[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Cas Number:

2409816-82-4

Molecular formula:

C26H42O4P2

IUPAC Name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537)

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):

All Salmonella typhimurium strains were obtained from Trinova BioChem GmbH (batch: TA98: 5508D, TA100: 5526D, TA102: 5524D, TA1535: 5504D, TA1537:5530D) and were stored as lyophilizates in the refrigerator at 2 – 8 °C.

On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate per strain at 3:30 pm.
These overnight cultures were placed in the heating chamber at 37 ± 1 °C for 16.5 hours.
For the last two hours the overnight cultures were shaken on an orbital shaker (150 rpm) at 37 ± 1 °C.
Afterwards, the overnight cultures were ready for use in the experiment.
During the test, the overnight cultures were stored at room temperature (20 ± 5 °C) to pre-vent changes in the titre.

Metabolic activation:

with and without

Metabolic activation system:

The test was performed in two valid experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.

Experiment 1:
In the first experiment, the test item (dissolved in demineralized water, demin. water) was tested up to concentrations of 5 µL/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxicity could be observed in the presence and the absence of metabolic acti-vation.
The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2:
Based on the results of exp. 1,the test item was tested up to concentrations of 5 µL/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method.
The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxicity could be observed in the presence and the absence of metabolic acti-vation.
The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Test concentrations with justification for top dose:

It is not specificed in the final report.

Vehicle / solvent:

Demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

On the day of the start of each experiment, a stock solution containing 50 mL/L of the test item in demin. water was prepared.

The stock solution was used to prepare the geometric series of the concentrations to be tested.

The following nominal test item concentrations were prepared for experiment 1:
5, 1.5, 0.5, 0.15 and 0.05 µL/plate.

The following nominal test item concentrations were prepared for experiment 2:
5, 2.5, 1.25, 0.63, 0.313 and 0.156 µL/plate.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other: 4-Nitro-1,2-pheny-lene diamine, 2-Amino-anthracene

Details on test system and experimental conditions:

Per bacteria strain and concentration, three plates with (+S9) and three plates without me-tabolic activation (-S9) were used.

For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histi-dine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.

Experiment 1
Tested strains: TA98, TA100, TA102, TA1535, TA1537
Test item concentrations: 5, 1.5, 0.5, 0.15, 0.05 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C
Method: plate incorporation method

Experiment 2
Tested strains: TA98, TA100, TA102, TA1535, TA1537
Test item concentrations: 5, 2.5, 1.25, 0.63, 0.313, 0.156 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C
Method: pre-incubation method

Plate incorporation method:
The following materials were gently vortexedin a test tube and poured onto the selective agar plates:
1. 100 µL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control).
2. 500 µL S9-mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
3. 100 µL bacteria suspension (test system, culture of the strains)
4. 2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
1. 100 µL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control).
2. 500 µL S9-mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
3. 100 µL bacteria suspension (test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.

Evaluation criteria:

Five different analysable concentrations were used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A result is considered as clearly positive if all following criteria are fulfilled:
• A concentration-related increase, in revertants
• a clear biological relevant increase in at least one concentration compared to the con-current solvent control
• at least one concentration with an increase above the distribution of historical solvent control data (mean ± 3 SD).

A biologically relevant increase is described as follows:
• if in the bacteria strains TA98, TA100, TA102the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
• if in the bacteria strainsTA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

A test result is considered as clearly negative, if it does not meet the criteria above.

Results and discussion

Test results

Test results 1

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

All strains met the criterion of at least 109 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.

In exp. 1, the test item showed no precipitateson the plates in all tested concentrations.
The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced. Thus, no signs of toxicity towards the bacteria strains could be observed.
No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed.
To verify this result, a further experiment with adapted conditions (pre-incubation method) was performed (exp. 2).
The mean revertant values of experiment 1 are shown in Table 8.1 a.

In exp. 2, the test item showed no precipitateson the plates in all tested concentrations.
The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced. Thus, no signs of toxicity towards the bacteria strains could be observed.
No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed.
The mean revertant values of experiment 2 are shown in Table 8.2 a.

Any other information on results incl. tables

Table 8.1‑a    Mean Revertants Experiment 1

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	11	18	54	54	169	175	7	6	3	4
	sd	1.5	2.0	3.6	6.2	8.3	12.9	1.5	0.6	0.6	0.6
DMSO	Mean	13	16	46	45	160	160	8	9	4	5
	sd	1.2	1.0	1.2	1.5	6.9	10.6	1.2	1.2	0.6	0.6
Positive Controls*	Mean	456	128	205	1021	749	1115	132	167	62	102
	sd	52.5	11.7	16.7	92.7	53.3	85.5	10.6	28.1	4.2	8.7
	f(I)	35.08	8.00	3.80	22.69	4.43	6.97	18.86	18.56	15.50	20.40
5µL/plate	Mean	14	14	50	55	167	155	8	8	4	5
	sd	3.8	2.1	1.0	1.7	12.2	14.0	0.6	0.0	0.6	0.6
	f(I)	1.27	0.78	0.93	1.02	0.99	0.89	1.14	1.33	1.33	1.25
1.5µL/plate	Mean	13	15	50	51	164	165	7	7	4	4
	sd	2.0	2.1	2.3	4.0	10.6	6.1	1.5	1.0	1.2	1.0
	f(I)	1.18	0.83	0.93	0.94	0.97	0.94	1.00	1.17	1.33	1.00
0.5 µL/plate	Mean	12	15	51	53	155	153	7	7	4	5
	sd	1.5	3.1	2.5	2.0	10.1	10.1	1.0	0.6	0.6	1.2
	f(I)	1.09	0.83	0.94	0.98	0.92	0.87	1.00	1.17	1.33	1.25
0.15 µL/plate	Mean	14	16	49	49	159	159	8	9	3	4
	sd	3.8	2.3	4.5	4.9	10.1	8.3	1.5	1.2	0.6	0.6
	f(I)	1.27	0.89	0.91	0.91	0.94	0.91	1.14	1.50	1.00	1.00
0.05 µL/plate	Mean	14	17	53	55	157	149	9	7	5	4
	sd	4.0	3.2	3.5	3.5	12.2	10.1	0.6	0.6	0.6	1.0
	f(I)	1.27	0.94	0.98	1.02	0.93	0.85	1.29	1.17	1.67	1.00

sd = standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 13

f(I) = increase factor, calculation see chapter 7.4, page 20

bold marked values = relevant increase in the number of revertants

 

Table 8.2‑a    Mean Revertants Experiment 2

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	15	21	68	74	159	157	9	10	4	5
	sd	1.5	2.5	3.5	6.0	10.1	10.1	1.0	1.0	1.0	0.6
DMSO	Mean	18	25	61	69	163	159	8	9	4	4
	sd	2.0	3.5	4.6	5.7	8.3	6.1	2.5	1.5	1.0	0.0
Positive Controls*	Mean	589	124	499	984	500	596	293	101	60	285
	sd	39.5	8.0	12.2	68.4	21.2	17.4	24.4	6.1	10.6	9.2
	f(I)	32.72	4.96	7.34	14.26	3.14	3.75	32.56	11.22	15.00	71.25
5µL/plate	Mean	11	19	59	76	163	159	6	5	5	6
	sd	1.5	1.5	4.9	4.5	6.1	16.2	0.0	0.6	2.9	0.0
	f(I)	0.73	0.90	0.87	1.03	1.03	1.01	0.67	0.50	1.25	1.20
2.5µL/plate	Mean	17	21	63	75	161	157	7	7	6	6
	sd	0.0	2.6	5.9	1.5	12.2	10.1	2.1	1.0	1.5	1.2
	f(I)	1.13	1.00	0.93	1.01	1.01	1.00	0.78	0.70	1.50	1.20
1.25 µL/plate	Mean	19	20	73	71	163	169	9	8	4	6
	sd	2.1	2.6	5.9	6.0	14.0	12.2	2.6	2.1	0.6	1.0
	f(I)	1.27	0.95	1.07	0.96	1.03	1.08	1.00	0.80	1.00	1.20
0.63 µL/plate	Mean	20	21	70	67	156	165	8	9	4	5
	sd	2.5	3.1	6.0	6.7	6.9	12.2	1.5	2.1	1.0	1.5
	f(I)	1.33	1.00	1.03	0.91	0.98	1.05	0.89	0.90	1.00	1.00
0.313 µL/plate	Mean	18	25	68	68	169	159	9	8	5	5
	sd	2.1	1.5	2.5	5.7	4.6	12.2	1.7	1.2	2.3	0.6
	f(I)	1.20	1.19	1.00	0.92	1.06	1.01	1.00	0.80	1.25	1.00
0.156 µL/plate	Mean	19	21	64	59	155	163	8	8	5	4
	sd	3.5	5.0	3.1	2.1	12.2	8.3	1.2	2.0	1.0	1.5
	f(I)	1.27	1.00	0.94	0.80	0.97	1.04	0.89	0.80	1.25	0.80

sd = standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 13

f(I) = increase factor, calculation see chapter 7.4, page 20

bold marked values = relevant increase in the number of revertants

Applicant's summary and conclusion

Conclusions:

Tributyl(ethyl)phosphonium diphenyl phosphate is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.