5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

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:

2016-09-16 to 2016-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

EC Number:

817-187-7

Cas Number:

1803088-15-4

Molecular formula:

C54 H73 O5 P

IUPAC Name:

5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

Details on test material:

- State of aggregation: Solid / white
- Storage Condition: room temperature under light exclusion

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; under light exclusion ; no direct sunlight

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:

induced rat liver S9 mix

Test concentrations with justification for top dose:

1st experiment and 2nd experiment:
0; 33; 100; 333; 1000; 2500; 5000 µg/plate
Top dose of 5 mg/plate was selected in agreement with the current guidelines.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO has been demonstrated to be suitable for bacterial reverse mutation tests and historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 4-nitro-o-phenylenediamine (NOPD) without S9, strain: TA 98; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) without S9, strains: TA 1535 and TA 1537; 2-aminoanthracene (2-AA) with S9 all strains and without S9, strains: TA1537

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

SELECTION AGENT: -his and -trp deficient medium

NUMBER OF REPLICATIONS: 3 plates per dose

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants (factor <= 0.6)
- clearing or diminution of the background lawn

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: DMSO was used as vehicle
- Precipitation: was found from 33 µg/plate onward with and without S9 mix

HISTORICAL CONTROL DATA
- Positive historical control data: please refer to "Any other information on results" table 4
- Negative (solvent/vehicle) historical control data: please refer to "Any other information on results" table 4

CYTOTOXICITY
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward. In the preincubation assay weak bacteriotoxicity (slight decrease in the number of his+ revertants) was occasionally observed depending on the strain and test conditions from about 33 μg/plate onward. Please also refer to "Any other information on results" table 3.

Any other information on results incl. tables

Table 1. Experiment 1 standard plate incorporation test

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli
	TA 100	TA 98	TA 1535	TA 1537	WP2 uvrA
	Results without S9
DMSO	103.0 ± 14.0	21.7 ± 4.2	8.3 ± 1.5	8.0 ± 1.0	19.7 ± 1.2
33	107.0 ± 7.0	28.3 ± 6.4	12.7 ± 2.1	6.0 ± 1.0	13.0 ± 4.4
100	107.3 ± 15.0	25.0 ± 9.5	9.0 ± 3.5	9.0 ± 2.0	13.3 ± 3.2
333	103.3 ± 6.0	28.0 ± 11.3	9.0 ± 2.6	7.0 ± 1.0	15.0 ± 1.0
1000	94.7 ± 8.6	24.0 ± 1.0	7.3 ± 2.3	3.3 ± 1.5	12.7 ±4.5
2500	82.7 ± 15.0	10.0 ± 4.0	6.0 ± 3.6	3.7 ± 2.1	15.7 ± 1.5
5000	85.0 ± 5.6	8.3 ± 2.5	7.7 ± 1.5	4.3 ± 2.5	17.7 ± 6.1
MNNG (5)	4149.7 ± 147.0	-	4984.7 ± 175.6	-	-
NOPD (10)	-	909.7 ± 30.1	-	-	-
AAC (100)	-	-	-	1997.7 ± 133.0	-
4-NQO (5)	-	-	-	-	1130.3 ± 48.2
	Results with S9
DMSO	112.0 ± 16.0	25.7 ± 5.5	9.0 ± 1.7	9.7 ± 3.5	26.3 ± 8.5
33	101.7 ± 7.5	28.3 ± 9.1	7.7 ± 2.3	12.0 ± 4.0	23.0 ± 9.6
100	95.3 ± 4.7	29.3 ± 6.7	8.0 ± 3.6	11.0 ± 2.6	25.0 ± 4.6
333	92.0 ± 27.6	28.7 ± 1.5	10.3 ± 2.1	7.7 ± 4.0	29.7 ± 8.1
1000	78.7 ± 7.5	22.7 ± 4.5	8.7 ± 2.1	6.7 ± 2.1	25.0 ± 1.7
2500	68.3 ± 8.4	12.7 ± 2.1	5.3 ± 0.6	6.0 ± 2.6	17.0 ± 3.6
5000	109.0 ± 14.2	21.7 ± 7.2	8.3 ± 2.1	8.3 ± 4.2	18.7 ± 3.5
2- AA (2.5)	1467.3 ± 116.7	1234.3 ± 41.5	149.3 ± 12.4	118.7 ± 3.8	93.3 ± 21.2

Table 2. Experiment 2 Pre-incubation test

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli
	TA 100	TA 98	TA 1535	TA 1537	WP2 uvrA
	Results without S9
DMSO	94.7 ± 6.1	20.3 ± 6.4	15.3 ± 3.8	9.7 ± 3.1	20.0 ± 7.5
33	84.0 ± 3.5	19.0 ± 4.4	7.7 ± 1.2	9.0 ± 2.0	14.3 ± 2.5
100	92.0 ± 3.6	15.0 ± 3.6	7.7 ± 2.9	7.7 ± 2.1	13.7 ± 3.1
333	90.7 ± 8.1	18.0 ± 2.6	10.3 ± 6.5	11.0 ± 3.0	14.0 ± 2.6
1000	84.7 ± 12.9	13.0 ± 5.3	8.7 ± 2.1	11.7 ± 4.0	15.0 ± 5.6
2500	90.3 ± 14.6	17.7 ± 1.2	7.0 ± 2.6	11.0 ±4.4	13.0 ± 6.1
5000	76.0 ±8.7	13.0 ± 4.0	8.7 ± 1.5	6.3 ± 2.5	13.3 ± 3.1
MNNG (5)	2189.0 ± 191.8	-	2491.7 ± 96.4	-	-
NOPD (10)	-	811.0 ±41.4	-	-	-
AAC (100)	-	-	-	1201.7 ± 148.5	-
4-NQO (5)	-	-	-	-	322.0 ± 18.1
	Results with S9
DMSO	124.0 ±16.7	22.3 ± 3.6	12.0 ± 3.5	11.0 ± 5.3	22.0 ± 3.6
33	71.3 ± 1.2	16.7 ± 3.8	8.7 ± 1.2	6.7 ± 3.8	16.7± 3.8
100	76.0 ± 8.0	16.0 ± 1.0	9.0 ± 4.4	9.0 ± 1.7	16.0 ± 1.0
333	67.0 ± 2.6	17.0 ± 2.6	8.0 ±4.6	10.3 ± 3.1	17.0 ± 2.6
1000	73.3 ± 18.5	14.7 ± 4.2	9.0 ± 4.6	9.0 ± 0.0	14.7 ± 4.2
2500	73.3 ± 8.3	16.7 ± 3.1	8.3 ± 2.3	9.0 ± 1.0	16.7 ± 3.1
5000	82.0 ± 11.3	14.7 ± 4.6	4.3 ± 0.6	5.3 ± 1.5	14.7 ± 4.6
2- AA (2.5)	1187.7 ± 57.6	111.7 ± 6.4	167.7 ± 10.7	156.7 ±17.6	111.7 ± 6.4

Table 3. Decreased revertant numbers (cytotoxicity) observed at following concentrations (µg/plate)

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E. coli
1st - SPT	Without	-	-	1000 - 5000	2500 - 5000	-
	With	2500
2nd - PIT	Without	1000 - 5000	-	-	5000	-
	With	5000	33 - 2500	5000	-	-

Table 4. Historical control data

		Bacterial strains
Historical control data of DMSO control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	18	95	10	7	22
		SD	3.1	9.9	2.2	1.5	4.0
		Minimum	12	69	6	5	12
		Maximum	30	140	17	12	33
	+S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	24	100	10	8	23
		SD	4.7	12.3	2.1	2.2	4.0
		Minimum	13	76	6	5	13
		Maximum	35	150	18	16	35
Historical control data of positive control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	486	3331	4063	975	884
		SD	183.3	1021.5	1308.8	339.9	442.4
		Minimum	271	1125	1174	253	133
		Maximum	431	5557	6612	1858	1552
	+S9 (2-AA		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	1427	1792	217	130	98
		SD	361.9	448.9	50.0	33.6	26.6
		Minimum	431	361	108	56	54
		Maximum	2427	2819	367	241	169

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains in a reverse mutation assay. Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were used as tester strains with a dose range of 33 μg - 5000 μg/plate in both standard plate test and preincubation test with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance GSID 1212253 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.