Reaction products of N,N'-ethane-1,2-diylbis(1,3-propanediamine), cyclohexane, peroxidized 4-butylamino-2,2,6,6-tetramethylpiperidine and 2,4,6-trichloro-1,3,5-triazine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance (as described in section 1.2) was negative in Ames and HPRT tests and did not induce chromosomal aberrations in CHO cells with and without metabolic activation.

Link to relevant study records

Reference

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:

August 31, 2012 - January 03, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

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, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt (hypoxanthine-guanine phosphoribosyl transferase)

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) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneus mutant frequency: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I:
4 hours, with and without S9 mix: 0.7, 2.0, 6.0, 18.0, 54.0, (162.0) µg/ml
Experiment II:
24 hours, without S9 mix: (0.7), 2.0, 6.0, 18.0, 54.0, 162.0 µg/ml
4 hours, with S9 mix: 0.7, 2.0, 6.0, 18.0, 54.0, (162.0) µg/ml
numbers in parantheses: these cultures were discontinued.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

without S9: EMS, 0.15 mg/ml; with S9: DMBA, 1.1 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 - 20 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density

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 was 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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation, visible to the unaided eye, was noted in experiment I at 18.0 μg/mL and above with and without metabolic activation. In experiment II precipitation occurred at 54.0 μg/mL and above without and at 18.0 μg/mL and above with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. An isolated reduction of the cloning efficiency noted at 1155 μg/mL following 4 hours treatment without metabolic activation was judged as precipitation artefact as there was no dose dependent cytotoxicity. 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. Precipitation occurred at 18.0 μg/mL and above with and without metabolic activation after 4 and 24 hours treatment. In the pre-experiment there was no relevant shift of osmolarity and pH values of the medium even at the maximum concentration of the test item. Based on precipitation of the test item noted in the pre-experiment, the concentration range of the main experiments was selected.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency did not exceed the historical range of solvent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in experiment I and II up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of Results

	concentration (µg/ml)	P	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 (DMSO)			-	100.0	100.0	100.0	15.0	1.0	100.0	100.0	100.0	18.6	1.0
positive control (EMS)	150.0		-	81.8	103.1	85.3	144.7	9.6	83.7	140.4	105.6	67.6	3.6
test item	0.7		-	98.5	110.7	98.0	18.9	1.3	105.0	134.2	101.2	9.9	0.5
test item	2.0		-	85.1	100.2	93.6	15.5	1.0	95.7	126.3	103.3	14.1	0.8
test item	6.0		-	86.0	94.5	98.7	31.9	2.1	94.4	134.0	103.2	11.2	0.6
test item	18.0	P	-	89.1	166.1	106.4	13.4	0.9	102.8	137.8	100.5	14.9	0.8
test item	54.0	P	-	77.1	108.7	93.0	14.8	1.0	70.3	132.1	102.3	13.9	0.7
test item	162.0	P	-	36.6	culture was not continued#				45.5	culture was not continued#
solvent control (DMSO)			+	100.0	100.0	100.0	13.6	1.0	100.0	100.0	100.0	9.7	1.0
positive control (DMBA)	1.1		+	52.1	109.8	88.2	764.5	56.1	53.9	112.0	83.6	647.7	66.7
test item	0.7		+	103.7	92.6	97.6	16.1	1.2	95.0	92.4	92.2	18.7	1.9
test item	2.0		+	98.9	90.0	107.3	10.2	0.7	98.9	108.8	98.2	31.1	3.2
test item	6.0		+	96.3	100.2	87.4	13.4	1.0	100.0	113.1	91.6	18.2	1.9
test item	18.0	P	+	107.8	100.5	97.0	12.3	0.9	99.7	115.0	90.9	16.8	1.7
test item	54.0	P	+	89.4	77.2	92.8	9.7	0.7	99.2	105.7	88.2	19.3	2.0
test item	162.0	P	+	98.3	culture was not continued#				90.1	culture was not continued#
Experiment II / 24h treatment
solvent control (DMSO)			-	100.0	100.0	100.0	12.3	1.0	100.0	100.0	100.0	8.9	1.0
positive control (EMS)	150.0		-	92.8	79.9	89.0	394.0	31.9	96.9	249.6	76.8	222.8	25.0
test item	0.7		-	98.9	culture was not continued##				96.4	culture was not continued##
test item	2.0		-	99.2	79.8	83.3	10.9	0.9	97.2	252.6	74.7	23.3	2.6
test item	6.0		-	99.4	99.6	101.8	17.3	1.4	91.3	214.1	77.1	14.0	1.6
test item	18.0		-	86.8	59.7	94.4	15.1	1.2	79.7	192.5	85.6	12.0	1.3
test item	54.0	P	-	82.5	87.1	101.9	6.6	0.5	75.8	231.8	87.0	17.6	2.0
test item	162.0	P	-	58.6	46.5	98.5	19.4	1.6	60.4	144.2	81.6	13.2	1.5
Experiment II / 4h treatment
solvent control (DMSO)			+	100.0	100.0	100.0	18.8	1.0	100.0	100.0	100.0	12.0	1.0
positive control (DMBA)	1.1		+	56.8	83.7	82.6	418.8	22.2	55.4	49.0	77.6	554.9	46.3
test item	0.7		+	97.7	87.3	78.5	11.1	0.6	99.8	92.6	100.7	11.6	1.0
test item	2.0		+	97.9	96.1	107.4	15.1	0.8	98.0	78.0	100.0	9.2	0.8
test item	6.0		+	96.5	97.5	89.0	12.9	0.7	99.8	82.1	89.4	13.0	1.1
test item	18.0	P	+	98.2	102.7	88.5	18.3	1.0	96.3	104.0	95.9	14.8	1.2
test item	54.0	P	+	95.2	73.9	83.3	12.0	0.6	94.8	66.3	93.0	9.5	0.8
test item	162.0	P	+	94.4	culture was not continued#				85.2	culture was not continued#

P Precipitation

# culture was not continued to avoid analysis of too many precipitating concentrations

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

Conclusions:

Interpretation of results (migrated information):
negative

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 and is therefore considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a GLP-compliant genotoxicity study according to OECD guideline 476 the test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. 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. The highest concentration of 2310 μg/mL used in the pre-experiment was limited by the solubility properties of the test item. The concentration range of the main experiments (0.7 – 162 µg/ml) was limited by precipitation of the test item. The test item was dissolved in THF. Precipitation, visible to the unaided eye, was noted in experiment I at 18.0 μg/mL and above with and without metabolic activation. In experiment II precipitation occurred at 54.0 μg/mL and above without and at 18.0 μg/mL and above with metabolic activation.

No relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in experiment I and II up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the second culture of the first experiment at 2.0 μg/mL with metabolic activation. The effect however, was based on a rather low solvent control of 9.7 mutant colonies/10⁶ cells and has no biological relevance as it was neither dose dependent as indicated by the lacking statistical significance nor reproduced in the parallel culture. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.9 up to 18.8 mutants per 10⁶ cells; the range of the groups treated with the test item was from 6.6 up to 31.9 mutants per 10⁶ 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. 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 and is therefore 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

Additional information

Additional information from genetic toxicity in vitro:

Ames Test

The mutagenic potential of the test substance was investigated in a GLP-compliant Ames test (Covance, 1997), performed on Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli strain WP2 uvrA in the presence and absence of a metabolic activation system (S9 mix). None of the tested concentrations (33.3, 100, 333, 1000, 3333 and 5000 µg/plate) led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control either with or without metabolic activation. This result was confirmed in a second independent experiment. Concentrations of 333 - 5000 µg/plate resulted in precipitation of the test substance; no cytotoxicity was noted up to the limit concentration. The positive controls induced the appropriate responses. Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

This result was confirmed in a second GLP-compliant Ames test (Corning, 1996) performed with Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli strain WP2 uvrA. In this study, test article concentrations of 33.3, 100, 333, 1000, 3333 and 5000 µg/plate were tested both in the presence and absence of a metabolizing activation system. All concentrations were tested in duplicates. Similar to the results above, Concentrations of 333 - 5000 µg/plate resulted in precipitation of the test item and no cytotoxicity was noted up to the limit concentration. An increase in the number of his+ or trp+ revertants was not observed either with or without S9 mix. Overall, based on the presented data, the test substance is considered to be not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.

Chromosome Aberration Test

In a GLP compliant mammalian cell cytogenetic assay (Covance, 1997) the test substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) in Chinese hamster ovary (CHO) cells in vitro both in the presence and in the absence of a metabolizing system (S9-Mix). The concentrations of the test substance used in the experiments were 15.7, 31.3, 62.5, 125, 250, and 499 µg/mL in the initial trial and 31.3, 62.5, 125, 250, and 499 µg/mL in the confirmatory trial. Clastogenic activity was assessed at concentrations of 31.3, 125, 250, and 499 µg/mL. In studies performed without metabolic activation the cells were treated with the test article for 18 and 42 hours; with S9-Mix incubation was limited to 3 hours followed by a 21 hour recovery time. In all experiments performed, no biologically significant increase in the number of specific chromosome aberrations was observed. No evidence of a clastogenic potential was observed. The test item is therefore considered negative for inducing chromosomal aberrations in CHO cells with and without metabolic activation.

HPRT Test

In a GLP-compliant genotoxicity study according to OECD guideline 476 the test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster (Harlan, 2013). The study was performed in two independent experiments, using identical experimental procedures. 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. The concentration range of the main experiments (0.7 – 162 µg/ml) was limited by precipitation of the test item. The test item was dissolved in THF. Precipitation, visible to the unaided eye, was noted in experiment I at 18.0 μg/mL and above with and without metabolic activation. In experiment II precipitation occurred at 54.0 μg/mL and above without and at 18.0 μg/mL and above with metabolic activation. No relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in experiment I and II up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. Appropriate reference mutagens (EMS and DMBA), 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 reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Justification for selection of genetic toxicity endpoint
Most recent GLP-compliant guideline study

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.