Sodium salicylate

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:

17-04-2018 - 10-05-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium salicylate

EC Number:

200-198-0

EC Name:

Sodium salicylate

Cas Number:

54-21-7

Molecular formula:

C7H6O3.Na

IUPAC Name:

sodium salicylate

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report):sodium salicylate
- Molecular formula :C7H6O3.Na
- Molecular weight : 160.1035 g/mol
- Substance type: organic
- Physical state: solid
- Smilies notation: c1(c(cccc1)O)C(=O)[O-].[Na+]
- InChI: 1S/C7H6O3.Na/c8-6-4-2-1-3-5(6)7(9)10;/h1-4,8H,(H,9,10);/q;+1/p-1

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was soluble in RO water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

RO water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

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 vehicle/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 control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test. In TA 98 and TA 100, cyto-toxicity was observed in the treated concentrations 1.582 and 5 mg/plate (T7 to T8), moderate inhibition was observed in the treated concentrations 0.501 mg/plate (T6) and there was no reduction in colony count as well as background lawn in any of the following concentrations tested; 0.002, 0.005, 0.016, 0.050 and 0.158 ( T1 to T5) mg/plate both in absence and in the presence of metabolic activation, when compared to that of the negative control group. Based on the results of pre-experiment following doses were selected for the main study trials: 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	122	24	126	25
	R2	118	20	119	20
	R3	124	19	121	22
T1 (0.002)	R1	106	17	108	19
	R2	110	19	112	17
	R3	111	17	106	17
T2 (0.005)	R1	108	19	112	21
	R2	102	20	108	23
	R3	104	18	110	20
T3 (0.016)	R1	114	20	108	18
	R2	106	22	110	16
	R3	102	19	114	18
T4 (0.050)	R1	116	21	106	21
	R2	112	17	114	23
	R3	118	18	108	19
T5 (0.158)	R1	102	18	100	17
	R2	98	17	96	18
	R3	100	15	104	17
T6 (0.501)	R1	36 (+ + +)	2 (+ + +)	42 ( + + + )	4 (+ + +)
	R2	26 (+ + +)	5 (+ + +)	34 (+ + +)	3 (+ + +)
	R3	24 (+ + +)	3 (+ + +)	30 ( + + +)	3 (+ + +)
T7 (1.582)	R1	0 (+)	0 (+)	0 (+)	0 (+)
	R2	0 (+)	0 (+)	0 (+)	0 (+)
	R3	0 (+)	0 (+)	0 (+)	0 (+)
T8 (5)	R1	0 (+)	0 (+)	0 (+)	0 (+)
	R2	0 (+)	0 (+)	0 (+)	0 (+)
	R3	0 (+)	0 (+)	0 (+)	0 (+)
PC	R1	1088	960	1584	1242
	R2	1136	992	1616	1180
	R3	1168	1008	1600	1208

NC           =     Negative control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	16	25	126	280
	R2	6	15	20	119	272
	R3	8	14	22	121	260
T1 (0.002)	R1	4	13	19	108	242
	R2	5	11	17	112	238
	R3	5	10	17	106	232
T2 (0.005)	R1	5	12	21	112	240
	R2	4	13	23	108	248
	R3	4	13	20	110	252
T3 (0.016)	R1	6	14	18	108	256
	R2	5	12	16	110	248
	R3	4	13	18	114	264
T4 ((0.050)	R1	6	15	21	106	260
	R2	5	12	23	114	254
	R3	5	14	19	108	268
T5 (0.158)	R1	6	14	17	100	270
	R2	6	15	18	96	266
	R3	5	14	17	104	258
PC	R1	168	480	1242	1584	1384
	R2	186	452	1180	1616	1336
	R3	170	492	1208	1600	1312

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	16	24	122	276
	R2	6	14	20	118	264
	R3	7	13	19	124	258
T1 (0.002)	R1	5	12	17	106	232
	R2	4	14	19	110	240
	R3	5	11	17	111	236
T2 (0.005)	R1	6	13	19	108	242
	R2	4	15	20	102	238
	R3	5	13	18	104	246
T3 (0.016)	R1	5	14	20	114	240
	R2	5	14	22	106	252
	R3	6	15	19	102	236
T4 ((0.050)	R1	6	14	21	116	254
	R2	5	12	17	112	250
	R3	6	13	18	118	262
T5 (0.158)	R1	6	15	18	102	256
	R2	6	15	17	98	260
	R3	6	14	15	100	252
PC	R1	180	1320	960	1088	1824
	R2	174	1272	992	1136	1840
	R3	170	1344	1008	1168	1888

NC= Negative Control,T=Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                  2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                        Sodium azide [10μg/plate]: TA 1535, TA 100                                                 

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]   Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	8	16	28	128	272
	R2	6	14	25	123	258
	R3	7	13	23	124	260
T1 (0.002)	R1	5	11	20	120	230
	R2	4	10	20	118	238
	R3	5	13	19	121	244
T2 (0.005)	R1	5	13	21	123	250
	R2	4	12	23	120	246
	R3	4	12	24	124	240
T3 (0.016)	R1	6	10	19	122	238
	R2	5	14	25	119	246
	R3	5	15	26	125	254
T4 ((0.050)	R1	6	13	25	123	248
	R2	6	12	24	124	256
	R3	5	15	23	124	250
T5 (0.158)	R1	7	15	26	125	258
	R2	6	14	22	123	264
	R3	6	14	25	123	260
PC	R1	162	380	1344	1440	1680
	R2	174	440	1360	1472	1704
	R3	180	420	1384	1504	1712

 

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	15	26	126	260
	R2	6	13	23	123	252
	R3	7	13	22	120	248
T1 (0.002)	R1	4	10	21	108	232
	R2	5	13	23	102	228
	R3	5	11	19	104	236
T2 (0.005)	R1	4	14	18	106	234
	R2	4	11	20	112	230
	R3	4	12	23	110	242
T3 (0.016)	R1	5	13	24	114	248
	R2	6	14	22	108	240
	R3	4	12	19	111	252
T4 ((0.050)	R1	6	13	23	114	250
	R2	5	13	24	116	254
	R3	5	14	20	120	246
T5 (0.158)	R1	6	14	24	123	258
	R2	6	14	23	120	248
	R3	5	13	25	121	250
PC	R1	180	1168	890	1248	1552
	R2	178	1184	924	1280	1520
	R3	182	1216	916	1304	1568

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.00	1.00	15.00	1.00	22.33	2.52	122.00	3.61	270.67	10.07
T1 (0.002)	4.67	0.58	11.33	1.53	17.67	1.15	108.67	3.06	237.33	5.03
T2 (0.005)	4.33	0.58	12.67	0.58	21.33	1.53	110.00	2.00	246.67	6.11
T3 (0.016)	5.00	1.00	13.00	1.00	17.33	1.15	110.67	3.06	256.00	7.02
T4 (0.050)	5.33	0.58	13.67	1.53	21.00	2.00	109.33	4.16	260.67	8.00
T5 (0.158)	5.67	0.58	14.33	0.58	17.33	0.58	100.00	4.00	264.67	6.11
PC	174.67	9.87	474.67	20.53	1210.00	31.05	1600.00	16.00	1344.00	36.66

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.67	0.58	14.33	1.53	21.00	2.65	121.33	3.06	266.00	9.17
T1 (0.002)	4.67	0.58	12.33	1.53	17.67	1.15	109.00	2.65	236.00	4.00
T2 (0.005)	5.00	1.00	13.67	1.15	19.00	1.00	104.67	3.06	242.00	4.00
T3 (0.016)	5.33	0.58	14.33	0.58	20.33	1.53	107.33	6.11	242.67	8.33
T4 (0.050)	5.67	0.58	13.00	1.00	18.67	2.08	115.33	3.06	255.33	6.11
T5 (0.158)	6.00	0.00	14.67	0.58	16.67	1.53	100.00	2.00	256.00	4.00
PC	174.67	5.03	1312.00	36.66	986.67	24.44	1130.67	40.27	1850.67	33.31

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100 Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                  

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.00	1.00	14.33	1.53	25.33	2.52	125.00	2.65	263.33	7.57
T1 (0.002)	4.67	0.58	11.33	1.53	19.67	0.58	119.67	1.53	237.33	7.02
T2 (0.005)	4.33	0.58	12.33	0.58	22.67	1.53	122.33	2.08	245.33	5.03
T3 (0.016)	5.33	0.58	13.00	2.65	23.33	3.79	122.00	3.00	246.00	8.00
T4 (0.050)	5.67	0.58	13.33	1.53	24.00	1.00	123.67	0.58	251.33	4.16
T5 (0.158)	6.33	0.58	14.33	0.58	24.33	2.08	123.67	1.15	260.67	3.06
PC	172.00	9.17	413.33	30.55	1362.67	20.13	1472.00	32.00	1698.67	16.65

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.67	0.58	13.67	1.15	23.67	2.08	123.00	3.00	253.33	6.11
T1 (0.002)	4.67	0.58	11.33	1.53	21.00	2.00	104.67	3.06	232.00	4.00
T2 (0.005)	4.00	0.00	12.33	1.53	20.33	2.52	109.33	3.06	235.33	6.11
T3 (0.016)	5.00	1.00	13.00	1.00	21.67	2.52	111.00	3.00	246.67	6.11
T4 (0.050)	5.33	0.58	13.33	0.58	22.33	2.08	116.67	3.06	250.00	4.00
T5 (0.158)	5.67	0.58	13.67	0.58	24.00	1.00	121.33	1.53	252.00	5.29
PC	180.00	2.00	1189.33	24.44	910.00	17.78	1277.33	28.10	1546.67	24.44

NC= Negative Control, T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the given test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.