2-(chloromethyl)-4-methylquinazoline

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

476-280-7

EC Name:

-

Cas Number:

109113-72-6

Molecular formula:

C10 H9 Cl N2

IUPAC Name:

2-(chloromethyl)-4-methylquinazoline

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: Off-white with yellow spherical particles

Method

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

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.