2,4,8,10-tetra(tert-butyl)-6-hydroxy-12H-dibenzo[d,g][1,3,2]dioxaphosphocin 6-oxide, sodium salt

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

First Addendum to OECD Guidelines No. 4 71, "Salmonella typhimurium, Reverse Mutation Assay", adopted May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 84/449, L 251, B 14, p. 143-145

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4,8,10-tetra(tert-butyl)-6-hydroxy-12H-dibenzo[d,g][1,3,2]dioxaphosphocin 6-oxide, sodium salt

EC Number:

286-344-4

EC Name:

2,4,8,10-tetra(tert-butyl)-6-hydroxy-12H-dibenzo[d,g][1,3,2]dioxaphosphocin 6-oxide, sodium salt

Cas Number:

85209-91-2

Molecular formula:

C29H43O4P.Na

IUPAC Name:

sodium 5,7,13,15-tetra-tert-butyl-10-oxo-9,11-dioxa-10λ⁵-phosphatricyclo[10.4.0.0³,⁸]hexadeca-1(12),3,5,7,13,15-hexaen-10-olate

Method

Target gene:

Histidine locus in selected strains

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: nutrient medium: 8 g Difco Nutrient Broth, 5 g NaCl in 20 ml (for 0,5 ml bacterial suspension)
- Storage: stock cultures in ampoules with nutrient broth and 5 % DMSO in liquid nitrogen
- Properly maintained: yes

Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in C C R according to Ames et al. (1970, 1977) . In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254 induced rat liver)

Test concentrations with justification for top dose:

Pre-study with strain TA 98 and TA 100: 1 – 5000 µg/plate

Main-experiments:
Exp. I : TA 1535, TA 1537, TA 98, TA 100:
10.0; 33.3; 100.0; 333.3; 1000.0 and 5000.0 µg /plate
Exp. II : TA 1535, TA 98, TA 100:
10.0; 33.3; 100.0; 333.3; 1000.0 and 5000.0 µg/plate
Exp. II: TA 1537:
10.0; 33.3; 66.6; 333.3; 100.0; 666.6 and 1000.0 µg/plate

Vehicle / solvent:

Solvent: Methanol. On the day of experiment, the test article was dissolved.
Justification: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

sodium azide for strains: TA 1535, TA 100 without metabolic activation, 10 µg/plate, dissolved in aqua dest..

Controls 2

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

4-nitro-o-phenylene-diamine for strains TA 1537, TA 98 without metabolic activation, dissolved in DMSO, 50 µg/plate.

Controls 3

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2-aminoanthracene for all strains with metabolic activation, dissolved in DMSO, 10 µg /plate.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72h

NUMBER OF REPLICATIONS: 3/strain/dose for each of the two experiments.

Evaluation criteria:

The test material is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.

A significant response is described as follows:
The test material is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test material regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method was available.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

partial or complete reduction in the number of revertants, at the higher dose levels with and without metabolic activation in experiment I and II in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration- dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Table 1: Results WITHOUT S9 mix (/ = not performed)

Dose µg/plate	Strains Experiment	TA 1535		TA 1537		TA 98		TA 100
		I	II	I	II	I	II	I	II
Neg. control		9	9	10	6	24	21	107	96
Solvent control		8	7	6	7	23	16	99	94
10.0		10	13	5	6	24	19	111	98
33.3		8	6	6	7	32	16	99	99
66.6		/	/	/	10	/	/	/	/
100.0		8	7	6	9	21	15	78	76
333.3		8	8	5	7	23	17	75	44
666.6		/	/	/	2	/	/	/	/
1000.0		5	5	3	0	11	9	29	8
5000.0		3	2	0	/	0	1	24	4
Pos. contr.: Sodium azid 10 µg/plate		122	964					29	8
Pos. control: 4-Nitro-o-phenylene-diamine 50 µg/plate				193	318	1981	1913

Table 2: Results WITH S9 mix (/ = not performed)

Dose µg/plate	Strains Experiment	TA 1535		TA 1537		TA 98		TA 100
		I	II	I	II	I	II	I	II
Neg. control		11	13	5	8	43	20	95	93
Solvent control		9	13	7	11	43	32	99	115
10.0		11	13	12	11	44	27	128	120
33.3		10	10	11	14	46	27	147	102
66.6		/	/	/	9	/	/	/	/
100.0		11	12	8	10	30	28	112	122
333.3		12	15	7	10	38	21	132	126
666.6		/	/	/	6	/	/	/	/
1000.0		7	11	4	5	27	18	123	86
5000.0		4	1	0	/	1	0	27	13
Pos. contr.: 2-Amino-anthracene 10 µg/plate		126	117	111	132	772	753	783	926

Applicant's summary and conclusion

Conclusions:

Interpretation of results : negative

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.