Benzyltriphenylphosphonium chloride

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:

From 22-Jun-2017 To 01 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Benzyltriphenylphosphonium chloride

EC Number:

214-154-3

EC Name:

Benzyltriphenylphosphonium chloride

Cas Number:

1100-88-5

Molecular formula:

C25H22P.Cl

IUPAC Name:

benzyltriphenylphosphonium chloride

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 180739
- Expiration date of the lot/batch: 02 March 2022
- Purity test date (Purity): 02 March 2017 (99.5%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in dimethyl sulfoxide. In the dose range finding test, the stock solution was treated with ultrasonic waves until the test item had completely dissolved. In the first and second mutation experiment, the test item was dissolved after a vortexing step only.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Test concentrations: 17, 52, 164, 512 and 1600 μg/plate (Based on the results of the dose-range finding test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (Merck, Darmstadt, Germany)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First Experiment: Direct Plate Assay
Benzyltriphenylphosphonium Chloride was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the
dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512 and 1600 μg/plate. The results are shown in Table 1 and Table 2. The individual data are presented in Appendix 3.
Precipitate
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
Toxicity
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. The definitions are stated in Appendix 2.
Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
Mutagenicity
In tester strain WP2uvrA in the absence of S9-mix, the test item induced up to 2.4-fold increases in the number of revertant colonies compared to the solvent control. However, these increases were within the historical control data range, coupled to a low solvent control value and only observed in one experiment. Therefore, these increases are considered to be not biologically relevant.
In all other tester strains, no increases in the number of revertants were observed upon treatment with Benzyltriphenylphosphonium Chloride under all conditions tested.

Second Experiment: Pre-Incubation Assay
To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, Benzyltriphenylphosphonium Chloride was tested up to the dose level of 5000 µg/plate in the tester strain WP2uvrA and up to 1600 µg/plate in the tester strain TA1535, TA1537, TA98 and TA100. The results are shown in Table 3, the individual data are presented in Appendix 3.
Precipitate
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
Toxicity
Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
 
Mutagenicity
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Any other information on results incl. tables

Table1: Dose-Range Finding Test: Mutagenic Response of Benzyltriphenylphosphonium Chloride in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay

Direct plate assay Mean number of revertant colonies/3 replicate plates (±S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain.
Dose (µg/plate)	TA100	WP2uvrA	TA100	WP2uvrA
	Without S9-mix		With S9-mix
Positive control	1039 ± 62	1374 ± 85	1234 ± 53	164 ± 27
Solvent control	123 ± 9	8 ± 3	103 ± 14	21 ± 4
1.7	119 ± 9	18 ± 3	100 ± 24	24 ± 7
5.4	107 ± 18	15 ± 8	110 ± 18	17 ± 5
17	103 ± 11	18 ± 6	108 ± 11	17 ± 6
52	97 ± 7	17 ± 6	92 ± 9	21 ± 2
164	127 ± 40n	19 ± 7	109 ± 22 n	22 ± 7
512	106 ± 8 ^s	18 ± 4	76 ± 5 ^s	25 ± 10
1600	e MC	10 ± 5 n	e MC	24 ± 10 ^n
5000	0 ± 0 aNP	e NP MC	e NP MC	e NP MC

 

MC : Microcolonies

NP : No precipitate

a: Bacterial background lawn absent

e: Bacterial background lawn extremely reduced

n: Normal bacterial background lawn

s: Bacterial background lawn slightly reduced

 

Table 2: Experiment 1: Mutagenic Response of Benzyltriphenylphosphonium Chloride in theSalmonella typhimuriumReverse Mutation Assay

Direct plate assay Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimurium.
Dose (µg/plate)	TA11535	TA1537	TA98	TA11535	TA1537	TA98
	Without S9-mix			With S9-mix
Positive control	904 ± 103	1136 ± 96	1419 ± 96	203 ± 19	268 ± 52	770 ± 101
Solvent control	9 ± 3	5 ± 3	11 ± 5	9 ± 1	6 ± 2	24 ± 7
17	14 ± 3	5 ± 3	14 ± 3	14 ± 7	3 ± 2	25 ± 4
52	10 ± 4	7 ± 4	10 ± 2	10 ± 7	4 ± 3	14 ± 2
164	7 ± 2	3 ± 2	14 ± 4	11 ± 7	6 ± 3	15 ± 6
512	6 ± 2 ^n	3 ± 1^n	12 ± 3 ^n	9 ± 6 ^n	1 ± 2 ^n	18 ± 6 ^n
1600	e NP MC	e NP MC	e NP MC	e NP MC	e NP MC	e NP MC

MC : Microcolonies

NP : No precipitate

e: Bacterial background lawn extremely reduced

n: Normal bacterial background lawn

Table 3: Experiment 2: Mutagenic Response of Benzyltriphenylphosphonium Chloride in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay

Pre-incubation assay Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain.
Dose (µg/plate)	TA11535	TA1537	TA98	TA100	WP2uvrA	TA11535	TA1537	TA98	TA100	WP2uvrA
	Without S9-mix					With S9-mix
Positive control	805 ± 39	112 ± 23	1172 ± 25	857 ± 108	101 ± 16	188 ± 23	185 ± 18	509 ± 11	1332 ± 71	464 ± 19
Solvent control	6 ± 3	5 ± 3	9 ± 3	126 ± 9	23 ± 7	9 ± 1	7 ± 3	14 ± 4	115 ± 4	40 ± 12
5.4	8 ± 4	3 ± 2	7 ± 1	128 ± 22	-	7 ± 3	7 ± 3	18 ± 2	125 ± 19	-
17	8 ± 1	10 ± 3	8 ± 4	104 ± 8	18 ± 5	9 ± 2	9 ± 4	12 ± 2	118 ± 22	35 ± 12
52	5 ± 1^n	3 ± 3^n	12 ± 2	120 ± 10^n	26 ± 10	10 ± 6	3 ± 4	20 ± 3	126 ± 24	32 ± 5
164	4 ± 2 ^s	2± 2^s	12 ± 8^n	85 ± 5^s	21 ± 9^n	8 ± 4^n	3 ± 2^n	12 ± 4	115 ± 16^n	28 ± 7
512	e MC	e MC	e MC	e MC	13 ± 5^m	5 ± 2 ^m	1 ± 1^m	11 ± 4^n	96 ± 9^m	19 ± 2^n
1600	e MC NP	e MC NP	e MC NP	0 ± 0 a NP	0 ± 0 a	e MC NP	e MC NP	e MC NP	0 ± 0 a NP	eMC
5000	-	-	-	-	0 ± 0 a NP	-	-	-	-	0 ± 0 a NP

 

MC : Microcolonies

NP : No precipitate

a: Bacterial background lawn absent

e: Bacterial background lawn extremely reduced

m: Bacterial background lawn moderately reduced

n: Normal bacterial background lawn

s: Bacterial background lawn slightly reduced

-: Not tested

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that Benzyltriphenylphosphonium Chloride is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Benzyltriphenylphosphonium Chloride and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonellatyphimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichiacoli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 180734 of Benzyltriphenylphosphonium Chloride was an off-white powder. The test item was dissolved in dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP₂uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 at dose levels of 512 μg/plate and upwards and in tester strain WP₂uvrA at the dose level of 5000 μg/plate. 

In the first mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to concentrations of 5000 µg/plate in WP₂uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Benzyltriphenylphosphonium Chloride is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.