Phenethyl salicylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria

Based on the available results and applying the weight of evidence approach the test chemical can be considered to be non-mutagenic to 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.

 

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:

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.

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

Target gene:

Histidine

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:

Type and composition of metabolic activation system:
Aroclor 1254-induced S9 was procured from Defence Research and Development Organization and stored at -60°C to -80°C inside the deep freezer. The protein concentration in the S9 fraction was 36.2 mg/mL. Each batch of S9 mix was tested with 2-Aminoanthracene as well as benzo (a) pyrene for its efficiency. Thus, requirements of Ames were fulfilled, and the results of efficiency testing were archived at RCC Laboratories India Private Limited.
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

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 [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: RO water
- Justification for choice of solvent/vehicle: The test chemical was soluble in RO water

Untreated negative controls:

yes

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

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

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

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.

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from a peer reviewed journal.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

To evaluate the mutagenic potential of the test chemical in bacterial cell lines

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

No Data Available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No Data Available

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Aroclor 1254-induced rat- and hamster-liver S9.

Test concentrations with justification for top dose:

0.000, 3.300, 10.000, 33.300, 100.000, 333.300 ug/plate were used in this study. Dose levels were selected based on the results of a pre-experiment using the TA100 strain. In the pre-experiment, cytotoxicity, as defined by a decrease in the number of revertant colonies or a clearing in the density of the background lawn, was observed at ≥333.3 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: DMSO was used as a vehicle


- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine and 2-aminoanthracene

Remarks:

Positive controls in the absence of S9 were sodium azide (TA1535 and TA 100), 9-aminoacridine (TA97 and TA 1537), and 4-nitro-o-phenylenediamine (TA98). The positive control in the presence of S9 was 2-aminoanthracene for all strains.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : TRIPLICATE
- Number of independent experiments : SINGLE

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk : Pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 37"C
- Exposure duration/duration of treatment: 20 minutes
- Harvest time after the end of treatment (sampling/recovery times):

- Other: The test chemical was tested for mutagenicity as per the the preincubation method. The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37"C, without shaking, for 20 min. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel- Bonner medium. The histidine-revertant (his') colonies arising on these plates were counted following 2 days incubation at 37°C. The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used. At least five doses of the chemical were tested in triplicate. Experiments were repeated at least 1 wk following the initial trial. Each chemical was tested initially at half-log doses up to a dose that elicited toxicity.

Evaluation criteria:

An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen. The distinctions between a weak mutagenic response and a mutagenic response, or between a weak mutagenic response and a questionable mutagenic response are highly subjective.

Statistics:

Mutagenic responses of Salmonella tester strains to the test chemical concentrations were reported (mean+/- SEM; three plates)

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The positive controls produced distinct increases in the number of revertant colonies, thus confirming the validity of the assay.

Ames test:
- Signs of toxicity
- Individual plate counts : No trend of increased number of revertant colonies with increased dosing of the test chemical was observed. The positive controls produced distinct increases in the number of revertant colonies, thus confirming the validity of the assay.
- Mean number of revertant colonies per plate and standard deviation

Remarks on result:

other: not mutagenic

Conclusions:

The test chemical was concluded to be non-mutagenic in TA98, TA100, TA1535 and TA1537 in the presence and absence of metabolic activation.

Executive summary:

The chemical was tested in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 0 (solvent control), 1, 3.3, 10.0, 33.0, 100.0 and 333.3 µg/plate with and without metabolic activation (Aroclor 1254-induced rat- and hamster-liver S9). Dose levels were selected based on the results of a pre-experiment using the TA100 strain. In the pre-experiment, cytotoxicity, as defined by a decrease in the number of revertant colonies or a clearing in the density of the background lawn, was observed at ≥333.3 µg/plate. Therefore, 333 µg/plate was selected as top dose in the main experiment. Sodium azide, 9-aminoacridine, 4-nitro-o-phenylenediamine and 2-aminoanthracene served as positive controls. The test chemical failed to produce any significant increase in the number of revertant colonies. No trend of increased number of revertant colonies with increased dosing of the test chemical was observed. The positive controls produced distinct increases in the number of revertant colonies, thus confirming the validity of the assay. Based on the data, the test chemical was concluded to be non-mutagenic in TA98, TA100, TA1535 and TA1537 in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria

Various studies have been performed to evaluate the mutagenic potential of the test chemical. The results include in vitro studies performed on various bacterial strains for the test chemical which are mentioned as follows:

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.

 

This is supported by another Salmonella Mutagenicity Tests carried out to determine the degree of mutation response obtained after bacterial strains were exposed to the test chemical.The chemical was tested in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 0 (solvent control), 1, 3.3, 10.0, 33.0, 100.0 and 333.3 µg/plate with and without metabolic activation (Aroclor 1254-induced rat- and hamster-liver S9). Dose levels were selected based on the results of a pre-experiment using the TA100 strain. In the pre-experiment, cytotoxicity, as defined by a decrease in the number of revertant colonies or a clearing in the density of the background lawn, was observed at ≥333.3 µg/plate. Therefore, 333 µg/plate was selected as top dose in the main experiment. Sodium azide, 9-aminoacridine, 4-nitro-o-phenylenediamine and 2-aminoanthracene served as positive controls. The test chemical failed to produce any significant increase in the number of revertant colonies.No trend of increased number of revertant colonies with increased dosing of the test chemical was observed. The positive controls produced distinct increases in the number of revertant colonies, thus confirming the validity of the assay. Based on the data, the test chemical was concluded to be non-mutagenic in TA98, TA100, TA1535 and TA1537 in the presence and absence of metabolic activation.

 

Based on the available results and applying the weight of evidence approach the test chemical can be considered to be non mutagenic to 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.

Justification for classification or non-classification

Based on the available results, the test chemical cannot be expected to produce mutagenic reactions to bacterial and mammalian cell lines when tested under in vitro conditions. Hence, the test chemical can be classified under the category "Not Classified' as per CLP Regulation.