Bis(2,4,4-trimethylpentyl)phosphinic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From December 19th 1994 to January 31st 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP guideline study with some restrictions: no certificate of analysis, only one main experiment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: no certificate of analysis, only one main experiment

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2,4,4-trimethylpentyl)phosphinic acid

EC Number:

280-445-7

EC Name:

Bis(2,4,4-trimethylpentyl)phosphinic acid

Cas Number:

83411-71-6

Molecular formula:

C16H35O2P

IUPAC Name:

bis(2,4,4-trimethylpentyl)phosphinic acid

Test material form:

other: clear colorless liquid

Method

Target gene:

Each strain derived from S. typhimurium contains one mutation in the histidine operon, resulting in a requirement for histidine. The Escherichia coli strain WP2 contains a mutation in the trp gene, resulting in a requirement of tryptophan.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: additional mutations in rfa and uvrB genes. For the strains TA98 and TA100 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens.

Species / strain 2

Species / strain / cell type:

S. typhimurium, other: TA1538

Additional strain / cell type characteristics:

other: additional mutations in rfa and uvrB genes. For the strains TA98 and TA100 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens.

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: deletion in the uvrA gene

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Range finding study (on TA100 and E. coli WP2): from 10 to 10000 µg/plate
Main study: 33; 100; 333; 1000; 3333 and 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all bacterial strain with S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-nitrofluorene (TA98, TA1538)

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (direct plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 to 72 hrs

NUMBER OF REPLICATIONS: Range-finding: one plate; Main test: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of the background bacterial lawn (thining, increase in the size of the microcolonies)

Evaluation criteria:

The mean of each positive control must exhibit at least three-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels are required to evaluate assay data.
A dose level is considered toxic if one or both of the following criteria are met: 1) A > 50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction mus be accompanied by an abrupt dose-dependent drop in the revertant count. 2) A reduction in the background lawn.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article. Data sets for strains TA1535, TA1537 and TA1538 will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2 uvrA will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: the results of the dose range-finding study and the main study indicate that no precipitate was observed up to the maximum dose level (10000µg/plate)
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: the results of the dose range-finding study indicate that no precipitate but toxicity was observed. Therefore the maximum dose that was plated in the mutagenicity assay was 10000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: the values obtained for the vehicle and positive control of each strain were in the range of the historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity was observed except in the E.coli WP2 strain. Without metabolic activation, cytoxicity is observed from concentration of 1000 µg/plate in TA98, TA100, TA1537 and TA1538 and from 3333 µg/plate in TA1535. With metabolic activation, cytoxicity is observed from concentration of 3333 µg/plate in TA98, TA100, TA1535 and from 1000 µg/plate in TA1537 and TA1538.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Number of revertants per plate in the absence of metabolic activation (direct plate incorporation method)

Concentration (µg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	29	5	157	4	12	1	7	2	7	1	16	6
33	30	5	164	10	8	4	7	2	7	4	17	1
100	28	4	153	11	10	3	5	4	7	4	16	4
333	28	6	147	5	13	4	5	4	8	1	18	6
1000	21	4	140	15	10	3	4	2	4	3	18	5
3333	13	6	106	11	10	3	1	2	3	0	14	6
10000	9	2	87	16	7	3	4	2	1	1	18	5
Positive control**	362	39	653	93	626	58	102	46	560	26	145	11

$: Mean of triplicate

*Solvent control = 50 µl ethanol

**Mutagens positive controls:

- 2-nitrofluorene (1 µg/plate) in TA98 and TA1538 strains

- Sodium azide (1 µg/plate) in TA100 and TA1535 strains

- 9-aminoacridine (75 µg/plate) in TA1537 strain

- Methyl methanesulfonate (1000 µg/plate) in E.coli WP2 strain

Number of revertants per plate in the presence of metabolic activation (direct plate incorporation method)

Concentration (µg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	45	6	192	6	14	4	8	3	19	3	15	1
33	44	14	183	52	17	7	8	3	17	2	17	4
100	41	7	189	13	12	3	8	4	14	4	22	1
333	46	4	192	6	13	2	7	3	19	3	23	3
1000	46	9	183	10	15	5	7	3	7	1	20	2
3333	23	2	129	15	13	5	6	2	5	1	14	4
10000	25	3	106	11	8	4	2	2	2	1	16	4
Positive control**	755	29	873	4	89	11	89	9	958	31	395	10

$: Mean of triplicate

*Solvent control = 50 µL ethanol

**Mutagens positive controls:

- 2-Aminoanthracene (1 µg/plate) in TA98, TA100, TA1535, TA1537 and TA1538 strains

- 2-Aminoanthracene (10 µg/plate) in E.coli WP2 strain

Applicant's summary and conclusion

Conclusions:

negative with and without metabolic activation

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD guideline No.471, and in compliance with the GLP, strains TA 98, TA 100, TA1535, TA1537, TA1538 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to the test material diluted in ethanol in the presence or absence of mammalian metabolic activation (fraction of S9 from the liver of rats treated with Aroclor 1254). The method of direct incorporation was tested.

The results of the dose range-finding study performed on TA100 strain of Salmonella typhimurium and on Escherichia coli WP2 uvrA with 10 concentrations ranging from 10 to 10000 µg/plate indicate that no precipitate but toxicity was observed. Therefore the maximum dose that was plated in the main mutagenicity assay was10000 µg/plate. The substance was tested up to the highest recommended limit concentration (5000 µg/plate) and cytotoxicity was observed at concentrations equal and higher than 1000 or 3000 µg/plate depending on the bacteria strain and the metabolic activation condition (with or without).The positive controls induced the appropriate responses in the corresponding strains.

Under the test conditions, the test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium TA 98, TA 100, TA1535, TA1537 and TA1538 and with Escherichia coli WP2 uvrA as there was no evidence of induced mutant colonies over background.

This study was considered as acceptable as it satisfied the main criteria of the OECD guideline 471.