmetic means and standard deviation of the counted colonies were calculated.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

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:

valid

Positive controls validity:

valid

Remarks on result:

other: results from experiment I and II

Toxic effects, evident as incomplete or no bacterial lawn, were observed at the following concentrations of active ingredient:

 

Strain	Experiment I [μg/ plate]		Experiment I [μg/ plate]
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA 100	> 1600	> 1600	> 1600	5000
TA 1535	> 1600	5000	> 1600	5000
TA 1537	> 1600	5000	> 1600	5000
TA 98	> 1600	5000	> 1600	5000
WP2uvrA	5000	5000	5000	5000

Conclusions:

In a guideline study according to OECD TG 471 under GLP conditions, the test item showed no mutagenic activity in a plate incorporation as well as a preincubation experiment with and without metabolic activation.

Executive summary:

In a guideline study according to OECD TG 471 under GLP conditions mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with and without metabolic activation at concentrations of 0, 50, 160, 500, 1600 and 5000 μg/plate (active ingredient). The first experiment was conducted as a plate-incorporation test, the second as a preincubation test.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 JUN 2002 to 08 OCT 2002

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

19 May 2000

GLP compliance:

yes

Type of assay:

other: Chromosome aberration assay in mammalian cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:cell bank of "Genetic Toxicology", Aventis Pharma Germany GmbH, ProTox
- Suitability of cells: yes
- Methods for maintenance in cell culture if applicable: 175 cm2 plastic flasks.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) with Earle-salts and 10 % FCS, 4 % CO, in plastic flasks
- subcultered twice a week

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix (induced with Aroclor 1254)

Test concentrations with justification for top dose:

1. experiment: treatment time 3 h, with and without metabolic activation: 60.2, 120,5, 241.2, 482.1, 964.3, 1928.1, 3857.1, 7714.3 µg/mL
2. experiment: treatment time 20 h, without metabolic activation: 60.2, 120,5, 241.2, 482.1, 964.3, 1928.1, 3857.1, 7714.3 µg/mL
3. experiment: treatment time 3 h, with metabolic activation: 60.2, 120,5, 241.2, 482.1, 964.3, 1928.1, 3857.1, 7714.3 µg/mL

The concentrations correspond to the following concentrations of the active ingredient: 25.3, 50.6, 101.3, 202.5, 405, 810, 1620 and 3240 μg/mL

The highest dose level was determined by the solubility of the test compound up to the maximum of 10 mM

Vehicle / solvent:

cell culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
1. experiemtn: cells were treated for 3 hours in both the presence and absence of S9-mix
2. experiment: cells were treated for 20 hours in the absence of S9-mix.
3. experimetn: cells were treated for 3 hours in the presence of S9-mix.
In all experiments the cells were sampled 20 hours after the start of treatment as were the concurrent solvent and positive control cultures. Colcemide was added to each culture 2 hours before sampling in order to arrest cell division.

SPINDLE INHIBITOR (cytogenetic assays): colcemid (approx. 0.05 µg/mL/culture medium)

STAIN (for cytogenetic assays): orcein solution

NUMBER OF REPLICATIONS: two slides per concentration group and experiment were prepared

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 25 - 100 metaphases per experimental group and cell culture were examined.

CRITERIA FOR CA IDENTIFICATION:The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed and classified. Only metaphases with 22 +/- 2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges including intrachanges, chromosome exchanges including intrachanges, dicentrics, pulverization and ring formation. Furthermore the incidence of polyploid metaphases was determined in 100 metaphases of each cell culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (the number of cells undergoing mitosis in a total of 1000 cells)

Rationale for test conditions:

Study performed according to OECD TG 473

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are statistically significant and within the laboratory's normal range

Criteria for clastogenicity
A test substance is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data
and/or
- no significant increase in the number of structural chromosome aberrations is observed.
A test substance is classified as clastogenic if:
- the number of induced structural chromosome aberrations is above the range of our historical control data
and
- either a concentration-related or a significant increase in the number of structural chromosome aberrations is observed.
Statistical significance is confirmed by means of the Fisher's exact test. However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

Statistical evaluation was not conducted if all the means of the dose groups were in the range of the solvent controls.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> =1620 µg/mL

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- In the first and third main experiments no reduction of mitotic index was observed.
- In the second experiment the mitotic index was reduced (indication of toxicity) after treatment with the highest dose levels.
- After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls
- Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any obvious effect on the osmolality, but did influence the pH-value of the treatment medium at the concentrations of 3240 µg/mL active ingredient. The treatment medium was re-adjusted to approximately pH 7.3 with concentrated HCL.

The evaluated experimental points were the following:
Experiment I:
without metabolic activation: 810, 1620 and 3240 μg/mL
with metabolic activation: 810, 1620 and 3240 μg/mL

Experiment II:
without metabolic activation: 405, 810, 1620 µg/mL

Experiment III:
with metabolic activation: 810, 1620 and 3240 μg/mL

Remarks on result:

other:

Remarks:

Results of experiment I

No relevant reproducible enhancement of metaphases with aberrations outside the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Conclusions:

In a guideline study according to OECD TG 473 under GLP conditions the test item did not induce structural chromosome abberations in V79 cells with and without metabolic activation.

Executive summary:

In a guideline study (OECD 473 as of 1997, GLP conditions) the test item, suspended in cell culture medium, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster V79 cells with and without metabolic activation (induced rat liver S9 mix).

Three experiments were performed with and without metabolic activation. The exposure times were 3 h (experiment I and III and 20 h (experiment II) The concentrations of active ingredient were 0, 25.3, 50.6, 101.3, 202.5, 405, 810, 1620 and 3240 μg/mL

In total 25 - 100 metaphases per experimental group and cell culture were examined for structural chromosome aberrations in each experiment. In all three independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They produced markedly positive results.It was concluded that the test substance is not clastogenic in Chinese hamster V79 cells under the experimental conditions described in the report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 NOV 2017 to 18 JAN 2018

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:

29. July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, P.O.Box 1549, Manassas, VA 20108,
USA

- Suitability of cells: Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time.

- Cell cycle length, doubling time or proliferation index: Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 12 to 14 hours was used.
This cell line is capable of developing resistance to 6-thioguanine (6TG) resulting from lack of hypoxanthine guanine phosphoribosyl transferase (hprt) enzyme activity as a result of mutation at the X chromosomes.
The cell line was tested for mycoplasma in the test facility. The karyotype analysis of this cell line is periodically performed and documented.
Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics, and L-glutamine was the basic medium.
Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells.
Basic medium supplemented with 5% fetal bovine serum (FBS) was the treatment medium and was used for target cell exposure to the test item and controls.
Cloning medium was basic medium supplemented with 20 % FBS and was used for the determination of cell viability or plating/cloning efficiency.
Selective medium was basic medium supplemented with 20 % FBS and the selective agent 6-Thioguanine (6-TG) at 35 µM and was used for the selection of mutants.
Dulbecco’s Phosphate buffered saline (PBS) (pH: 7.4)
Trypsin: EDTA solution

- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes)

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice. Batches stored in a deep freezer maintained at -68 to -86 ºC.

Test concentrations with justification for top dose:

For the preliminary cytotoxicity test the following concentrations were selected:
0, 25, 50, 100, 200, 400, 800 and 1550 µg/mL. Since the purity of the test item is 15.5 %, undiluted test item at 155 mg/mL (w/v) was used as the top concentration equivalent to approximately 1.5 mg/mL which is close to the top concentration of 2 mg/mL as indicated in the OECD test guideline 476.

Based on the results of the preliminary cytotoxicity test, the following test concentrations were selected for testing in the gene mutation test:
Experiments 1 and 2 (Presence and Absence of Metabolic Activation, respectively)
A) 194 B) 388 C) 775 and D) 1550 µg/mL (factor of 2)

Vehicle / solvent:

- Vehicle(s) used: One hundred fifty microliters (150 µL) of sterile Water (SW) was used per 15 mL of treatment medium as the vehicle control in each of the experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; Exponentially growing CHO-KI cells were plated in two replicates and each replicate has two flasks containing 15 mL of complete medium at a density of approximately 4.5 x 10exp6 cells / 75 cm2 flask and incubated for approximately 24 hours.
Two parallel cultures were also kept along with the vehicle control and treatment groups. Cell counts were made from these cultures at the 0-hour treatment to obtain the baseline cell count for estimation of Relative Survival (RS).

- Cell density at seeding (if applicable): 4.5 x 10exp6 cells / 75 cm2

DURATION
- Period: All treatment mixtures and positive control were prepared immediately before use in sterile test tubes. The target cells were exposed to the vehicle, positive control and various concentrations of the test item for 3 hours in the presence and absence of metabolic activation.
The medium from each target cells flask was removed by aspiration and replaced with 15 mL of the respective treatment mixture for the experiment in the presence and absence of metabolic activation, respectively.
One hundred fifty microliters (150 µL) each of the vehicle control and the positive control, were transferred to respective flasks and gently mixed and kept for incubation to expose the cells to treatment.

- Exposure duration: 3 hours
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): Replicate cultures from controls and each treatment level were trypsinized, and the cells suspended in 10 mL complete medium, pooled, and counted using a hemocytometer.
For selection of the 6-Thioguanine (6-TG) resistant phenotype, cells from each of the replicate cultures were plated in to 5 flasks at a density of approximately 2 x 10exp5 cells/25 cm2 flask (total of 10exp6 cells/replicate) in selective medium and incubated for 10 days.
For cloning efficiency determination at the time of selection, cells from each of the replicate cultures were plated at approximately 200 cells/25 cm2 flask in triplicate in cloning medium and incubated for 10 days.

STAIN (for cytogenetic assays): The colonies were stained with 0.5 % methylene blue and counted for both cloning efficiency and mutant selection after 10 days of incubation.

NUMBER OF REPLICATIONS: Two

S9 MIX: S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice. Batches stored in a deep freezer maintained at -68 to -86 ºC.
Each batch of S9 homogenate was characterized for its ability to metabolize the pro-mutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain and for the Protein content using modified Lowry Assay (Sword and Thomson, 1980). To check the sterility, the liver homogenate was streaked onto nutrient agar plates, in duplicate, and incubated for approximately 48 hours at 37 ± 1 °C.

Evaluation criteria:

When all the validity criteria are fulfilled:
1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

2. A test chemical is considered to be clearly negative if, in all experimental conditions examined:

• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed using the formula:

Y = (X + A) B

where,
Y = transformed mutant frequency
X = observed mutant frequency
and A, B = constants.

Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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: At the end of 3-hour exposure, the pH of the test medium in the presence of metabolic activation ranged from 6.98 to 7.06 with 7.00 in the sterile water control while in the absence of metabolic activation it was between 7.03 and 7.14 with 7.09 in the sterile water control.
- Effects of osmolality: At the end of the 3-hour exposure period, the osmolality of the test medium, at the highest test item concentration level (1550 mg/mL) was 0.286 and 0.298 OSMOL/kg. in the presence and absence of metabolic activation, respectively. The corresponding osmolality in the sterile water control was 0.311 and 0.291 OSMOL/kg in the presence and absence of metabolic activation, respectively.
- Precipitation: no

CRITERIA FOR ACCEPTABILITY OF THE TEST
The assay will be considered valid if the following criteria are met:
a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b)The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.

RANGE-FINDING/SCREENING STUDIES:
The Relative Survival at 1550 iag/mL was 54 and 49 %, in the presence and absence of metabolic activation, respectively.
Based an these observations a maximum of 1550 µg/mL was tested in the gene mutation assay.

 

TABLE 1.       Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6ClonableCells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
SW	3	2	3	1	2	22	0.000011	184	188	191	0.937	11.74
	2	2	3	1	3			185	188	188
194	2	2	1	0	3	17	0.0000085	180	186	183	0.908	9.36
	1	2	2	3	1			179	184	177
388	2	2	2	3	1	17	0.0000085	178	171	175	0.871	9.76
	2	1	1	1	2			169	174	178
775	2	2	2	1	2	17	0.0000085	169	165	171	0.848	10.02
	2	1	2	2	1			172	167	173
1550	3	2	2	2	1	18	0.000009	164	158	166	0.808	11.14
	0	3	2	2	1			166	161	155
3-MCA	35	33	38	34	29	334	0.000167	158	162	151	0.774	215.76^+
	33	34	30	32	36			147	158	153

Positive Control: 8 µg/mL 3-MCA             Vehicle Control: Sterile Water (SW)                          CE: Cloning Efficiency               MF: Mutant Frequency

* calculated from the mean values of the replicates of each group and rounded off to three decimal places

  Mutant frequency of 6-TG mutants is significantly higher than the concurrent vehicle control value (p < 0.05)

 

CE	=	Total No. of colonies		MF	=	CE of mutant colonies in selective medium	x 10^6
		No. of cells plated				CE in non-selective medium

 

 

 

TABLE 2.       Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6ClonableCells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
SW	2	3	3	3	0	23	0.0000115	184	187	189	0.937	12.27
	2	3	2	2	3			189	189	186
194	3	3	2	2	1	19	0.0000095	178	173	180	0.902	10.53
	2	1	1	2	2			188	179	184
388	2	2	1	1	2	19	0.0000095	173	170	168	0.845	11.24
	3	2	3	1	2			168	171	164
775	4	1	1	1	2	19	0.0000095	159	163	155	0.793	11.98
	2	1	3	2	2			165	159	151
1550	2	2	2	4	2	22	0.000011	158	149	161	0.783	14.05
	2	2	2	2	2			159	157	155

Vehicle Control: Sterile Water (SW)      CE: Cloning Efficiency                    MF: Mutant Frequency                                                              

* calculated from the mean values of the replicates of each group and rounded off to three decimal places

 

 

CE	=	Total No. of colonies		MF	=	CE of mutant colonies in selective medium	x 10^6
		No. of cells plated				CE in non-selective medium

 

 

 

 

Conclusions:

It is concluded that the test item does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The mutagenic potential of the test item to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 54 and 49 % at the 1550 µg/mL, in the presence and absence of metabolic activation, respectively. There was no precipitation of the test item in the test medium at any of the tested concentrations, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of   1550 µg/mL was tested in the gene mutation assay. 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 194, 388, 775 and 1550 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (SW) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at thehprtlocus with the test item indicated that the test item was non-mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a guideline study according to OECD TG 471 under GLP conditions mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with and without metabolic activation at concentrations of 0, 50, 160, 500, 1600 and 5000 μg/plate (active ingredient). The first experiment was conducted as a plate-incorporation test, the second as a preincubation test.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagens showed distinct positive mutagenic effects.

In a guideline study (OECD 473 as of 1997, GLP conditions) the test item, suspended in cell culture medium, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster V79 cells with and without metabolic activation (induced rat liver S9 mix).

Three experiments were performed with and without metabolic activation. The exposure times were 3 h (experiment I and III) and 20 h (experiment II). The concentrations of active ingredient were 0, 25.3, 50.6, 101.3, 202.5, 405, 810, 1620 and 3240 μg/mL

In total 25 - 100 metaphases per experimental group and cell culture were examined for structural chromosome aberrations in each experiment. In all three independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They produced markedly positive results. It was concluded that the test substance is not clastogenic in Chinese hamster V79 cells under the experimental conditions described in the report.

In a guideline study (OECD 476 under GLP conditions) the mutagenic potential of the test item to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells. In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 194, 388, 775 and 1550 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (SW) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions

The results of the forward gene mutation test at thehprtlocus with the test item indicated that the test item was non-mutagenic under the conditions of this study.

Justification for classification or non-classification

As no effects were observed in the genetic toxicity testing, no classification is required according to Regulation (EC) No. 1272/2008 (CLP).