Dibenzo[d,f][1,3,2]dioxaphosphepin, 6,6'-[[3,3',5,5'-tetrakis(1,1-dimethylethyl)[1,1'-biphenyl]-2,2'-diyl]bis(oxy)]bis-

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: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

yes

Remarks:

2-Aminoanthracene is used as the sole indicator of the efficacy of the S9-mix.

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid - liquid: suspension

Details on test material:

- Name of test material (as cited in study report): L4-Ligand
- Physical state: Solid, white
- Analytical purity: 97.0 g/100 g
- Lot/batch No.: 0005473663
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period is guaranteed until 01 Feb 2013
- Storage condition of test material: Room temperature (protected from humidity; N2 conditions)

Method

Target gene:

his and trp operon

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital i.p. and β-naphthoflavone orally.

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2500 and 5 000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in ultrapure water, ethanol was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

with S9 mix for all strains

Controls 2

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

without S9 mix for strains TA 1535, TA 100

Controls 3

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Remarks:

without S9 mix for strain TA 98

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix for strain 1537

Controls 5

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix for strain E. coli WP2 uvrA

Details on test system and experimental conditions:

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

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

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:

other: A weak bacteriotoxic effect was occasionally observed in the SPT depending on the strain and test conditions from about 1000 μg/plate onward. In the PIT no bacteriotoxicity was observed up to the highest required concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 2 500 μg/plate onward only without S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (standard plate test).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	E. coli	TA98	TA1537
–	0	78 ± 7	11 ± 1	56 ± 8	31 ± 3	9 ± 3
–	33	73 ± 21	11 ± 1	54 ± 10	28 ± 7	7 ± 2
–	100	78 ± 1	11 ± 1	52 ± 3	23 ± 3	10 ± 4
–	333	80 ± 8	11 ± 1	52 ± 8	30 ± 3	7 ± 2
–	1000	83 ± 15	11 ± 2	50 ± 7	26 ± 1	5 ± 2
–	2500	72 ± 7P	11 ± 1P	47 ± 11P	30 ± 9 P	5 ± 4 P
-	5000	76 ± 16P	10 ± 2P	41 ± 3 P	24 ± 5 P	6 ± 3 P
Positive controls, –S9	Name	MNNG	MNNG	4NQO	4NOPD	AAC
	Concentrations (μg/plate)	5	5	5	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	655 ± 57	625 ± 54	940 ± 24	636 ± 67	446 ± 31
+	0	80 ± 10	11 ± 1	49 ± 10	36 ± 8	8 ± 2
+	33	86 ± 17	13 ± 2	43 ± 6	30 ± 3	8 ± 1
+	100	73 ± 4	13 ± 3	53 ± 5	42 ± 3	10 ± 6
+	333	82 ± 13	12 ± 2	47 ± 10	35 ± 8	8 ± 1
+	1000	75 ± 12	12 ± 1	44 ± 7	34 ± 5	7 ± 5
+	2500	89 ± 5P	12 ± 1P	50 ± 7 P	38 ± 6 P	8 ± 2 P
+	5000	82 ± 15P	11 ± 1P	54 ± 8 P	30 ± 3 P	7 ± 1 P
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	1198 ± 125	146 ± 31	255 ± 13	968 ± 11	155 ± 7

 MNNG = N-methyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

4NOPD = 4-nitroquinoline-N-oxide

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

P = Precipitate

Table 2: Test results of experiment 2 (preincubation test).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	E. coli	TA98	TA1537
–	0	74 ± 7	12 ± 1	37 ± 4	19 ± 1	7 ± 2
–	33	82 ± 7	12 ± 3	39 ± 6	20 ± 5	7 ± 1
–	100	76 ± 8	13 ± 2	36 ± 8	20 ± 3	7 ± 3
–	333	76 ± 7	11 ± 3	35 ± 10	21 ± 5	7 ± 2
–	1000	76 ± 3	12 ± 2	37 ± 7	19 ± 2	7 ± 1
–	2500	73 ± 7P	12 ± 1P	37 ± 6P	21 ± 2 P	6 ± 2 P
-	5000	73 ± 3P	12 ± 2P	38 ± 6P	20 ± 4 P	6 ± 2 P
Positive controls, –S9	Name	MNNG	MNNG	4NQO	4NOPD	AAC
	Concentrations (μg/plate)	5	5	5	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	869 ± 36	758 ± 24	648 ± 88	541 ± 29	367 ± 28
+	0	83 ± 11	12 ± 2	44 ± 4	24 ± 3	8 ± 2
+	33	86 ± 9	12 ± 2	48 ± 3	27 ± 4	7 ± 2
+	100	83 ± 6	12 ± 3	44 ± 4	25 ± 4	8 ± 2
+	333	79 ± 5	11 ± 3	40 ± 3	25 ± 4	6 ± 2
+	1000	79 ± 4	14 ± 1	45 ± 8	21 ± 4	8 ± 1
+	2500	71 ± 7P	11 ± 2P	40 ± 6P	23 ± 3 P	7 ± 2 P
+	5000	75 ± 8P	12 ± 3P	41 ± 3P	21 ± 3 P	7 ± 0 P
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	890 ± 97	154 ± 20	272 ± 14	687 ± 44	149 ± 27

 

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

4NOPD = 4-nitroquinoline-N-oxide

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

P = Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative