Diisobutyl hydrogen 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:

23 July 2020 - 10 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment

Version / remarks:

31 March 2011

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diisobutyl hydrogen phosphate

EC Number:

228-602-0

EC Name:

Diisobutyl hydrogen phosphate

Cas Number:

6303-30-6

Molecular formula:

C8H19O4P

IUPAC Name:

bis(2-methylpropoxy)phosphinic acid

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium: histidine
Escherichia coli: tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight)
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- volume of S9 mix and S9 in the final culture medium: 0.5 mL S9-mix

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
First experiment (direct plate assay): 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (pre-incubation assay): 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Solvent used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 6-Chloro-9-(3-N-(2-Chloroethylamino) propylamino-2-methoxyacridine dihydrochloride ICR-191

Remarks:

Without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

With metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: 10^9 cells/mL
- Test substance added in agar (plate incorporation) (experiment 1) and pre-incubated (experiment 2)

TREATMENT AND HARVEST SCHEDULE:
Experiment 1 (the dose-range finding study with two tester strains is reported as part of the direct plate assay, experiment 1)
- Duration of treatment: 48 ± 4 h
- Temperature during treatment: 37.0 ± 1.0°C

Experiment 2
- Preincubation period: 30 ± 2 minutes
- Temperature during preincubation period: 37 ± 1°C
- Harvest time after the end of treatment: 48 ± 4 h
- Temperature after end of treatment: 37.0 ± 1.0°C

CYTOTOXICITY:
- 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.

FORMULATION ANALYSIS
- Sample collection: See under 'Any other information on materials and methods incl. tables'.
- Concentration and homogeneity analysis:
Storage conditions: At room temperature
Acceptance criteria: For concentration: mean sample concentration results within or equal to ± 10% of theoretical concentration. For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group.
- Stability analysis:
Conditions tested: 4 h at room temperature
Storage conditions: Temperature set to maintain room temperature
Acceptance criteria: Mean sample concentration results after storage that is within or equal to ±10% of the initial mean sample concentration results.

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

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

A test item is considered positive (mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
- 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:

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

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Positive controls validity:

valid

Test results 3

Key result

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

Positive controls validity:

valid

Test results 4

Key result

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

Positive controls validity:

valid

Test results 5

Key result

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test item on the plates was not observed in any tester strain.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : See table 1-3

Ames test:
EXPERIMENT 1
- Signs of toxicity : In strain TA100 (absence and presence) a slight reduction of the bacterial background lawn was observed. No biologically relevant decreases in the number of revertants were observed.
- Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

EXPERIMENT 2
- Signs of toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested 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.

HISTORICAL CONTROL DATA
- The negative and strain-specific positive control values were within our laboratory historical cont rol data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly (see table 4 and 5).

FORMULATION ANALYSIS
- Accuracy: In the vehicle, no test item was detected. The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
- Homogeneity: The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).
- Stability: Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

Any other information on results incl. tables

Table 1 Dose-Range Finding Test: Mutagenic Response of Diisobutyl hydrogen phosphate in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay
Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain.
	TA100	WP2uvrA

 Without S9-mix

Positive control	798	±	70		1234	±	44
Solvent control	100	±	15		11	±	6
1.7	107	±	11		13	±	2
5.4	99	±	13		11	±	3
17	99	±	9		15	±	3
52	95	±	18		21	±	8
164	120	±	2		16	±	5
512	100	±	9		15	±	1
1600	101	±	12	^n	21	±	5
5000	93	±	10	^s NP	17	±	8	^n NP

 With S9-mix

Positive control	1935	±	562		400	±	69
Solvent control	96	±	11		18	±	5
1.7	93	±	10		15	±	4
5.4	89	±	19		18	±	5
17	108	±	21		24	±	3
52	89	±	9		22	±	6
164	92	±	14		18	±	1
512	106	±	1		19	±	11
1600	102	±	16	^n	19	±	4
5000	80	±	6	^s NP	23	±	9	^n NP

 

Table 2 Experiment 1: Mutagenic Response of Diisobutyl hydrogen phosphate in theSalmonella typhimuriumReverse Mutation Assay

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimurium.
	TA1535	TA1537	TA98

 Without S9-mix

 

Positive control	863	±	118		968	±	37		1377	±	149
Solvent control	8	±	3		3	±	2		18	±	4
52	8	±	1		3	±	1		16	±	4
164	10	±	3		6	±	4		14	±	6
512	9	±	1		5	±	2		15	±	5
1600	11	±	3		4	±	3		15	±	4
5000	10	±	2	^n NP	4	±	0	^n NP	16	±	2	^n NP

 With S9-mix

 

Positive control	248	±	22		416	±	39		1182	±	156
Solvent control	11	±	4		5	±	2		21	±	5
52	14	±	1		4	±	1		17	±	5
164	10	±	5		5	±	2		17	±	6
512	12	±	1		8	±	3		19	±	1
1600	22	±	3		4	±	1		21	±	7
5000	19	±	6	^n NP	4	±	2	^n NP	21	±	2	^n NP

Table 3 Experiment 2: Mutagenic Response of Diisobutyl hydrogen phosphate in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay

Pre-incubation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

 Without S9-mix

 

Positive control	936	±	73		198	±	21		1697	±	133		703	±	30		1715	±	67
Solvent control	10	±	5		4	±	1		14	±	3		109	±	12		14	±	6
52	11	±	4		5	±	1		14	±	3		106	±	13		17	±	3
164	8	±	2		8	±	4		17	±	10		85	±	8		14	±	2
512	14	±	6		2	±	1		10	±	2		84	±	12		17	±	6
1600	13	±	3		4	±	1		15	±	10		87	±	7		17	±	6
5000	10	±	4	^n NP	5	±	3	^n NP	14	±	2	^n NP	93	±	8	^n NP	16	±	2	^n NP

 

 With S9-mix¹

 

Positive control	259	±	15		130	±	6		709	±	15		1560	±	61		453	±	49
Solvent control	11	±	7		5	±	5		19	±	1		95	±	13		18	±	11
52	15	±	0		6	±	5		19	±	1		93	±	13		25	±	7
164	8	±	4		4	±	3		18	±	10		98	±	8		19	±	8
512	14	±	6		7	±	4		20	±	2		96	±	10		21	±	2
1600	12	±	5		3	±	2		25	±	10		92	±	14		19	±	8
5000	10	±	5	^n NP	3	±	2	^n NP	18	±	9	^n NP	98	±	11	^n NP	19	±	1	^n NP

Table 4 HCD Solvent control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	4 – 25	2 – 24	2 – 18	5 – 61	8 – 60	61 – 188	55 - 176	11 – 61	12 - 68
Mean	10	11	5	6	14	19	110	103	25	31
SD	3	3	2	2	5	6	18	21	8	10
n	2733	2694	2707	2712	2717	2754	2806	2724	2622	2596

Table 5 HCD Positive control

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	107 – 1530	78 – 1481	58 – 1467	48 – 1239	365 – 2118	258 – 2033
Mean	955	282	811	290	1220	903
SD	174	114	372	138	221	367
n	2598	2587	2223	2630	2670	2621

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1993	397 – 2666	93 – 1999	109 - 1968
Mean	879	1371	1167	421
SD	170	412	527	169
n	2654	2634	2516	2528

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles, Diisobutyl hydrogen phosphate was found not to be mutagenic with or without metabolic activation.

Executive summary:

In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles. The test item did not induce a 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 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. In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrAin the pre-incubation assay. In all three experiments the test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 at the dose level of 5000μg/platein the absence and presence of S9-mix in the dose-range finding study. No toxicity was observed in any of the other strains at all conditions tested. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that diisobutyl hydrogen phosphate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation. Since all acceptability criteria were met, the study is considered valid.