2-(chloromethyl)-4-methylquinazoline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxic potential of the substance was investigated in an in vitro Ames test according to OECD 471 and in an in vitro chromosome aberration test according to OECD 473. The substance was tested positive in the Ames test in S. typhimurium strains TA98 and TA 100. Additionally the compound induced chromosome aberrations in CHO cells. Based on animal welfare and the available data the substance will be classified worst case as Mutagen Cat. 2 (H341).

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:

2006-11-24 to 2007-01-25

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.5100 - Escherichia coli WP2 and WP2 uvrA Reverse Mutation Assays (June 1996 (Public Draft))

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium
TA1537: his C 3076; rfa; uvrB: frameshift mutations
TA98: his D 3052; rfa; uvrB; R-factor: frameshift mutations
TA1535: his G 46; rfa; uvrB: base-pair substitutions
TA100: his G 46; rfa; uvrB; R-factor: base-pair substitutions

Escherichia coli
WP2 uvrA: trp E; uvrA: base-pair substitutions

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Main study:
Experiment I (Initial Mutation Test, plate incorporation test): 5000; 1250; 1000; 500; 250; 125; 62.5 and 31.3 µg/plate
Experiment II (Confirmatory Mutation Test, preincubation test): 5000; 1250; 1000; 500; 250; 125 and 62.5 µg/plate
Repeat Experiment I ( plate incorporation test only with TA 98 and TA 100): 5000; 1250; 1000; 500; 250; 125 and 62.5 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: This solvent was compatible with the survival of the bacteria and the S9 activity. The solvent applied in the experiments was chosen according to the result of the solubility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar; in experiment I (Initial Mutation Test) plate incorporation, experiment II (Confirmatory Mutation Test) pre-incubation, Repeat Experiment I (only with TA 98 and TA 100) plate incorporation

DURATION
- Preincubation period: 20 min
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: background growth, revertant colony numbers, pinpoint colony

Evaluation criteria:

The test is considered acceptable if for each strain:
– the bacteria demonstrate their typical responses to crystal violet and ampicillin
– the control plates without S9 mix are within the historical control data range
– corresponding background growth on both negative control and test plates occurs
– the positive controls show a distinct enhancement over the control plate

A test item is considered mutagenic if:
– a dose–related increase in the number of revertants occur and/or
– a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

A biologically relevant increase is described as follows:
– if in strain TA 100 the number of reversions is at least twice as high when compared to the spontaneous reversion rate of the solvent control plates,
– if in strains TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher as compared to the spontaneous reversion rate of the solvent control plates.

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Pre-Experiment for Toxicity:
In the case of Salmonella typhimurium TA 98 the revertant colony numbers were significantly increased in a dose related manner, in the concentration range of 1250-78.13 µg/plate, without metabolic activation, and in the range of 625-78.13 µg/plate, with addition of metabolic activation [additionally at 39.06 µg/plate (±S9 Mix) and at 19.53 µg/plate (+S9 Mix) slight increases were observed]. In the case of Salmonella typhimurium TA 100 dose-related increases were obtained in the concentration range of 625-39.06 µg/plate, without metabolic activation, and in the range of 625-78.13 µg/plate, with addition of metabolic activation. The observed increases were biologically relevant, with mutation rates above the threshold for being positive in Salmonella typhimurium TA 98 in the concentration range of 1250-156.25 µg/plate (–S9 Mix). There was no bacterial growth at 5000 and 2500 µg/plate with and without metabolic activation in TA 98 and TA 100 and additionally at 1250 µg/plate in Salmonella typhimurium TA 100 (–S9 Mix; in Salmonella typhimurium TA 98 one-three colonies were detected at 2500 µg/plate (+S9 Mix)). In both Salmonella typhimurium strains at 1250 µg/plate, with metabolic activation, small, pinpoint colonies were observed.

Pre-Experiment for Toxicity:
TA98 and TA100
5000 and 2500 µg/plate with and without metabolic activation: Reduced background lawn development
1250 µg/plate with metabolic activation: Appearance of pinpoint colonies

Experiment I (Initial Mutation Test, plate incorporation test):

In Experiment I for Salmonella typhimurium TA 98 positive results were obtained. The increases of revertant colony numbers, compared to the solvent control plates, were significant and dose-dependent in the concentration range of 1250-250 µg/plate (slight increase was observed at 250 µg/plate), in absence of metabolic activation. The mutation rates reached the biologically relevant threshold at the concentrations of 1250 and 1000 µg/plate (MF = 3.96 and 4.62, respectively). Dose dependent increases below the threshold level were observed for this strain in presence of metabolic activation as well (in the concentration range of 1250-500 and additionally at 31.3 µg/plate).

In the case of Salmonella typhimurium TA 100, the highest increases were observed at concentrations of 1000 µg/plate (–S9 Mix) and 500 µg/plate (±S9 Mix), but increases remained below the biological threshold level.

For Salmonella typhimurium TA 1535, TA 1537 and Escherichia coli WP2 uvrA sporadically increased revertant colony numbers observed were considered to reflect the biological variability of the test.

In Experiment I inhibitory effects of the test item were observed in all examined bacterial strains. The signs of the inhibitory effects included reduced revertant colony numbers, compared to the solvent control plates, and reduced background lawn development. No bacterial growth was observed in all Salmonella typhimurium strains at the highest concentration level of 5000 µg/plate, without and with addition of metabolic activation, and additionally in Salmonella typhimurium TA 1537 at 1250 µg/plate (growth of 0-1 colony/plate was observed with metabolic activation).

In Salmonella typhimurium TA 1535 revertant colony numbers were reduced compared to the solvent control plates in the concentration range of 1250- 500 µg/plate (±S9 Mix), in TA 1537 at 1000 µg/plate (–S9 Mix), and in Escherichia coli WP2 uvrA at 5000 µg/plate (±S9 Mix). The slightly reduced revertant colony numbers, observed in Escherichia coli WP2 uvrA at 31.3µg/plate (–S9 Mix) and 250 µg/plate (+S9 Mix) reflect the biological variability of this test.

Repeat Experiment I ( plate incorporation test only with TA 98 and TA 100):

Because of the positive and partially equivocal results observed in Experiment I (Initial Mutation Test, plate incorporation test) the plate incorporation test was repeated with Salmonella typhimurium TA 98 and TA 100 (Repeat Experiment I), using the same concentration levels as in Experiment I (Intitial Mutation Test), the lowest concentration level of Experiment I (Initial Mutation Test) 31.3 µg/plate was left out. Significantly, dose-related increased revertant colony numbers were observed in both examined strains with and without addition of metabolic activation. In this repeated experiment the positive results obtained in Experiment I (Initial Mutation Test) in Salmonella typhimurium TA 98 at the concentrations of 1250 and 1000 µg/plate (–S9 Mix) was confirmed. In contrast to Experiment I (Initial Mutation Test) , revertant colony numbers were above the genotoxicological threshold in Salmonella typhimurium TA 98 at 500 µg/plate and in TA 100 at 1250 -500 µg/plate without metabolic activation and in both strains at 1250 and 1000 µg/plate, with metabolic activation. Further, a borderline increase of revertant colony numbers was detected in TA 100 with S9 Mix at 500 µg/plate (MF=1.98). Similar to the results of the Experiment I (Initial Mutation Test) no bacterial growth was observed in both strains with and without metabolic activation at 5000 µg/plate indicating the strong inhibitory effect of the test item.

Experiment II (Confirmatory Mutation Test, preincubation test):

In Experiment II (Confirmatory Mutation Test) no positive result was obtained for Salmonella typhimurium TA 98, but in the case of TA 100, with addition of metabolic activation at 500 µg/plate.

In the case of Salmonella typhimurium TA 98, TA 100 and TA 1537 the increases in revertant colony numbers remained below the genotoxicological threshold level. However, dose-related increases, compared to the solvent control plates, were observed in the concentration range of 500-62.5µg/plate, without metabolic activation. In Salmonella typhimurium TA 1535 additional increases were observed at 125 µg/plate, without metabolic activation, and in Escherichia coli WP2 uvrA at 500 µg/plate, without metabolic activation. No dose dependence occurred at the sporadically increased revertant colony numbers obtained in presence of metabolic activation. In Salmonella typhimurium TA 1537 the highest increase (MF=2.30) was observed at 500 µg/plate, with addition of metabolic activation.

In Experiment II (Confirmatory Mutation Test) strong inhibitory effects of the test item were observed in all examined bacterial strains. The inhibitory effects were reduced revertant colony numbers, compared to the solvent control plates, reduced background lawn development and pinpoint colony appearance.

Inhibition was observed with and without metabolic activation down to and including the concentration level of 1000 µg/plate in the Salmonella typhimurium strains and at the highest concentration level at 5000 µg/plate in case of Escherichia coli WP2 uvrA.

In the case of Salmonella typhimurium TA 100 (±S9 Mix) and TA 1535 (+S9 Mix) at 1000 µg/plate the revertant colony numbers were increased, but reduced background lawn development was observed and the colonies were small, pinpoint colonies. No bacterial growth (or only growth of several colonies) was observed in the case of Salmonella typhimurium strains at the concentrations of 5000 µg/plate (+/- S9 Mix) and additionally at 1250 µg/plate in TA 100 (–S9 Mix), in TA 1537 (+S9 Mix).

Summary Tables of the Results of Experiment I

Experiment I.: Initial Mutation Test (Plate Incorporation Test)
Concentrations (µg/plate)	Mean values of revertants per plate Mutation factor (MF)	Salmonella typhimurium test strains								Escherichia coli WP2uvrA
		TA 98		TA 100		TA 1535		TA 1537
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
5000	Mean	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	3.3	21.3
	MF	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.11	0.67
1250	Mean	101.7	44.0	223.7	124.7	12.7	8.0	0.0	0.7	37.3	28.3
	MF	3.96	1.78	1.34	0.95	0.54	0.46	0.00	0.07	1.23	0.89
1000	Mean	118.7	40.3	284.3	145.0	16.3	9.0	6.0	8.0	30.3	33.0
	MF	4.62	1.64	1.70	1.10	0.70	0.52	0.72	0.80	1.00	1.04
500	Mean	47.0	37.0	307.0	243.7	18.3	11.0	13.7	15.7	33.7	32.7
	MF	1.83	1.50	1.84	1.86	0.79	0.63	1.64	1.57	1.11	1.03
250	Mean	36.0	26.7	217.7	200.7	29.7	14.7	12.3	10.0	32.0	24.3
	MF	1.40	1.08	1.30	1.53	1.27	0.85	1.48	1.00	1.05	0.77
125	Mean	20.7	23.3	191.3	153.3	30.3	16.0	7.7	12.3	34.3	32.3
	MF	0.81	0.95	1.15	1.17	1.30	0.92	0.92	1.23	1.13	1.02
62.5	Mean	28.7	27.3	182.3	154.0	24.7	20.3	10.0	10.3	29.0	33.7
	MF	1.12	1.11	1.09	1.17	1.06	1.17	1.20	1.03	0.96	1.06
31.3	Mean	25.0	35.0	168.7	123.0	23.3	15.7	9.0	10.7	21.7	26.0
	MF	0.97	1.42	1.01	0.94	1.00	0.90	1.08	1.07	0.71	0.82

 

Summary Tables of the Results of Experiment I

Experiment I.: Initial Mutation Test (Plate Incorporation Test)
Controls	Mean values of revertants per plate Mutation factor (MF)	Salmonella typhimurium test strains								Escherichia coli WP2uvrA
		TA 98		TA 100		TA 1535		TA 1537
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	21.0	27.3	189.7	138.7	26.0	16.7	7.0	8.7	26.7	41.3
	MF	0.82	1.11	1.14	1.06	1.11	0.96	0.84	0.87	0.88	1.31
Distilled water control	Mean	-	-	172.7	115.7	26.7	18.7	-	-	34.0	40.3
	MF	-	-	1.03	0.88	1.14	1.08	-	-	1.12	1.27
DMSO control	Mean	25.7	24.7	167.0	131.3	23.3	17.3	8.3	10.0	30.3	31.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
NOPD (4 µg)	Mean	422.3	-	-	-	-	-	-	-	-	-
	MF	16.45	-	-	-	-	-	-	-	-	-
SAZ (2 µg)	Mean	-	-	836.0	-	792.3	-	-	-	-	-
	MF	-	-	4.84	-	29.71	-	-	-	-	-
9AA (50 µg)	Mean	-	-	-	-	-	-	316.3	-	-	-
	MF	-	-	-	-	-	-	37.96	-	-	-
MMS (2µL)	Mean	-	-	-	-	-	-	-	-	664.7	-
	MF	-	-	-	-	-	-	-	-	19.55	-
2AA (2 µg)	Mean	-	1603.0	-	1162.0	-	183.0	-	195.7	-	-
	MF	-	64.99	-	8.85	-	10.56	-	19.57	-	-
2AA (50 µg)	Mean	-	-	-	-	-	-	-	-	-	428.3
	MF	-	-	-	-	-	-	-	-	-	13.53

 

Summary Tables of the Results of Repeat Experiment I

Experiment I.: Repeat Experiment I (Plate Incorporation Test)
Concentrations (µg/plate)	Mean values of revertants per plate Mutation factor (MF)	Salmonella typhimurium test strains
		TA 98		TA 100
		-S9	+S9	-S9	+S9
5000	Mean	0.0	0.0	0.0	0.0
	MF	0.00	0.00	0.00	0.00
1250	Mean	242.0	211.0	356.7	288.0
	MF	9.68	6.09	2.71	2.14
1000	Mean	194.0	185.7	404.3	322.7
	MF	7.76	5.36	3.07	2.40
500	Mean	75.7	68.3	325.7	266.7
	MF	3.03	1.97	2.47	1.98
250	Mean	44.0	50.7	254.7	195.0
	MF	1.76	1.46	1.93	1.45
125	Mean	34.7	31.7	188.7	181.0
	MF	1.39	0.91	1.43	1.34
62.5	Mean	23.0	31.7	147.3	135.7
	MF	0.92	0.91	1.12	1.01

Summary Tables of the Results of Repeat Experiment I

Experiment I.: Repeat Experiment I (Plate Incorporation Test)
Controls	Mean values of revertants per plate Mutation factor (MF)	Salmonella typhimurium test strains
		TA 98		TA 100
		-S9	+S9	-S9	+S9
Untreated control	Mean	20.0	30.3	128.0	134.3
	MF	0.80	0.88	0.97	1.00
Distilled water control	Mean	-	-	137.3	147.7
	MF	-	-	1.04	1.05
DMSO control	Mean	25.0	34.7	131.7	134.7
	MF	1.00	1.00	1.00	1.00
NOPD (4 µg)	Mean	413.0	-	-	-
	MF	16.52	-	-	-
SAZ (2 µg)	Mean	-	-	976.0	-
	MF	-	-	7.11	-
2AA (2 µg)	Mean	-	1454.7	-	1474.7
	MF	-	41.96	-	10.95

 

Conclusions:

The reported data of this mutagenicity assay shows that, under the experimental conditions reported, the test item induces gene mutations by frameshift and base-pair substitution in the genome of the strains used. Therefore, the test item is considered mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was dissolved in Dimethyl sulfoxide (DMSO). In Experiment I (Initial Mutation Tests, plate incorporation test) the tested concentrations were: 5000; 1250; 1000; 500; 250; 125; 62.5 and 31.3 µg/plate. In Experiment II Confirmatory Mutation Tests and Repeat Experiment I (preincubation test and additional repeated plate incorporation test) the concentration levels investigated were slightly modified, the lowest concentration level of the Initial Mutation Test 31.3µg/plate was left out.

Bacteria Salmonella typhimurium TA 98, TA 1537, TA 1535 and TA 100 strains and Escherichia coli WP2 uvrA were used.

A plate incorporation test (Experiment I, Initial Mutation Test) and a preincubation test (Experiment II, Confirmatory Mutation Test) were performed to investigate the potential mutagenic effect of the test item CD 605. Because positive (Salmonella typhimurium TA 98) and inconclusive (Salmonella typhimurium TA 100) results were obtained in Experiment I, the plate incorporation test were repeated for TA 98 and TA 100 (Repeat Experiment I). Each assay was conducted with and without metabolic activation (S9 mix). For each experiment, concentration and controls were run in triplicate.

In Experiment I positive results were obtained for Salmonella typhimurium TA 98, at the concentrations of 1250 and 1000µg/plate, without S9 Mix. In the Repeat Experiment I positive results were obtained for Salmonella typhimurium TA 98 and TA 100 in the concentration range of 1250- 500µg/plate, without S9 Mix and at the concentrations of 1250 and 1000µg/plate with addition of S9 Mix.

In Experiment II positive results were obtained for Salmonella typhimurium TA 100 at the concentration of 500µg/plate, with metabolic activation.

The bacterial growth was strongly inhibited especially in the Salmonella typhimurium strains, at the highest concentration level of 5000µg/plate (plate incorporation tests) and at 5000-1000µg/plate (pre-incubation test), without and with addition of metabolic activation.

The revertant colony numbers of solvent control plates without S9 mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies.

The reported data of this mutagenicity assay shows that, under the experimental conditions reported, the test item induces gene mutations by frameshift and base-pair substitution in the genome of the strains used.

Therefore, CD 605 is considered mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-02-06 to 2007-03-13

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Sub-line (KI)
- doubling time of cells (12-14h)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, once before freezing

Metabolic activation:

with and without

Metabolic activation system:

rodent S9 mix

Test concentrations with justification for top dose:

The results of an experimental pre-test on toxicity were used to select the concentrations of test item for the Chromosome Aberration Assays.
Experiment A:
4.88; 9.76; 19.53; 39.06 µg/ml (without S9)
2.44; 4.88; 9.76; 14.64 µg/ml (with S9)
Experiment B:
2.44; 4.88; 9.76; 14.64 µg/ml (without S9)
2.44; 4.88; 9.76; 14.64 µg/ml (with S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [ DMSO ]

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Remarks:

ethylmethanesulphonate (Experiment A and B without metabolic activation); N-dimethylnitrosamine (Experiment A and B with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hrs with/without metabolic activation (experiment A); 3 hrs with metabolic activation and 20 hrs without metabolic activation (experiment B)
- Expression time (cells in growth medium): 20 hrs (experiment A); 28 hrs (experiment B)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hrs (experiment A); 26 hrs (experiment B)

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: 2 per concentration

NUMBER OF CELLS EVALUATED: 200 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cell counts in relation to solvent control

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The criteria for determining a positive result are:
– a concentration-related increase or a reproducible increase in the number of cells with aberrations.
– biological relevance of the results should be considered first, however, for the interpretation of the data both biological and statistical significance should be considered together.
– an increase in the number of polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
– an increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle progression.

A test item for which the results do not meet the above criteria is considered nonmutagenic in this system.

Statistics:

CHI^2 Test

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Chinese hamster ovary (CHO-KI) cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: CD 605 was dissolved in DMSO. A clear solution was obtained up to a concentration of 25 mg/ml.
- Precipitation: There was no precipitation in the medium at any concentration tested.


RANGE-FINDING/SCREENING STUDIES: The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system (rodent S9-mix). Toxicity was determined by cell counting and results noted as % cells in relation to the solvent control. These results were used to select concentrations of CD 605 for the Chromosome Aberration Assays.


COMPARISON WITH HISTORICAL CONTROL DATA: As in Experiment A, in Experiment B in the absence and in the presence of S9-mix increases in structural chromosome aberrations without gaps were above the historical control data range of the laboratory.

The cytotoxicity at the highest concentrations was adequate in the studies as indicated by reduction of % cell survival of at least 50 %.

In Experiment A, CD 605 induced statistically significant increases in the number of cells with aberrations without gaps at 39.06 µg/ml (P<0.01) at a relative survival rate of 31 % in the absence of metabolic activation, and moderate increase at 14.64 µg/ml at a relative survival rate of 34 % in the presence of metabolic activation, up to and including cytotoxic concentrations. These increases were above the historical solvent control data range of the laboratory.

In Experiment B, with CD 605 examined without S9 mix, over a prolonged treatment period (20 hours), the frequency of the cells with structural chromosome aberrations without gaps showed a statistically significant increase at 9.76 µg/ml (P>0.01) at a relative survival rate of 34 %, when compared to the concurrent solvent control.

In Experiment B, in the presence of S9 mix, the three-hour treatment with CD 605 resulted in a moderate but statistically no significant increases in the number of cells with structural chromosome aberrations without gaps at 4.88, 9.76 and 14.64 µg/ml and at a relative survival rates of 73, 42 and 30%, further indicating that the findings in Experiment A were above normal biological variation. As in Experiment A, in Experiment B in the absence and in the presence of S9-mix increases in structural chromosome aberrations without gaps were above the historical control data range of our laboratory.

No biologically relevant increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of CD 605.

In the control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, proving the suitability of the cell line used.

The positive controls Ethylmethane sulphonate (0.4 µl/ml) and N-Nitrosodimethylamine (0.4 µl/ml) caused the expected biologically relevant increases of cells with structural chromosome aberrations. The studies are, therefore, considered valid.

Table 1: Results (mean) of chromosome analysis Experiment A, with and without activation (total count from 2 cultures per test group/100 metaphases cored per culture = 200 per test group)

Experiment A treatment period 3h	Metabolic activation	Relative Survive%	Mean Number of aberrant cells		Mean Number of Aberrations
			incl. Gaps	excl. gaps	incl. Gaps	excl. gaps
Solvent control	-	100	6	3	7	3
Pos control	-		31**	27**	63**	36**
Test item
4.88 µg/ml	-	92	9	3	9	3
9.76 µg/ml	-	84	11	5	11	5
19.53 µg/ml	-	52	16*	6	16*	7
39.06 µg/ml	-	31	25**	18**	51**	36**
Solvent control	+	100	6	2	6	2
Pos control	+		29**	23**	46**	26**
Test item
2.44 µg/ml	+	93	12	5	18**	9*
4.88 µg/ml	+	85	9	4	10	4
9.76 µg/ml	+	48	8	4	10	4
14.64 µg/ml	+	34	19**	8	24**	10*

* p<0.05 CHI^2

** p<0.01 CHI^2

Table 2: Results (mean) of chromosome analysis Experiment B, with and without activation (total count from 2 cultures per test group/100 metaphases cored per culture = 200 per test group)

Experiment B treatment period 20h	Metabolic activation	Relative Survive%	Number of aberrant cells		Number of Aberrations
			incl. Gaps	excl. gaps	incl. Gaps	excl. gaps
Solvent control	-	100	5	2	6	2
Pos control	-		40**	35**	83**	54**
Test item
2.44 µg/ml	-	88	14*	6	17*	6
4.88 µg/ml	-	65	12	6	13	7
9.76 µg/ml	-	34	27**	13**	38**	14**
14.64 µg/ml	-	a)
treatment period 3 h
Solvent control	+	100	6	2	6	2
Pos control	+		29**	24**	50**	31**
Test item
2.44 µg/ml	+	93	9	4	10	4
4.88 µg/ml	+	73	13	7	14	7
9.76 µg/ml	+	42	9	6	11	6
14.64 µg/ml	+	30	17*	8	19**	9*

a) concentration not evaluable due to excessive toxicity

* p<0.05 CHI^2

** p<0.01 CHI^2

Table 3: Historical control data (number of experiments 33 per treatment group)

	aberration rate (phases with aberration in %)
	Solvent control		Positive control
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
3 -4h/20h treatment/sampling time without S9 –mix*
Range	3-11	1-5	23-43	19-35
3 -4h/20h treatment/sampling time with S9 –mix**
Range	4-11	1-4	21-42	18-30
20h/28h treatment/sampling time without S9 –mix*
Range	4-12	1-4	23-55	20-48
3-4h/28h treatment/sampling time with S9 –mix**
Range	4-11	1-4	16-43	11-31

* positive control: Ethylmethane sulphonate

**positive control: N-Nitrosodimethylamine

Range = min.-max. values

Conclusions:

In conclusion, the test item, tested both with and without metabolic activation, induced structural chromosome aberrations in this test using Chinese Hamster ovary cell. Therefore, the test item is considered clastogenic in this system.

Executive summary:

The test item, test item was tested in a Chromosome Aberration Assay in CHO-KI cells. The test item was dissolved in DMSO and the concentrations used in the test were selected on the basis of cytotoxicity investigations made in a preliminary study.

In two independent experiments (both run in duplicate) at least 200 well-spread metaphases cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50% survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 4.88, 9.76, 19.53 and 39.06 µg/ml test item.

with S9 mix: 2.44, 4.88, 9.76 and 14.64 µg/ml test item.

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 2.44, 4.88, 9.76 and 14.64* µg/ml test item.

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 2.44, 4.88, 9.76 and 14.64 µg/ml test item.

* concentration not evaluable due to excessive toxicity

In Experiment A, the three-hour treatment with the test item in the absence of S9 mix caused statistically significant increase in the number of cells with structural chromosome aberrations without gaps at 39.06 µg/ml (P < 0.01) at a relative survival rate of 31 %. In Experiment A with S9 mix, the frequency of the cells with structural chromosome aberrations without gaps showed a moderate increase at 14.64 µg/ml at a relative survival rate of 34 %, compared to the concurrent control. These increases were above the historical control data range of the laboratory. In Experiment B, with the test item examined without S9 mix, over a prolonged treatment period (20 hours), the frequency of the cells with structural chromosome aberrations without gaps showed a statistically significant increase (P < 0.01) at 9.76 µg/ml, when compared to the concurrent control, at a relative survival rate of 34 %. In Experiment B, in the presence of S9 mix, the three-hour treatment with the test item resulted in moderate, but statistically not significant increases in the number of cells with structural chromosome aberrations without gaps at 4.88, 9.76 and 14.64 µg/ at a relative survival rates of 73, 42 and 30%, further indicating that the findings in Experiment A were above normal biological variation. As in Experiment A, in Experiment B in the absence and in the presence of S9-mix increases in structural chromosome aberrations without gaps were above the historical control data range of the laboratory. There was no biologically relevant increase in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The validity of the test was shown using Ethylmethane sulphonate (0.4 µl/ml) and N-Nitrosodimethylamine (0.4 µl/ml) as positive controls.

In conclusion, the test item, tested both with and without metabolic activation, induced structural chromosome aberrations in this test using Chinese Hamster ovary cells. Therefore, the test item is considered clastogenic in this system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

According to ECHA Guidance on the Application of the CLP Criteria (v. 5.0, 2017) a substance which is positive in in vitro mammalian mutagenicity assays, and which also shows a chemical structure activity relationship similar to known germ cell mutagens, shall be considered for classification as a Category 2 mutagen. Profiling the substance for mechanisms related to mutagenicity using OECD Toolbox v. 3.4, shows that the ´DNA binding by OECD´ profiler identifies potential for covalent binding to DNA by SN2 electrophilic mechanism. The mechanistic profiling results from the ´DNA binding by OECD´ profiler is supported by the results of the endpoint profiling that shows there to be an alert supported by toxicological data for simple aliphatic halogenic. These consistent profiling results indicate a mutagenic mechanism of action.  Thus, and based on the available positive in vitro data and to avoid unnecessary testing on animals, no further in vivo testing is required. There are no structurally similar molecules with suitable data for further QSAR evaluation using the OECD Toolbox.

Justification for classification or non-classification

Based on a worst case approach the substance is classified as Mutagen Category 2, H 341 (Suspected of causing genetic defects) in accordance with the CLP Regulation (1272/2008).