2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen 3-chloro-2-hydroxypropylphthalate

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:

26 June 2018 - 20 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2018

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen 3-chloro-2-hydroxypropylphthalate

EC Number:

259-130-3

EC Name:

2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen 3-chloro-2-hydroxypropylphthalate

Cas Number:

54380-33-5

Molecular formula:

C17H19ClO7

IUPAC Name:

2-(3-chloro-2-hydroxypropyl)-6-({2-[(2-methylprop-2-enoyl)oxy]ethoxy}carbonyl)benzoic acid

Test material form:

liquid

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

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:

Type and composition of metabolic activation system:
- source of S9
S9 fraction of Phenobarbital and β-naphthoflavone induced rat liver, Supplier: Trinova Biochem GmbH (Rathenau Str. 2); D-35394 Giessen, Germany
- method of preparation of S9 mix
The preparation of the S9 Mix was performed according to Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium
The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M Sodium phosphate-buffer, pH 7.4 (500.0 mL)
Rat liver homogenate (S9) (100.0 mL)
Salt solution for S9 Mix (400.0 mL)

Test concentrations with justification for top dose:

Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test.
±S9 Mix: 5000, 1600, 500, 160; 50 and 16 μg/plate
In experiment I (initial mutation test) of the main study the plate incorporation method was used. In experiment II (confirmatory mutation test) the pre-incubation method was applied.
+S9 Mix: 5000, 4500, 4000, 3000, 2500, 1600, 500 and 160 μg/plate (additional confirmatory mutation test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, ultrapure water
Dimethyl sulfoxide (DMSO) for Test Item, NPD (4-Nitro-1,2-phenylenediamine), 9AA (9-Aminoacridine), 2AA (2-Aminoanthracene)
Ultrapure water for SAZ (Sodium azide) and MMS (Methyl methanesulfonate)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours in the dark
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each.

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs 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.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle
control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least
three times higher than the reversion rate of the vehicle control.
Conditions for the Validity of the Test
The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and
the deletion in the uvrB gene.
- The TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The E. coli WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the
vehicle controls.
- The tester strain culture titer is in the 10E9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revert
ant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-tox
ic dose levels is required to evaluate assay data).
A dose level is considered toxic if
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value
and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.

Statistics:

none

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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: Precipitate was noticed on the plates at the concentration of 5000 μg/plate (±S9 Mix). The obtained precipitate did not disturb the scoring in any case.

RANGE-FINDING/SCREENING STUDIES
Based on the solubility test, a 50 mg/mL test item solution was prepared in dimethyl sulfoxide (DMSO) and diluted in six steps. The number of revertant colonies and the background lawn of auxotrophic cells of two test strains (S. typhimurium TA98 and S. typhimurium TA100) were determined using seven different test item concentrations (5000; 1600; 500; 160, 50, 16 and 5 μg/plate), in presence and absence of a metabolic activation system (S9). In the informatory toxicity test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case. All of the noticed slightly lower or higher revertant colony numbers (when compared to that of the vehicle control) remained within the corresponding historical control data ranges, within the biological variability range of the applied test system.

HISTORICAL CONTROL DATA
see table 4

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (µg/plate)	Salmonella typhimurium tester strains																Escherichia coli WP2 uvrA
	TA 98				TA100				TA 1535				TA 1537
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	19.0	1.30	19.3	0.88	70.7	1.05	102.7	1.03	9.3	0.97	13.0	0.85	7.7	0.85	8.3	1.25	33.0	1.13	35.7	1.03
DMSO Control	14.7	1.00	22.0	1.00	67.0	1.00	100.0	1.00	9.7	1.00	15.3	1.00	9.0	1.00	6.7	1.00	29.3	1.00	34.7	1.00
Ultrapure Water Control	-	-	-	-	74.3	1.00	-	-	13.0	1.00	-	-	-	-	-	-	27.7	1.00	-	-
5000	12.0	0.82	25.7	1.17	104.0	1.55	139.3	1.39	20.7	2.14	44.0	2.87	7.7	0.85	9.0	1.35	32.3	1.10	43.0	1.24
1600	15.7	1.07	25.0	1.14	86.7	1.29	116.7	1.17	17.3	1.79	25.0	1.63	9.3	1.04	7.7	1.15	37.3	1.27	38.0	1.10
500	17.0	1.16	23.3	1.06	83.0	1.24	91.3	0.91	18.7	1.93	20.0	1.30	8.0	0.89	9.7	1.45	33.7	1.15	45.7	1.32
160	17.3	1.18	25.0	1.14	85.3	1.27	112.0	1.12	12.7	1.31	16.0	1.04	7.0	0.78	7.0	1.05	29.7	1.01	45.7	1.32
50	15.7	1.07	22.7	1-03	71.7	1.07	110.0	1.10	9.0	0.93	13.7	0.89	8.0	0.89	8.0	1.20	36.0	1.23	42.0	1.21
16	17.3	1.18	17.7	0.80	79.7	1.19	97.3	0.97	10.7	1.10	15.3	1.00	8.0	0.89	8.3	1.25	31.3	1.07	36.7	1.06
NPD (4 µg/plate)	436.0	29.73	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
SAZ (2 µg/plate)	-	-	-	-	701.3	9.43	-	-	743.3	57.18	-	-	-	-	-	-	-	-	-	-
9AA (50 µg/plate)	-	-	-	-	-	-	-	-	-	-	-	-	599.3	66.59	-	-	-	-	-	-
MMS (2 µL/plate)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	858.7	31.04	-	-
2AA (2 µg/plate)	-	-	997.3	45.33	-	-	1245.3	12.45	-	-	186.7	12.17	-	-	131.3	19.70	-	-	-	-
2AA (50 µg/plate)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	184.7	5.33

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoactidine; MMS: Methyl methanesulfonate; 2.4A: 2-aminoanthracene
Remarks:Dimethyl sulfoxide (DMSO) was applied as vehicle for the test item, for NPD, 9AA and 2AA and ultrapure water was applied as vehicle of the positive control substances SAZ and MMS. The mutation rate of the test item, the untreated control, NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of SAZ and MMS is given referring to ultrapure water.

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-incubation Test)

Concentrations (µg/plate)	Salmonella typhimurium tester strains																Escherichia coli WP2 uvrA
	TA 98				TA 100				TA 1535				TA 1537
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertauts per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	12.7	0.86	17.7	0.70	69.3	1.07	91.0	1.07	11.0	1.03	11.3	0.94	6.7	1.11	8.7	0.93	26.7	1.11	37.3	1.06
DMSO control	14.7	1.00	25.3	1.00	65.0	1.00	85.3	1.00	10.7	1.00	12.0	1.00	6.0	1.00	9.3	1.00	24.0	1.00	35.3	1.00
Ultrapure Water Control	-	-	-	-	63.0	1.00	-	-	14.0	1.00	-	-	-	-	-	-	26.3	1.00	-	-
5000	12.7	0.86	21.7	0.86	60.3	0.93	110.3	1.29	29.3	2.75	45.3	3.78	2.0	0.33	4.7	0.50	42.0	1.75	44.0	1.25
1600	21.0	1.43	25.3	1.00	63.7	0.98	115.0	1.35	22.0	2.06	26.3	2.19	2.3	0.39	7.0	0.75	28.7	1.19	36.7	1.04
500	17.7	1.20	25.7	1.01	80.0	1.23	107.7	1.26	15.7	1.47	21.0	1.75	5.3	0.89	7.7	0.82	26.0	1.08	34.3	0.97
160	18.7	1.27	22.0	0.87	73.0	1.12	103.0	1.21	13.7	1.28	14.7	1.22	6.0	1.00	8.0	0.86	28.3	1.18	35.7	1.01
50	10.3	0.70	18.7	0.74	59.7	0.92	103.3	1.21	13.0	1.22	12.7	1.06	6.7	1.11	8.7	0.93	27.7	1.15	28.3	0.80
16	20.3	1.39	24.7	0.97	68.7	1.06	108.3	1.27	11.3	1.06	14.0	1.17	10.3	1.72	12.7	1.36	22.0	0.92	30.3	0.86
NPD (4 µg/plate)	460.7	31.41	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
SAZ (2 µg/plate)	-	-	-	-	625.3	9.93	-	-	1157.3	82.67	-	-	-	-	-	-	-	-	-	-
9AA (50 µg/plate)	-	-	-	-	-	-	-	-	-	-	-	-	364.7	60.78	-	-	-	-	-	-
MMS (2 µL/plate)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	1258.7	47.80	-	-
2AA (2 µg/plate)	-	-	677.3	26.74	-	-	726.7	8.52	-	-	107.0	8.92	-	-	145.7	15.61	-	-	-	-
2AA (50 µg/plate)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	156.3	4.42

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine, SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: Dimethyl sulfoxide (DMSO) was applied as vehicle for the test item, for NPD, 9AA and 2AA and ultrapure water was applied as vehicle of the positive control substances SAZ and MMS. The mutation rate of the test item, the untreated control, NPD, 9AA and 2AA is given referring to the DMSO, and the mutation rate of SAZ and MMS is given referring to ultrapure water.

Table 3: Summary Table of the Results of the Additional Confirmatory Mutation Test

 

Additional Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (µg/plate)	Salmonella typhimurium tester strain
	TA 1535
	+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR
Untreated Control		0.92
DMSO Control	8.3	1.00
5000	57.0	6.84
4500	42.3	5.08
4000	44.7	5.36
3000	40.3	4.84
2500	30.3	3.64
1600	22.3	2.68
500	10.0	1.20
160	11.3	1.36
2AA (2 µg/plate)	176.0	21.12

MR: Mutation Rate

2AA: 2-aminoanthracene

Remarks: Dimethyl sulfoxide (DMSO) was applied as vehicle for the test item and for the positive control item: 2AA. The mutation rate of the test item, the untreated control and 2AA is given referring to the DMSO.

 

Table 4: Historical Control Values for Revertants Plate (for the Period of 2015-2017)

			Bacterial strains
Historical control data of untreated control			TA98	TA100	TA1535	TA1537	E. coli
		Average	19.7	94.6	10.9	8.9	25.2
	-S9	SD	1.7	2.4	0.4	1.3	5.2
		Minimum	8	67	4	3	11
		Maximum	40	133	21	20	52
			TA98	TA100	TA1535	TA1537	E. coli
		Average	24.5	112.9	11.0	9.2	31.7
	+S9	SD	1.4	6.1	0.5	1.2	6.4
		Minimum	11	74	3	3	13
		Maximum	43	159	20	20	60
			Bacterial strains
Historical control data of DMSO control			TA98	TA100	TA1535	TA1537	E. coli
		Average	18.1	87.0	10.8	8.5	24.6
	-S9	SD	1.1	3.6	0.6	1.3	3.3
		Minimum	9	58	4	3	10
		Maximum	36	131	23	20	54
			TA98	TA100	TA1535	TA1537	E. coli
		Average	23.0	102.4	11.0	9.2	31.4
	+S9	SD	0.8	7.7	0.4	1.3	5.8
		Minimum	11	69	3	3	12
		Maximum	42	14S	23	21	59
			Bacterial strains
Historical control data of Water control			TA98	TA100	TA1535	TA1537	E. coli
		Average	19.5	92.8	11.3	9.1	26.6
	-S9	SD	1.4	5.0	0.5	1.8	6.6
		Minimum	12	62	4	3	10
		Maximum	30	139	22	IS	52
			TA98	TA100	TA1535	TA1537	E. coli
		Average	24.4	109.9	11.2	9.5	34.0
	+S9	SD	1.2	6.7	0.8	1.9	6.1
		Minimum	13	83	5	4	16
		Maximum	37	149	18	18	63
			Bacterial strains
Historical control data of positive controls			TA98	TA100	TA1535	TA1537	E. coli
		Average	285.9	1214.0	1024.3	594.4	858.4
	-S9	SD	25.5	80.0	120.3	36.4	164.7
		Minimum	152	609	407	136	330
		Maximum	598	2272	2597	2048	1760
			TA98	TA100	TA1535	TA1537	E. coli
		Average	1395.7	1727.4	160.7	145.8	204.9
	+S9	SD	261.4	244.1	30.0	18.9	3.9
		Minimum	286	712	91	70	133
		Maximum	3211	3435	328	315	367

Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100. TA1535. TA1537;

E. coli: Escherichia coli WP2 uvrA
SD: Standard deviation

 

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item (acting as promutagen, in presence of exogenous metabolic activation) induced gene mutations by base-pair substitution in the genome of the Salmonella typhimurium TA1535 tester strain investigated. Therefore, the test item is considered mutagenic in this bacterial reverse mutation assay.

Executive summary:

A study according OECD TG 471 (Ames test) was performed. The test item was dissolved in dimethyl sulfoxide (DMSO). In the initial and confirmatory mutation tests the following concentrations were examined:

±S9 Mix: 5000, 1600, 500, 160; 50 and 16 μg/plate.

Because of the positive results observed in the confirmatory mutation test an additional confirmatory mutation test was performed (in Salmonella typhimurium TA1535, +S9 Mix) with following concentration levels:

+S9 Mix: 5000, 4500, 4000, 3000, 2500, 1600, 500 and 160 μg/plate.

In the initial and confirmatory mutation tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. In the additional confirmatory mutation test the Salmonella typhimurium TA1535 strain was investigated.

Five bacterial strains were used to investigate the mutagenic potential in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). For confirmation and repetition of the positive results obtained in the confirmatory mutation test an additional confirmatory mutation test was carried out. Each assay was conducted with and without metabolic activation (±S9 Mix); however in the additional confirmatory mutation test the Salmonella typhimurium TA1535 strain was investigated in the presence of exogenous metabolic activation (+S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

In the performed experiments all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled.

In the confirmatory mutation test following treatment with the test item significant, biological relevant revertant colony number increases, positive result was noticed at the concentration of 5000 μg/plate in S. typhimurium TA1535 in the presence of exogenous metabolic activation (+S9 Mix). The revertant colony number increases showed clear dose-relationship at the further, lower concentration levels. The dose related tendencies, the unequivocal positive results were repeated in a subsequent experiment: in the additional pre-incubation test (additional confirmatory mutation test).

In the confirmatory mutation test slight, rather equivocal inhibitory effect of the test item was observed in Salmonella typhimurium strains at 5000 μg/plate (in TA1537 also at 1600 μg/plate) in absence of exogenous metabolic activation (-S9 Mix). The slight inhibitory effect of the test item was indicated mainly by affected (slightly reduced) background lawn development. Microdrops (colloid-chemical phenomenon) were noticed in the examined strains at the highest examined concentration of 5000 μg/plate, in the absence (-S9 Mix) of exogenous metabolic activation following the pre-incubation procedure (confirmatory mutation test).

The reported data of this mutagenicity assay shows that under the experimental conditions reported, the test item (acting as promutagen, in presence of exogenous metabolic activation) induced gene mutations by base-pair substitution in the genome of the Salmonella typhimurium TA1535 tester strain investigated.