Ethyltriphenylfosfonium bromide

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:

July 20, 2018 to September 10, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental 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:

The experiment was performed to assess the potential of the test item to induce gene mutations in the Bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The experiment was performed as a plate incorporation assay, Negative results were obtained in this experiment; hence the second experiment was performed as a pre-incubation assay. .

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylfosfonium bromide

EC Number:

216-223-3

EC Name:

Ethyltriphenylfosfonium bromide

Cas Number:

1530-32-1

Molecular formula:

C20H20P.Br

IUPAC Name:

bromo(ethyl)triphenylphosphorane

Test material form:

solid: crystalline

Remarks:

White Crystalline Powder

Details on test material:

Name of test material: Ethyltriphenylphosphonium Bromide
- CAS No. 1530-32-1
- EC No. 216-223-3
- Molecular formula: C20-H20-P.Br
- Molecular weight: 371.256 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: 98.60%

Method

Target gene:

Histidine Operon

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

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Arochlor induced S9 was procured from Defence Research and Development Organization.
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution which contains D-glucose-6-phosphate 0.8 g, β-NADP 1.75 g, MgCl2 1.0 g, KCl 1.35g, Na2HPO4 6.4 g, NaH2PO4.H2O 1.4 g in 500 ml of distilled water
- concentration or volume of S9 mix and S9 in the final culture medium : 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

0.0 (NC), 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate doses were selected based on the result obtained from pre-experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test item was found to be soluble in distilled water at 50 mg/ml.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Triplicate
- Number of independent experiments : The test chemical was tested in two independent experiments (Trial I and Trial II). Trial I: plate incorporation method and Trial II: preincubation method.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min at 37°C
- Exposure duration/duration of treatment: 48 hours at 37°C for both Trial I and Trial II
- Harvest time after the end of treatment (sampling/recovery times): No data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction in revertant count and inhibition of background lawn
- Any supplementary information relevant to cytotoxicity: The pre-experiment was performed with TA 98 and TA 100 strain of Salmonella typhimurium and eight concentrations spaced by half log intervals in triplicates. 5 mg/plate were selected as the highest dose in the pre-experiment based on the solubility and precipitation test, Concentration 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 study.

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER: No data

Rationale for test conditions:

No data available.

Evaluation criteria:

A test item was 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 was observed.
A dose-dependent increase was considered biologically relevant if the threshold exceeded at more than one concentration.
An increase exceeding the threshold of biological significance at only one concentration was judged as biologically relevant if it was reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold was 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 such an increase was not considered biologically relevant.

Statistics:

Microsoft Office Excel based calculations were used for descriptive statistical analysis.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA1537, TA98, TA100, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: Test item was found to be soluble in water at 50 mg/ml .
- Precipitation and time of the determination: Slight precipitation was observed which was assumed to be non interfering with the scoring. Therefore, 5 mg/plate was selected as the highest concentration for pre-experiment.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed with TA 98 and TA 100 strain of Salmonella typhimurium and eight concentrations spaced by half log intervals in triplicates. 5 mg/plate were selected as the highest dose in the pre-experiment based on the solubility and precipitation test. 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate.

In TA 98 and TA 100 there was no reduction in colony count but reduction in background lawn was observed in treated concentrations 5 (T8) and 1.582 (T7) mg/plate and no reduction in backreduction in colony count as well as in background lawn in treated concentration 0.501 (T6) mg/plate - 0.002 (T1) mg/plate both in the absence and in the presence of metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The positive controls showed an unequivocal increase in revertant counts with all the five tester strains and the respective controls used. And the spontaneous reversion rates in the negative and positive controls were within the range of in house historical data

Ames test:
- Signs of toxicity : No substantial increase in Revertant colony count was observed
- Individual plate counts : Please refer the attached table
- Mean number of revertant colonies per plate and standard deviation : Please refer the table attached in any other information on result section.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer the table attached in remark section.
- Negative (solvent/vehicle) historical control data: Please refer the table attached in remark section.

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

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

Dose (mg/plate)	In the Presence (+S9) of Metabolic Activation
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.33	0.58	16.33	0.58	23.67	2.52	122.67	3.51	274.67	10.26
T1 (0.005)	4.67	0.58	10.67	1.53	21.00	1.00	107.67	2.08	274.67	6.11
T2 (0.016)	4.33	0.58	11.33	1.53	19.33	1.53	111.33	2.08	222.67	9.02
T3 (0.050)	5.00	1.00	13.00	1.00	21.67	0.58	112.00	3.61	231.00	6.24
T4 (0.158)	5.67	0.58	12.33	1.53	21.33	1.53	118.00	200	246.67	9.02
T5 (0.501)	5.33	0.58	15.67	2.08	23.00	1.00	118.67	2.52	272.00	7.21
PC	180.33	9.87	493.33	46.70	1186.67	33.31	1464.00	32.00	1309.33	51.43

Dose (mg/plate)	In the Absence (-S9) of Metabolic Activation
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.67	0.58	15.67	0.58	23.00	2.00	120.67	4.04	275.33	12.06
T1 (0.005)	5.00	1.00	12.33	3.21	16.00	1.00	104.67	2.08	218.00	4.00
T2 (0.016)	4.33	0.58	10.33	1.53	19.00	1.00	107.00	3.00	228.00	10.58
T3 (0.050)	5.67	0.58	11.67	1.15	20.33	1.53	112.00	3.61	246.33	5.51
T4 (0.158)	5.33	0.58	13.33	1.53	20.67	0.58	115.33	4.73	264.00	9.17
T5 (0.501)	6.00	0.00	14.67	0.58	19.00	2.65	113.33	3.51	262.00	10.58
PC	171.33	10.21	1192.67	49.00	986.67	20.13	1269.33	24.44	1717.33	142.29

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

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

Dose (mg/plate)	In the Presence (+S9) of Metabolic Activation
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.33	0.58	16.00	1.00	26.33	0.58	120.33	1.53	265.33	7.77
T1 (0.005)	4.67	0.58	11.67	2.08	21.33	0.58	102.67	1.53	229.33	9.07
T2 (0.016)	4.00	0.00	12.00	1.00	24.00	1.00	110.67	2.08	237.33	8.14
T3 (0.050)	5.33	1.53	13.67	1.53	25.00	1.00	115.33	2.08	244.33	7.37
T4 (0.158)	6.33	0.58	13.33	1.53	23.67	2.52	120.00	1.00	261.67	6.43
T5 (0.501)	5.67	0.58	15.67	0.58	25.67	0.58	117.00	1.00	259.33	7.23
PC	181.00	11.14	419.33	37.75	1325.33	48.88	1490.67	24.44	1469.33	51.43

 

Dose (mg/plate)	In the Absence (-S9) of Metabolic Activation
	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	15.67	0.58	27.33	1.53	119.33	3.79	264.33	10.41
T1 (0.005)	4.33	0.58	10.33	1.53	20.33	2.08	104.33	3.06	220.33	4.93
T2 (0.016)	5.00	1.00	12.67	1.53	21.00	3.46	116.00	2.00	215.67	3.06
T3 (0.050)	4.67	0.58	11.00	1.00	24.33	0.58	109.00	8.19	225.67	7.23
T4 (0.158)	5.67	0.58	13.00	1.00	23.00	2.00	117.67	2.08	236.33	2.52
T5 (0.501)	6.33	0.58	15.00	1.00	26.67	0.58	116.67	3.21	251.00	3.00
PC	174.00	9.17	1224.00	32.00	930.67	38.02	1122.67	44.06	1624.00	48.00

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

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.

 

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations either by base pair changes or frameshift in the genome of the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, TA 102 both in presence or in absence of metabolic activation system. Hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

The study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to negative control according to plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, TA102. The study was conducted as per OECD 471, Adopted July 21, 1997. The assay was performed in two independent experiments both with and without liver microsomal activation, each concentration, including the negative and positive controls was tested in triplicates. Based on the solubility and precipitation test result eight different concentrations 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. The pre-experiment was performed with TA100 and TA98 strain of salmonella typhimurium. In TA98 and TA100 there was no reduction in colony count but reduction in background lawn was observed in treated concentrations 5 (T8) and 1.582 (T7) mg/plate and no reduction in colony count as well as in background lawn in treated concentration 0.501 (T6) mg/plate – 0.002 (T1) mg/plate both in absence and in presence of metabolic activation. Based on the results, 0.501 mg/plate was selected as the highest dose for the main study trials both in the absence as well as in the presence of metabolic activation. Trial-I was performed with five concentration of test item along with the negative and concurrent positive control with the remaining three strains i.e. TA 1537, TA1535, TA 102 by plate incorporation method. For TA 98 and TA 100 revertant colony count were directly incorporated in the Trial-I from the pre-experiment up to the five concentrations [T2 (0.005 mg/plate) to T6 (0.501 mg/plate)]. Test item concentration 0.0 (NC), 0.005, 0.016, 0.050, 0.158 and 0.501mg/plate, were prepared with half log interval both in presence and in absence of metabolic activation. The plates were treated and incubated at 37±2⁰C for 48 hours. No increase in the revertant count in any of the five strains was reported at any of the test concentrations. Positive controls resulted in an unequivocal response in all the five tester strains compared to respective control used. Trial II was performed independently with all the five tester strains along with the negative and positive controls by pre-incubation method for the confirmation of the Trial-I results. The concentration 0.0 (NC), 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate were used both in the absence (-S9) as well as in the presence of metabolic activation (+S9). The concentration of positive controls used was same as used in the plate incorporation assay. In Trial II, the test item, negative and positive controls were pre-incubated along with 500µl of metabolic activation mix (+S9)/Buffer (-S9) and 100µl of bacterial culture for 60 minutes at 37±2^oC in an incubator. After pre-incubation, 2 mL of top agar was mixed with the pre-incubation mixture and poured on minimal glucose agar plates. The treaed plates were incubated for 48 hours in an incubator. No substantial increase in the revertant count was observed in any of the five tester strains pre-incubated with the test item. The positive controls showed an unequivocal increase in revertant counts with all the five tester strains and the respective controls used, hence confirming the non-mutagenic activity of the test item. Based on the results of this study, it is concluded that the test chemical was found to be non-mutagenic as it did not induce (point) gene mutations in the histidine operon by base-pair changes or frameshifts, in the presence or the absence of metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102). Hence, the test chemical is not likely to be classified as a gene mutant as per the criteria mentioned in the CLP regulation.