Cyclohexadecen-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay in vitro (Ames, OECD 471): negative

Chromosome Aberration assay in vitro (OECD 473): negative

Gene Mutation Assay in Chinese Hamster V 79 Cells in vitro (OECD476): negative

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:

2005-10-02

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/ß-Naphthoflavone induced rat liver

- method of preparation of S9 mix: prepared from 8 - 12 weeks old male Wistar Hanlbm rats (approx. 220 - 320 g) induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and ß-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week. The protein concentration in the S9 preparation was 34.5 mg/mL (lot no. R 300404) in the pre-experiment and experiment I (strains TA 98 and TA 100), and 32.8 mg/mL (lot no. R 151004) in experiment I and II.

- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
5 mM NADP

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, Ila: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Concentrations applied in the main experiment (I, II and IIa) werer based on the results of the Pre-Experiment.

Vehicle / solvent:

- Vehicle used: ethanol

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation, experiment I) and preincubation (experiment II and IIa)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Rationale for test conditions:

according to the test guidelines

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data is not required.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated weakly at different concentrations in the overlay agar. The undissolved particles of the test item had no influence on the data recording.


Ames test:
- Signs of toxicity : cytotoxicity was observed

HISTORICAL CONTROL DATA
- Positive historical control data: Please refer to "Any other information on results"
- Negative (solvent/vehicle) historical control data: Please refer to "Any other information on results"

Table 1 Summary of Results Pre-Experiment and Experiment I

 

Metabolic Activation	Test Item	Dose Level (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 98	TA 100
Without Activation	Ethanol		36 ± 6	92 ± 14
	Untreated		26 ± 6	99 ± 6
	Aurelione	3 µg	26 ± 3	91 ± 25
		10 µg	28 ± 4	83 ± 3
		33 µg	29 ± 7	74 ± 12
		100 µg	31 ± 7	86 ± 12
		333 µg	33 ± 6	70 ± 6
		1000 µg	33 ± 4	78 ± 11
		2500 µg	34 ± 7	79 ± 5
		5000 µg	31 ± 3	64 ± 7
	4-NOPD	10 µg	376 ± 5
	Sodium azide	10 µg		2211 ± 80
With Activation	Ethanol		49 ± 3	118 ± 10
	Untreated		37 ± 6	114 ± 5
	Aurelione	3 µg	49 ± 6	93 ± 11
		10 µg	44 ± 6	104 ± 6
		33 µg	51 ± 5	112 ± 30
		100 µg	41 ± 4	124 ± 3
		333 µg	43 ± 9	96 ± 9
		1000 µg	37 ± 7	44 ± 2
		2500 µg	30 ± 5	45 ± 5
		5000 µg	32 ± 8	48 ± 3
	2-AA	2.5 µg	2505 ± 191	2282 ± 179

 

4-NOPD      4-nitro-o-phenylene-diamine

2-AA            2-aminoanthracene

 

Table 2 Summary of Results Experiment I

 

Metabolic Activation	Test Item	Dose Level (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 102
Without Activation	Ethanol		30±7	17±6	450±15
	Untreated		31±2	12±3	416±27
	Aurelione	33 µg	33±8	20±7	439±24
		100 µg	31±9	15±6	453±28
		333 µg	39±6	8±3	365±62
		1000 µg	32±5	13±5	241±47
		2500 µg	30±15	10±2	167±25
		5000 µg	21±4	18±3	156±23
	Sodium azide	10 µg	1228±152
	4-NOPD	50 µg		94±12
	MMS	4.0 µL			5696±196
With Activation	Ethanol		33±8	17±3	554±37
	Untreated		32±2	17±5	482±69
	Aurelione	33 µg	35±8	18±1	520±76
		100 µg	32±4	18±3	529±69
		333 µg	32±2	9±4	498±10
		1000 µg	27±6	9±1	263±13
		2500 µg	30±12	14±3	189±13
		5000 µg	28±3	9±3	165±18
	2-AA	2.5 µg	270±12	219±33
	2-AA	10.0 µg			4004±238

 

4-NOPD      4-nitro-o-phenylene-diamine

MMS           methyl methane sulfonate

2-AA            2-aminoanthracene

 

 

 

Table 3 Summary of Results Experiment II

 

Metabolic Activation	Test Item	Dose Level (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98		TA 100	TA 102
					II	IIa
Without Activation	Ethanol		24 ± 2	11 ± 1	34 ± 3	21 ± 2	132 ± 4	301 ± 3
	Untreated		22 ± 6	12 ± 3	35 ± 11	27 ± 6	144 ± 22	292 ± 14
	Aurelione	10 µg	24 ± 8	9 ± 2	31 ± 4	26 ± 4	125 ± 14	325 ± 32
		33 µg	29 ± 6	11 ± 2	32 ± 12	24 ± 2	122 ± 7	327 ± 20
		100 µg	24 ± 7	8 ± 1	32 ± 6	21 ± 4	113 ± 13	305 ± 19
		333 µg	31 ± 2	11 ± 3	43 ± 5	7 ± 5	77 ± 5	243 ± 48
		1000 µg	23 ± 4	15 ± 8	52 ± 8	14 ± 5	72 ± 13	140 ± 3
		2500 µg	30 ± 6	9 ± 3	101 ± 13	n.a.	63 ± 12	84 ± 9
		5000 µg	27 ± 9	12 ± 2	80 ± 4	n.a.	56 ± 1	26 ± 5
	Sodium azide	10 µg	1291 ± 46				1773 ± 102
	4-NOPD	10 µg			458 ± 61
	4-NOPD	50 µg		100 ± 7		354 ± 14
	MMS	4.0 µL						1593 ± 130
With Activation	Ethanol		36 ± 7	31 ± 8	50 ± 1		151 ± 10	317 ± 16
	Untreated		35 ± 4	36 ± 5	60 ± 2		160 ± 17	402 ± 5
	Aurelione	10 µg	37 ± 7	34 ± 3	53 ± 6		138 ± 3	360 ± 12
		33 µg	30 ± 3	31 ± 4	58 ± 10		142 ± 16	213 ± 32
		100 µg	31 ± 2	33 ± 3	49 ± 3		129 ± 5	141 ± 22
		333 µg	39 ± 3	41 ± 5	61 ± 2		114 ± 12	57 ± 13
		1000 µg	37 ± 8	31 ± 2	56 ± 6		85 ± 17	32 ± 13
		2500 µg	35 ± 5	4 ± 1	35 ± 5		98 ± 20	19 ± 7
		5000 µg	27 ± 4	6 ± 2	33 ± 3		77 ± 8	16 ± 2
	2-AA	2.5 µg	376 ± 34	166 ± 19	1417 ± 81		1462 ± 127
	2-AA	10.0 µg						1628 ± 128

 

4-NOPD      4-nitro-o-phenylene-diamine

MMS           methyl methane sulfonate

2-AA            2-aminoanthracene

n.a.              not analysable due to reduced background growth

 

 

 

Table 4 Historical Control Data

 

Strain		Without S9 mix				With S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control Negative control Positive control	20 18 3042	6 5 756	9 10 1003	30 29 3618	18 18 357	10 10 111	7 9 172	39 38 476
TA 1537	Solvent control Negative control Positive control	11 12 97	7 6 21	4 5 52	29 29 191	18 18 141	9 6 47	6 8 94	31 29 380
TA 98	Solvent control Negative control Positive control	24 26 379	9 10 98	14 15 137	58 52 976	37 43 1239	13 15 510	21 17 229	57 64 5466
TA 100	Solvent control Negative control Positive control	121 141 2089	29 23 408	91 101 1262	198 189 2872	149 147 921	36 43 346	109 13 546	281 254 2589
TA 102	Solvent control Negative control Positive control	338 326 2764	77 53 1479	242 242 1220	430 390 5593	426 450 2104	70 60 752	332 280 872	514 556 3052

 

Mean = mean value of revertants/plate

SD = standard deviation

Min = minimal value

Max = maximal value

 

 

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102, according to OECD 471. The assay was performed in two independent experiments both with and without liver microsomal activation. An additional experiment was performed as pre-incubation with strain TA 98 without S9, only (reported as exp. IIa). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment 1: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, Ila: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background background growth in almost all strains with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants, occurred in strains TA 100 (with S9 mix) and TA 102 (with and without S9 mix) in experiment I and in strains TA 98 (IIa), 100, and TA 102 without S9 mix and in strains TA 1537 and TA 102 with S9 mix in experiment II. A seemingly dose dependent increase in revertant colony numbers was observed following treatment with the test item in strain TA 98 without metabolic activation in experiment II. The number of colonies reached or exceeded the threshold of twice the number of the corresponding solvent control at concentrations of 2500 µg/plate and above in the absence of metabolic activation. To verify the results an additional experiment was performed as pre-incubation test with TA 98 without S9 mix (exp. IIa). In this experiment an increase of the revertant colonies was not observed. However, reduced background growth was observed from 333 µg/plate up to 5000 µg/plate. Therefore, the observed large number of very small colonies in experiment II were judged to be based upon toxicity rather than indicating a possible mutagenic potential. A major reduction of the background growth results in less bacteria competing for the traces of histidine introduced by the top agar. The traces of histidine are sufficient to allow the surviving bacteria to grow into very small colonies until the histidine is depleted. All other strains did not show any mutagenic effect of the test item up to the maximal concentration applied. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-09-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: from healthy human donors

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: blood samples from human donors
- Suitability of cells: according to guideline
- Normal cell cycle time:

For lymphocytes:
- Sex, age and number of blood donors: blood samples were obtained from healthy donors not receiving medication:
Experiment I: two donors: 44-year-old male and 45 year-old female;
Experiment II 47-year old female)
- whole blood was used
- Whether blood from different donors were pooled or not: no
- mitogen: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media:
DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1, containing 10% FCS (fetal calf serum), antibiotic solution contains 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. The medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), the anticoagulant heparin (25,000 U.S.P.-U/mL) and HEPES (final concentration 10 mM)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : phenobarbital/betanaphthoflavone induced rat liver (8-12 weeks old male Wistar rats)
- method of preparation of S9 mix: The S9 fractions were produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g
- concentration or volume of S9 mix and S9 in the final culture medium : final protein concentration of 0.75 g/mL in cultures

Test concentrations with justification for top dose:

Please refer to table 1 bleow. Test concentrations in the main test were chosend based on the results of the cytotoxicity pre-test.

Using reduced mitotic indices as an indicator for cytotoxicity in Experiment I, clearly reduced mitotic indices were observed at 285.2 µg/mL after 4 hrs treatment in the absence of S9 mix. In the presence of S9 mix, no clear cytotoxicity could be observed up to the highest applied concentration. After continuous treatment in the absence of S9 mix, cytotoxicity was observed at 49 µg/mL and above. Therefore, 93.1 µg/mL were chosen as top treatment concentration in Experiment II, in the absence of S9 mix. In the presence of S9 mix, 2675.0 µg/mL were chosen as top treatment concentration with respect to the results obtained in Experiment I.

Vehicle / solvent:

- Vehicle/solvent used: ethanol

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h, 22 h and 46 h (without S9

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: colcemid
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): fixation of cells with methanol glacial acetic acid; staining according to Flurescent and Giemsa technique
- Number of cells spread and analysed per concentration: at least two slides per experimental group were prepared
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II, in the presence of metabolic activation, where only 50 metaphase plates were scored due to strong clastogenic effects. Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.

Rationale for test conditions:

according to the guidelines

Evaluation criteria:

A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher's exact test (11) (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria 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.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 0.8 % polyploid cells).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Table 2 Summary of results of the chromosomal aberration study

experiment	Preparation interval	Test item concentration µg/mL	Polyploid cells in %	Mitotic indices in % of control	Incl. gaps	Aberrant cells in % excl. gaps	With exchanges
Exposure period 4 hrs without S9
I	22 hrs	Solvent control	0.0	100.0	1.0	1.0	1.0
		Positive control	0.0	85.5	20.5	18.0	5.5
		53.2	0.2	113.9	2.0	1.5	0.0
		93.1	0.0	108.0	1.5	1.5	0.0
		163.0	0.4	97.6	0.5	0.5	0.0
Exposure period 22 hrs without S9
I	22 hrs	Solvent control	0.0	100.0	4.0	3.5	0.0
		Positive control	0.2	31.1	21.0	20.5	8.0
		16.0	0.2	83.9	2.5	2.0	0.0
		28.0	0.4	85.0	1.0	1.0	0.0
		49.0	0.0	30.7	2.5	2.5	0.0
Exposure period 46 hrs without S9
II	46 hrs	Solvent control	0.2	100.0	0.5	0.5	0.0
		Positive control	0.0	108.0	9.5	9.0	1.0
		9.9	0.2	118.2	1.5	1.5	0.0
		17.4	0.2	93.0	0.5	0.5	0.0
		30.4	0.2	79.3	2.0	2.0	0.0
Exposure period 4 hrs with S9
I	22 hrs	Solvent control	0.2	100.0	1.0	1.0	0.0
		Positive control	0.0	40.4	11.0	9.0	1.5
		53.2	0.0	86.8	2.0	1.5	0.0
		93.1	0.4	76.9	1.0	1.0	0.0
		163.0	0.0	76.0	3.0	2.5	0.0
II	46 hrs	Solvent control	0.0	100.0	0.5	0.5	0.0
		Positive control	0.0	26.2	37.0	37.0	10.0
		93.1	0.0	89.5	1.0	0.5	0.0
		163.0	0.0	86.9	2.0	1.5	0.0
		1528.6	0.0	88.5	4.5	3.5	0.0
		2675.0	0.2	34.0	2.5	1.5	0.0

Conclusions:

Under the experimental conditions of this study, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.

Executive summary:

The test item, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments according to OECD 473 TG. The following study design was performed:

	Without S9			With S9
	Exp I		Exp II	Exp I	Exp II
Exposure period	4 hrs	22 hrs	46 hrs	4 hrs	4 hrs
Recovery	18 hrs	-	-	18 hrs	42 hrs
Preparation interval	22 hrs	22 hrs	46 hrs	22 hrs	46 hrs

In each experimental group two parallel cultures were analysed at three to four test concentrations. Per culture 100 metaphase plates were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the presence of metabolic activation, where only 50 metaphase plates were scored due to strong clastogenic effects. The highest applied concentration in Experiment I (2675.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of test item precipitation in accordance to OECD Guideline 473. In Experiment I, in the absence of S9 mix, after pulse treatment, and in Experiment II, in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment I, after continuous treatment in the absence of S9 mix, and in Experiment II, in the presence of S9 mix, clear cytotoxic effects could be observed at the highest evaluated concentration. In Experiment I, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration being far in excess of test item precipitation. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment II, in the presence of S9 mix, a single significant increase in the number of aberrant cells was observed. This value was in the range of the laboratory's historical control data and thus regarded as being without biological relevance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-31 to 2019-04-17

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 using the Hprt and xprt genes)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: “Kanpoan No. 287 – Environment Protection Agency“ “Eisei No. 127 – Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 – Ministry of International Trade & Industry“

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hprt locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

For cell lines:
- Absence of Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: 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).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/beta-naphthoflavone induced rat liver S9
- concentration or volume of S9 mix and S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test

Test concentrations with justification for top dose:

Relevant cytotoxic effects, indicated by a relative cloning efficiency of 50% or below were observed at 15.7 µg/mL, and above without metabolic activation and at 125.0 µg/mL and above with metabolic activation. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Concentrations main test:
Exposure period 4 h, -S9
0.2; 0.5; 0.9; 1.9; 3.8; 7.5; 15.0; 30.0 µg/mL

Exposure period 4 h, +S9
1.9; 3.8; 7.5; 15.0; 30.0; 60.0; 120.0; 240.0 µg/mL

Vehicle / solvent:

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

- Justification for percentage of solvent in the final culture medium: final concentration of ethanol in the culture medium was 0.5% (v/v) as recommended by the guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Approximately 0.7 to 1.2 x 10^7
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 h
- Exposure duration/duration of treatment: 4h

FOR GENE MUTATION:
- Expression time: 6 days
- Selection time: 8 -11 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependency of mutant frequencies. The numbers of mutant colonies generated 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.

Linear regression:
Experimental group: -S9: p-value (calculated for the mean mutant frequencies of culture I and II): 0.083
Experimental group: +S9: p-value (calculated for the mean mutant frequencies of culture I and II): 0.127

A t-test was not performed since the 95% confidence interval was not exceeded at any experimental point.
However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 7.5 µg/mL and above (-S9); at 60.0 µg/mL and above (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: solvent control pH 7.13; test item (1000 µg/mL) pH 7.11
- Data on osmolality: solvent control: 413 mOsm; test item (1000 µg/mL): 391 mOsm
- Possibility of evaporation from medium: no
- Precipitation and time of the determination: precipitation was observed in the main test at 15 (-S9) and 60 (+S9) µg/mL, respectively

Table 1 Summary of results

	Concentration µg/mL	precipitation	S9	Relative cloning efficiency	Relative cell density	Rel. adjusted cloning efficiency	Mutant colonies/10^6 cells	95 % confidence interval
Main Experiment 4 h treatment				Mean values of culture I and II
Solvent control with ethanol			-	100.0	100.0	100.0	11.1	2.8 – 30.9
Positive control (EMS)	300.0		-	93.2	113.1	106.1	150.9	2.8 – 30.9
Test item	0.2		-	96.2	109.6	107.6	#
	0.5		-	78.8	107.1	84.7	#
	0.9		-	64.4	92.3	59.8	14.0	2.8 – 30.9
	1.9		-	63.2	110.0	72.3	10.2	2.8 – 30.9
	3.8		-	60.5	109.5	68.2	13.4	2.8 – 30.9
	7.5		-	24.8	104.3	24.4	9.6	2.8 – 30.9
	15.0	P	-	4.4	29.3	1.2	22.7	2.8 – 30.9
	30.0	P	-	Culture was discontinued due to exceedingly severe cytotoxicity
Solvent control with ethanol			+	100.0	100.0	100.0	13.0	3.1 – 30.7
Positive control (EMS)	2.3		+	93.6	96.0	89.8	125.6	3.1 – 30.7
Test item	1.9		+	99.3	107.1	106.0	#
	3.8		+	91.8	114.1	104.7	11.9	3.1 – 30.7
	7.5		+	91.2	97.6	88.9	8.7	3.1 – 30.7
	15.0		+	75.7	107.8	83.8	9.9	3.1 – 30.7
	30.0		+	74.9	98.5	73.7	11.7	3.1 – 30.7
	60.0	P	+	44.1	13.1	5.8	16.5	3.1 – 30.7
	120.0	P	+	Culture was discontinued due to exceedingly severe cytotoxicity
	240.0	P	+	Culture was discontinued due to exceedingly severe cytotoxicity

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

 

Table 2 Historical control values

2014 - 2018
Number of mutant colonies per 10^6 cells
without metabolic activation (4 hours treatment time)
	Positive control EMS 150 and 300 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF, EGDE)
Range:	53.9 – 872.3	3.4 – 41.0
Mean value:	223.3	16.9
Standard deviation:	101.1	7.0
95% confidence interval	--	2.8 – 30.9
Number of studies:	199	199
with metabolic activation (4 hours treatment time)
	Positive control DMBA 1.1 and 2.3 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF, EGDE)
Range:	55.6 – 739.9	2.4 – 40.4
Mean value:	188.0	16.9
Standard deviation:	98.6	6.9
95% confidence interval	--	3.1 – 30.7
Number of studies:	193	193

Conclusions:

The test item was found to be not mutagenic under the conditions of this study.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1000.0 µg/mL) was based on the solubility properties of the test item. The concentration range of the main experiment was limited by precipitation of the test item. No substantial and dose dependent increase of the mutation frequency 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 reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test item is 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

Bacterial reverse mutation assay (Ames), OECD 471

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102, according to OECD 471. The assay was performed in two independent experiments both with and without liver microsomal activation. An additional experiment was performed as pre-incubation with strain TA 98 without S9, only (reported as exp. IIa). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment 1: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, Ila: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth in almost all strains with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants, occurred in strains TA 100 (with S9 mix) and TA 102 (with and without S9 mix) in experiment I and in strains TA 98 (IIa), 100, and TA 102 without S9 mix and in strains TA 1537 and TA 102 with S9 mix in experiment II. A seemingly dose dependent increase in revertant colony numbers was observed following treatment with the test item in strain TA 98 without metabolic activation in experiment II. The number of colonies reached or exceeded the threshold of twice the number of the corresponding solvent control at concentrations of 2500 µg/plate and above in the absence of metabolic activation. To verify the results an additional experiment was performed as pre-incubation test with TA 98 without S9 mix (exp. IIa). In this experiment an increase of the revertant colonies was not observed. However, reduced background growth was observed from 333 µg/plate up to 5000 µg/plate. Therefore, the observed large number of very small colonies in experiment II were judged to be based upon toxicity rather than indicating a possible mutagenic potential. A major reduction of the background growth results in less bacteria competing for the traces of histidine introduced by the top agar. The traces of histidine are sufficient to allow the surviving bacteria to grow into very small colonies until the histidine is depleted. All other strains did not show any mutagenic effect of the test item up to the maximal concentration applied. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Chromosome Aberration test in vitro, OECD 473

The test item, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments according to OECD 473 TG. The following study design was performed:

	Without S9			With S9
	Exp I		Exp II	Exp I	Exp II
Exposure period	4 hrs	22 hrs	46 hrs	4 hrs	4 hrs
Recovery	18 hrs	-	-	18 hrs	42 hrs
Preparation interval	22 hrs	22 hrs	46 hrs	22 hrs	46 hrs

In each experimental group two parallel cultures were analysed at three to four test concentrations. Per culture 100 metaphase plates were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the presence of metabolic activation, where only 50 metaphase plates were scored due to strong clastogenic effects. The highest applied concentration in Experiment I (2675.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of test item precipitation in accordance to OECD Guideline 473. In Experiment I, in the absence of S9 mix, after pulse treatment, and in Experiment II, in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment I, after continuous treatment in the absence of S9 mix, and in Experiment II, in the presence of S9 mix, clear cytotoxic effects could be observed at the highest evaluated concentration. In Experiment I, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration being far in excess of test item precipitation. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment II, in the presence of S9 mix, a single significant increase in the number of aberrant cells was observed. This value was in the range of the laboratory's historical control data and thus regarded as being without biological relevance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.

Gene Mutation Assay in Chinese Hamster V 79 Cells in vitro, OECD 476

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.The treatment period was 4 hours with and without metabolic activation(S9).The maximum test item concentration of the pre-experiment (1000.0 µg/mL) was based onthe solubility properties of the test item. The concentration range of the main experiment was limited by precipitation of the test item. No substantial and dose dependent increase of the mutation frequency was observed in themain experiment.

 

Doses applied in the gene mutation assay
(concentrations given in bold letters were chosen for the mutation rate analysis)

exposure period	S9 mix	concentrations in µg/mL
		experiment I
4 hours	-	0.2	0.5	0.9	1.9	3.8	7.5	15.0 P	30.0 P
4 hours	+	1.9	3.8	7.5	15.0	30.0	60.0P	120.0P	240.0P

P = precipitation visible at the end of treatment

 

Appropriate reference mutagens, used as positive controls (ethylmethane sulfonate (-S9), and 7,12 -dimethylbenz(a)anthracene (+S9)), 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. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity, the test item does not require classifcation as mutagen according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.