Reaction product of phosphorus trichloride with reaction mass of 4-tert-butylphenol and phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline sudy

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Ll 362000, Annexe 4D, dated May 19, 2000

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: Kanpoan No. 287 - Environment Protection Agency; Eisei No. 127 - Ministry of Health & Welfare; Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-di-tert-butylphenyl diphenyl phosphite; 2-tert-butylphenyl diphenyl phosphite; 4-tert-butylphenyl diphenyl phosphite; triphenyl phosphite

EC Number:

941-802-9

Molecular formula:

Unspecified

IUPAC Name:

2,4-di-tert-butylphenyl diphenyl phosphite; 2-tert-butylphenyl diphenyl phosphite; 4-tert-butylphenyl diphenyl phosphite; triphenyl phosphite

Details on test material:

- Physical state: liquid
- Stability under test conditions: stable for hours in water, saline, polyethylene glycol and CMC
- Storage condition of test material: room temperature

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 S9 fraction

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Remarks:

with and without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Each concentration and the controls were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: backgrond growth, reduction in revertants

POSITIVE CONTROLS
- Without metabolic activation
TA 1535, TA100: sodium azide dissolved in water, 10 µg/plate
TA98: 4-nitro-o-phenyiene-diamine dissolved in DMSO, 10 µg/plate
TA1537: 4-nitro-o-phenyiene-diamine dissolved in DMSO, 50 µg/plate
WP2 uvrA: methyl methane sulfonate dissolved in water, 5 µl/plate
- with metabolic activation
TA 1535, TA 1537, TA 98, TA 100: 2-aminoanthracene dissolved in DMSO, 2.5 µg/plate
WP2 uvrA: 2-aminoanthracene dissolved in DMSO, 10 µg/plate

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP 2uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

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

Remarks:

starting from 2500 µg/plate in the preincubation assay

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. In the pre-experiment the concentration range of the test item was 3 - 5000 pg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 pg/plate were chosen as maximal concentration.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects, evident as a reduction in the number of revertants below 50 % of the corresponding solvent control, were observed during the preincubation assay (Experiment II) at the following concentrations:
TA 1535: 5000 µg/plate (with and without S9)
TA 1537: 2500-5000 µg/plate (with and without S9 mix)
TA 98: 2500 µg/plate (without S9) and 5000 µg/plate (with S9)
TA 100: 2500-5000 µg/plate (without S9)
WP2 uvrA: 5000 µg/plate (with S9)
The toxicity observed in the second experiment followed a rather shallow gradient leading to revertant colony counts around 50 % of the corresponding solvent control over broad dose ranges (e.g. in strain TA 1535 without metabolic activation). However, toxicity was not severe as indicated by the regular bacterial background growth and the plates were analysable at all concentrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

SUMMARY OF RESULTS

without S9 mix
	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Dose (µg/plate)	I	II	I	II	I	II	I	II	I	II
negative control	15	11	25	6	34	24	143	167	50	41
solvent control	18	9	25	6	39	23	122	127	44	42
positive control	852	665	245	47	181	200	964	908	830	173
33.3	23	8	30	7	38	25	135	146	42	41
100	21	7	41	5	32	25	136	143	41	41
333.3	20	5	27	4	27	24	142	116	47	30
1000	22	9	31	5	24	25	135	134	40	43
2500	17	6	37	2	31	10	142	54	38	37
5000	23	3	34	3	27	13	141	54	39	24

with S9 mix
	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Dose (µg/plate)	I	II	I	II	I	II	I	II	I	II
negative control	23	11	28	7	46	32	152	183	47	48
solvent control	22	12	28	10	49	27	145	149	51	43
positive control	114	106	245	61	351	598	515	872	162	169
33.3	21	13	31	8	42	33	133	162	57	40
100	21	7	31	6	45	34	110	156	53	41
333.3	23	5	34	4	46	33	113	169	56	36
1000	23	6	31	9	41	30	125	167	56	45
2500	23	6	33	5	50	23	122	131	47	30
5000	23	5	34	1	50	6	113	93	40	18

I = Plate Incorporation Test

II = Preincubation Test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and is therefore considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test article to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Distinct toxic effects, evident as a reduction in the number of revertants, occurred in the test groups of the second experiment with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.