2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012 -01-25 till 2012-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

acc. to §19 German Chemikaliengesetz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide

EC Number:

223-829-1

EC Name:

2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide

Cas Number:

4090-51-1

Molecular formula:

C10H20O5P2S2

IUPAC Name:

2,2'-oxybis(5,5-dimethyl-1,3,2-dioxaphosphinane) 2,2'-disulfide

Test material form:

solid: particulate/powder

Remarks:

migrated information: particulates

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
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.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: DMSO chosen because of solubility properties.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation and pre-incubation;

DURATION
- Pre-incubation period: 1 hour
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS:
3 plates

DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

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 WP2 uvrA) 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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed in the overlay agar in the test tubes and on the incubated agar plates from 1000 - 5000 µg/plate in both experiments.
COMPARISON WITH HISTORICAL CONTROL DATA:
performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
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.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

Remarks on result:

other: other: reverse mutation assay

Any other information on results incl. tables

1.1     Summary of Results Pre-Experiment and Experiment I

Study Name: 1455602	Study Code: Harlan CCR 1455602
Experiment: 1455602 VV Plate	Date Plated: 25/01/2012
Assay Conditions:	Date Counted: 30/01/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			22 ± 1	9 ± 3	24 ± 3	104 ± 20	49 ± 6
	Untreated			20 ± 4	10 ± 5	32 ± 11	102 ± 5	55 ± 6
	test item	3 µg		25 ± 7	10 ± 2	27 ± 7	98 ± 21	54 ± 10
	Labortrocknung	10 µg		19 ± 2	13 ± 3	28 ± 5	101 ± 3	46 ± 3
		33 µg		16 ± 3	10 ± 4	28 ± 1	88 ± 2	59 ± 14
		100 µg		25 ± 7	12 ± 2	23 ± 1	91 ± 10	56 ± 7
		333 µg		21 ± 1	12 ± 3	31 ± 5	95 ± 6	47 ± 3
		1000 µg		25 ± 9^P	16 ± 1^P	29 ± 4^P	84 ± 14^P	54 ± 4^P
		2500 µg		22 ± 3^P	10 ± 2^P	26 ± 6^P	91 ± 9^P	55 ± 7^P
		5000 µg		22 ± 3^P M	10 ± 2^P M	22 ± 4^P M	99 ± 11^P M	57 ± 7^P M
	NaN3	10 µg		1979 ± 56			2218 ± 118
	4-NOPD	10 µg				298 ± 22
	4-NOPD	50 µg			71 ± 11
	MMS	3.0 µL						898 ± 52
With Activation	DMSO			33 ± 3	18 ± 5	38 ± 3	101 ± 8	58 ± 6
	Untreated			28 ± 5	22 ± 7	37 ± 10	108 ± 12	67 ± 6
	test item	3 µg		34 ± 4	20 ± 7	46 ± 7	117 ± 5	64 ± 5
	Labortrocknung	10 µg		34 ± 4	20 ± 10	42 ± 6	108 ± 13	60 ± 8
		33 µg		37 ± 4	18 ± 7	44 ± 4	121 ± 5	64 ± 13
		100 µg		30 ± 10	19 ± 3	44 ± 4	120 ± 13	59 ± 14
		333 µg		39 ± 4	20 ± 4	50 ± 6	119 ± 13	72 ± 3
		1000 µg		37 ± 1^P	18 ± 3^P	40 ± 6^P	117 ± 4^P	65 ± 1^P
		2500 µg		34 ± 1^P	21 ± 6^P	38 ± 2^P	127 ± 13^P	67 ± 5^P
		5000 µg		32 ± 3^P M	18 ± 3^P M	43 ± 8^P M	116 ± 11^P M	61 ± 4^P M
	2-AA	2.5 µg		389 ± 16	323 ± 12	2304 ± 190	2562 ± 97
	2-AA	10.0 µg						386 ± 26

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

 

1.2     Summary of Experiment II

Study Name: 1455602	Study Code: Harlan CCR 1455602
Experiment: 1455602 HV2 Pre	Date Plated: 09/02/2012
Assay Conditions:	Date Counted: 15/02/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			22 ± 5	13 ± 2	29 ± 5	96 ± 9	46 ± 3
	Untreated			20 ± 5	14 ± 4	33 ± 4	113 ± 1	43 ± 6
	test item	33 µg		20 ± 5	14 ± 2	31 ± 4	94 ± 5	45 ± 8
	Labortrocknung	100 µg		22 ± 11	11 ± 4	31 ± 12	100 ± 1	46 ± 7
		333 µg		17 ± 3	15 ± 2	32 ± 4	87 ± 7	48 ± 9
		1000 µg		21 ± 8^P	14 ± 1^P	23 ± 3^P	77 ± 10^P	44 ± 12^P
		2500 µg		25 ± 6^P	14 ± 5^P	38 ± 3^P	86 ± 13^P	54 ± 14^P
		5000 µg		18 ± 4^P M	12 ± 3^P M	21 ± 3^P M	95 ± 9^P M	38 ± 4^P M
	NaN3	10 µg		1919 ± 37			2331 ± 42
	4-NOPD	10 µg				368 ± 16
	4-NOPD	50 µg			84 ± 7
	MMS	3.0 µL						287 ± 27
With Activation	DMSO			24 ± 3	20 ± 4	39 ± 4	126 ± 16	57 ± 6
	Untreated			29 ± 6	28 ± 8	52 ± 8	112 ± 3	60 ± 4
	test item	33 µg		25 ± 7	22 ± 8	41 ± 5	114 ± 6	57 ± 8
	Labortrocknung	100 µg		26 ± 10	24 ± 1	46 ± 6	110 ± 12	65 ± 7
		333 µg		28 ± 5	26 ± 4	43 ± 4	116 ± 6	52 ± 4
		1000 µg		26 ± 7^P	28 ± 5^P	38 ± 6^P	81 ± 22^P	52 ± 8^P
		2500 µg		31 ± 3^P	22 ± 2^P	43 ± 6^P	85 ± 17^P	52 ± 14^P
		5000 µg		25 ± 8^P M	19 ± 2^P M	35 ± 4^P M	83 ± 2^P M	54 ± 11^P M
	2-AA	2.5 µg		287 ± 9	143 ± 14	1319 ± 58	1473 ± 141
	2-AA	10.0 µg						362 ± 40

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

Applicant's summary and conclusion

Conclusions:

Negative with and without metabolic activation.

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.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and theEscherichia colistrain 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:

Pre-Experiment/Experiment I:          3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                    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.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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. They 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 and frameshifts in the genome of the strains used with and without metabolic activation.