Formaldehyde, reaction products with diethanolamine and 6(or 7)-methyl-1H-benzotriazole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 09, 2012 - November 12, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1H-Benzotriazole-1-methanamine, N,N-bis(2-ethylhexyl)-6-methyl- and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-5-methyl- and N,N-bis(2-ethylhexyl)-4-methyl-1H-benzotriazole-1-methylamine and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-4-methyl- and N,N-bis(2-ethylhexyl)-5-methyl-1H-benzotriazole-1-methylamine

EC Number:

939-700-4

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of 1H-Benzotriazole-1-methanamine, N,N-bis(2-ethylhexyl)-6-methyl- and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-5-methyl- and N,N-bis(2-ethylhexyl)-4-methyl-1H-benzotriazole-1-methylamine and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-4-methyl- and N,N-bis(2-ethylhexyl)-5-methyl-1H-benzotriazole-1-methylamine

Test material form:

liquid

Details on test material:

- State of aggregation: liquid
- Storage conditions: Room temperature; avoid temperatures < 0°C

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

See any other information on materials and methods incl. tables.

Vehicle / solvent:

On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol. The solvent was chosen based on solubility properties and its relative non-toxicity to the cell cultures. The final concentration of ethanol in the culture medium was 0.5 % (v/v).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10^5 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.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 7-10 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: The study was performed in two independent experiments, using identical experimental procedures.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
The data of this study comply with the above mentioned criteria.

Evaluation of Results
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 was considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A p-value below 0.05 was detected in culture I of the first experiment with metabolic activation and in culture II of the second experiment without metabolic activation. These trends however, were judged as biologically irrelevant as the mutation frequency remained well within the range of the historical solvent controls.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.8 up to 25.0 mutants per 10^6 cells; the range of the groups treated with the test item was from 3.8 up to 35.3 mutants per 10^6 cells.

EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 39.1 to 5000 μg/mL according to the current OECD guideline 476 to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects were observed at 78.1 μg/mL and above after 4 hours treatment without metabolic activation and at 156.3 μg/mL with metabolic activation. Following 24 hours treatment the cell growth was completely inhibited down to the lowest concentration.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation was observed at 625 μg/mL and above in the presence of metabolic activation following 4 hours treatment and at 312.5 mg/mL and above in the absence of metabolic activation following 24 hours treatment.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations generally spaced by a factor of 2 was applied. Narrower spacing was used at high concentrations with metabolic activation to cover the onset of cytotoxicity more closely.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I and/or a relative cell density below 50% in both cultures were observed in the first experiment at 78.0 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 10.0 μg/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered without metabolic activation. In the presence of metabolic activation moderate cytotoxic effects were noted in the first experiment with metabolic activation at the highest analysable concentration of 78 μg/mL with metabolic activation. Exceedingly severe cytotoxic effects occurred at the next higher, phase separating concentration of 156 μg/mL. In the second experiment with metabolic activation turbidity observed at the maximum concentration of 120 μg/mL indicated that the limit of solubility was reached.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental Result:

	concentration (µg/ml)	PST	S9 Mix	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor
Experiment I / 4h treatment				culture I					culture II
solvent control (ethanol)			-	100.0	100.0	100.0	8.8	1.0	100.0	100.0	100.0	25.0	1.0
positive control (EMS)	150.0		-	101.7	122.4	92.9	98.5	11.2	85.8	146.3	74.5	157.8	6.3
test item	0.16		-	110.4	culture was not continued#				95.1	culture was not continued#
test item	0.31		-	110.2	culture was not continued#				87.9	culture was not continued#
test item	0.63		-	119.0	117.1	83.0	14.7	1.7	87.9	102.1	128.4	14.4	0.6
test item	1.3		-	111.1	119.7	88.1	7.9	0.9	97.6	112.3	111.7	25.2	1.0
test item	2.5		-	114.4	97.7	77.6	19.5	2.2	98.2	72.9	123.4	10.1	0.4
test item	5.0		-	122.6	100.2	86.2	20.1	2.3	100.0	52.6	124.7	14.6	0.6
test item	10.0		-	96.7	56.5	78.8	19.5	2.2	66.8	45.2	99.2	25.6	1.0
solvent control (ethanol)			+	100.0	100.0	100.0	18.0	1.0	100.0	100.0	100.0	22.7	1.0
positive control (DMBA)	1.1		+	62.8	91.9	41.4	1579.1	87.7	59.3	104.1	73.7	1200.1	52.9
test item	9.8		+	102.2	106.8	84.3	15.5	0.9	98.4	93.5	89.1	22.0	1.0
test item	19.5		+	97.0	123.0	94.6	17.8	1.0	92.2	93.5	101.9	15.7	0.7
test item	39.0		+	99.7	113.1	98.4	19.3	1.1	96.8	85.8	82.0	23.2	1.0
test item	78.0		+	44.1	68.1	70.2	25.7	1.4	54.0	41.5	56.4	35.3	1.6
test item	156.0	PS	+	0.0	culture was not continued##				0.0	culture was not continued##
test item	234.0		+	0.0	culture was not continued##				0.0	culture was not continued##
Experiment II / 24h treatment
solvent control (ethanol)			-	100.0	100.0	100.0	12.5	1.0	100.0	100.0	100.0	14.3	1.0
positive control (EMS)	150.0		-	89.7	113.2	91.1	364.3	29.1	89.7	96.8	72.9	368.9	25.9
test item	0.31		-	98.8	culture was not continued#				101.3	culture was not continued#
test item	0.63		-	100.6	114.7	76.7	21.8	1.7	98.2	88.5	124.6	7.8	0.5
test item	1.3		-	98.9	109.6	178.7	3.8	0.3	98.5	83.5	95.2	7.0	0.5
test item	2.5		-	95.8	118.0	89.8	25.2	2.0	96.1	100.1	130.8	9.4	0.7
test item	5.0		-	77.6	107.9	122.0	17.6	1.4	72.9	75.8	79.1	11.4	0.8
test item	10.0		-	21.1	18.3	87.8	29.2	2.3	23.8	15.2	82.0	26.6	1.9
test item	20.0		-	0.0	culture was not continued##				0.0	culture was not continued##
Experiment II / 4h treatment
solvent control (ethanol)			+	100.0	100.0	100.0	14.5	1.0	100.0	100.0	100.0	14.7	1.0
positive control (DMBA)	1.1		+	83.9	80.9	87.3	544.1	37.6	79.3	78.5	71.8	577.2	39.3
test item	5.0		+	100.4	culture was not continued#				92.6	culture was not continued#
test item	10.0		+	101.1	86.0	103.7	15.0	1.0	89.6	93.6	91.5	18.5	1.3
test item	20.0		+	99.6	88.9	100.8	17.0	1.2	86.3	100.0	88.2	18.8	1.3
test item	40.0		+	93.8	78.5	108.7	15.6	1.1	93.5	108.0	54.8	20.5	1.4
test item	80.0		+	94.9	79.2	101.2	18.1	1.3	94.3	103.4	69.9	32.8	2.2
test item	120.0	T	+	94.9	75.6	113.8	16.6	1.1	88.2	100.0	70.4	24.3	1.7

PS = Phase Separation visible at the end of treatment

T = Turbidity

# culture was not continued as a minimum of only four concentrations is required

## culture was not continued due to exceedingly severe cytotoxic effects

Applicant's summary and conclusion

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.