Trihexyl phosphate

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:

16 Feb - 09 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovenska Narodna Akreditacna Sluzba, Bratislava, Slovenska Republika

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trihexyl phosphate

EC Number:

219-774-8

EC Name:

Trihexyl phosphate

Cas Number:

2528-39-4

Molecular formula:

C18H39O4P

IUPAC Name:

trihexyl phosphate

Method

Target gene:

his operon (for S. typhimurium strains), trp operon (for E. coli strain)

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor

Test concentrations with justification for top dose:

Range-finding study: 50, 150, 300, 500, 1500 and 5000 µg/plate in TA 97 and TA 100 without metabolic activation

First main experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second main experiment: 15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: DMSO was chosen due to the insolubility of the test substance in water. The vehicle used did not affect the spontaneous mutation level and it is recommended for use in this test.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Range Finding Test and first main experiment); preincubation (second main experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 replications each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: The bacterial growth was controlled in order to determine toxicity of the test substance.

Evaluation criteria:

Acceptance criteria
The study is considered valid if all of the following criteria are met:
- tester strains fulfil the criteria for sensitivity to UV light
- tester strains exhibit sensitivity to crystal violet
- tester Salmonella strains TA97, TA98, TA100 strain exhibit resistance to ampicillin
- all tester Salmonella strains and E.coli WP 2 uvrA exhibit a characteristic number of spontaneous revertant colonies when plated. The mean should be within the range of historical control values or within the published historical range
- all tester strains exhibit at least a three-fold increase in mutagen-induced revertant colonies when plated with positive control chemicals

Evaluation criteria
Considering biological relevance the test substance is considered positive if the assay is valid and the following conditions are met:
- concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- Mutation factor >2

The positive result indicates that the test substance induces mutations in Salmonella typhimurium or E.coli cells.

The test item for which results do not meet the above criteria is considered non-mutagenic in this test.
Negative results indicate that under the test conditions, the test substance does not produce mutations in Salmonella typhimurium or E.coli cells.

Statistics:

The mutation frequency at each dose concentration level of the test item was compared to the one observed in negative and positive controls. The statistical analysis was carried out using unpaired T-test.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: No inhibition of the bacterial growth was observed in the two tested Salmonella strains TA 97 and TA 100 in the concentration range 50 - 5000 µg/plate without metabolic activation. Therefore the same doses (except 300 µg/plate) were tested in all Salmonella strains (TA 97, TA 98, TA 100 and TA 1535) and in E. coli WP2 uvr A in the main tests, with and without metabolic activation.

HISTORICAL CONTROL DATA
The mean revertants for controls for Salmonella tester strains and E.coli WP2 uvr were within the range of historical control values (please refer to Table 3 in "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1:Summary of test results (experiment 1; Plate Incorporation Method)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Frameshift type		Base-pair substitution type
		TA 97	TA98	TA100	TA1535	WP2 uvr A
–	Solvent control (DMSO)	124 ± 3	18 ± 2	145 ± 12	17 ± 3	26 ± 7
	50	116 ± 3 *	17 ± 3	127 ± 3	16 ± 3	24 ± 5
	150	119 ± 2	19 ± 3	130 ± 2	14 ± 1	32 ± 3
	500	105 ± 5 *	18 ± 7	122 ± 18	15 ± 3	28 ± 9
	1500	128 ± 9	17 ± 3	148 ± 19	17 ± 3	25 ± 4
	5000	115 ± 8	18 ± 4	144 ± 7	18 ± 3	33 ± 5
	Positive controls (µg/plate)	9AA (75)	2NF (3)	SA (1.5)	SA (1.5)	4NQO (5)
	Mean No. of colonies/plate (average of 3 plates)	577 ± 38 *	432 ± 78 *	531 ± 28 *	448 ± 71 *	145 ± 8 *
+	Solvent control (DMSO)	152 ± 16	28 ± 9	133 ± 13	18 ± 5	39 ± 4
	50	136 ± 16	28 ± 2	134 ± 6	22 ± 7	44 ± 3
	150	149 ± 9	27 ± 1	124 ± 15	19 ± 3	43 ± 2
	500	153 ± 11	26 ± 8	113 ± 21	15 ± 2	38 ± 6
	1500	148 ± 4	26 ± 1	128 ± 7	17 ± 1	39 ± 8
	5000	159 ± 9	28 ± 2	118 ± 7	17 ± 2	43 ± 2
	Positive controls  (µg/plate)	2AA (5)
	Mean No. of colonies/plate (average of 3 plates)	549 ± 57 *	159 ± 12 *	772 ± 25 *	211 ± 8 *	328 ± 17 *

2AA = 2-aminoanthracene

2NF = 2-nitro-fluorene

4NQO = 4-nitroquinoline-1-oxide

9AA = 9-aminoacridine

SA = sodium azide

SD = standard deviation

* = p < 0.05

Table 2: Summary of test results (experiment 2; Preincubation Method)

	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Frameshift type		Base-pair substitution type
		TA 97	TA98	TA100	TA1535	WP2 uvr A
Without S9 Mix	Solvent control (DMSO)	159 ± 10	18 ± 5	130 ± 17	14 ± 3	34 ± 8
	15	145 ± 8	17 ± 7	129 ± 8	13 ± 3	28 ± 6
	50	151 ± 2	13 ± 4	119 ± 12	16 ± 1	28 ± 1
	150	151 ± 2	17 ± 5	116 ± 4	15 ± 2	30 ± 4
	500	153 ± 10	16 ± 2	117 ± 12	15 ± 3	34 ± 4
	1500	153 ± 12	13 ± 4	116 ± 17	14 ± 2	31 ± 4
	5000	155 ± 4	15 ± 1	127 ± 7	12 ± 4	33 ± 6
	Positive controls (µg/plate)	9AA (75)	2NF (3)	SA (1.5)	SA (1.5)	4NQO (5)
	Mean No. of colonies/plate (average of 3 plates)	657 ± 60 *	400 ± 17	531 ± 28 *	448 ± 71 *	145 ± 8 *

2AA = 2-aminoanthracene

2NF = 2-nitro-fluorene

4NQO = 4-nitroquinoline-1-oxide

9AA = 9-aminoacridine

SA = sodium azide

SD = standard deviation

* = p < 0.05

Table 3: Historical negative and positive control values (2015 -2018)

S9 Mix		Revertants per plate
		TA 100		TA 1535		TA 97		TA 98		WP2 uvr A
		Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos
-	Mean	194	698	23	624	163	1135	25	246	38	423
	SD	35	170	8	170	30	527	7	141	12	270
	Min	115	440	6	336	114	400	12	117	25	82
	Max	272	1148	43	964	227	2776	43	680	68	912
+	Mean	180	1041	23	192	195	893	36	779	46	397
	SD	29	443	8	130	36	369	12	438	10	145
	Min	123	398	11	34	127	370	19	256	31	194
	Max	231	2888	43	604	286	1616	85	1644	70	648

SD = standard deviation

Min = minimum value

Max = maximum value

Neg = negative control

Pos = positive control

Applicant's summary and conclusion

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells tested up to limit concentration.