[1,1'-Biphenyl]-2,2'-diol, 3,3'-bis(1,1-dimethylethyl)-5,5'-dimethoxy-

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:

Between 08 December 2008 and 02 January 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/08 Date of Signature: 04/03/09

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L17_Precursor

IUPAC Name:

L17_Precursor

Details on test material:

Sponsor's identification: L17_Precursor
Description : Pale yellow powder
Chemical name: 5,5’ –Dimethoxy-3,3`-di-tert.-butyl-2,2’ -biphenol
Purity : 93.4%
Batch number : DALA046312
Date received : 11 November 2008
Storage conditions: Room temperature in the dark

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test material was insoluble in acetone at 50 mg/ml but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With S9 mix at the following concentrations: 2AA at 1 µg/plate for TA100, 2AA at 2 µg/plate for TA1535 and TA1537 and 2AA at 10 µg/plate for WP2uvrA-

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix at the following concentration: BP at 5 µg/plate for TA98

Migrated to IUCLID6: : (BP)

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix at the following concentration: 0.2 µg/plate for TA98

Migrated to IUCLID6: : (4NQO)

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix at the following concentration: 80 µg/plate for TA1537

Migrated to IUCLID6: : (9AA)

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix at the following concentration: 3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA-

Migrated to IUCLID6: : (ENNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10E9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor.
- Precipitation: A powdery precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.


RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay are shown in the table below.




COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.


ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	69	78	77	71	60	64	79	80P	69P	61P	72P
+	TA100	72	129	90	78	85	85	66	112P	84P	66P	62P
-	WP2uvrA^-	36	37	17	41	29	20	24	39P	32P	27P	31P
+	WP2uvrA^-	40	36	40	36	32	24	43	31P	25P	25P	27P

P: precipitate

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A powdery precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1              Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
114		20		22		21		13
90	(99)	16	(18)	13	(18)	16	(17)	9	(12)
93		18		18		15		14

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
129		15		22		19		8
134	(133)	19	(17)	23	(23)	16	(17)	12	(10)
137		18		23		16		11

Table2              Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 13 December 2008					To: 16 December 2008
Without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
-	0	117 104 106	(109) 7.0#	16 22 20	(19) 3.1	23 20 26		(23) 3.0	23 16 20	(20) 3.5	12 15 13	(13) 1.5
-	50	112 110 98	(107) 7.6	15 20 13	(16) 3.6	27 22 16		(22) 5.5	15 13 18	(15) 2.5	11 8 14	(11) 3.0
-	150	110 104 88	(101) 11.4	21 23 16	(20) 3.6	20 18 21		(20) 1.5	19 15 20	(18) 2.6	9 13 11	(11) 2.0
-	500	92 P 95 P 97 P	(95) 2.5	23 P 15 P 19 P	(19) 4.0	22 P 19 P 22 P		(21) 1.7	23 P 14 P 15 P	(17) 4.9	9 P 14 P 8 P	(10) 3.2
-	1500	113 P 99 P 97 P	(103) 8.7	23 P 18 P 18 P	(20) 2.9	21 P 25 P 22 P		(23) 2.1	19 P 18 P 22 P	(20) 2.1	13 P 10 P 14 P	(12) 2.1
-	5000	101 P 93 P 98 P	(97) 4.0	23 P 22 P 20 P	(22) 1.5	24 P 25 P 23 P		(24) 1.0	19 P 22 P 19 P	(20) 1.7	11 P 11 P 12 P	(11) 0.6
Positive controls S9-Mix -	Name Concentration(μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		313 273 275	(287) 22.5	122 101 110	(111) 10.5	153 162 133		(149) 14.8	163 163 131	(152) 18.5	626 627 475	(576) 87.5

Table3              Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 13 December 2008					To: 16 December 2008
With S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
+	0	90 97 98	(95) 4.4#	8 13 11	(11) 2.5	19 21 25		(22) 3.1	21 20 13	(18) 4.4	10 12 13	(12) 1.5
+	50	117 84 99	(100) 16.5	10 10 10	(10) 0.0	19 19 21		(20) 1.2	20 21 18	(20) 1.5	16 12 11	(13) 2.6
+	150	77 107 86	(90) 15.4	9 7 9	(8) 1.2	24 16 22		(21) 4.2	19 14 20	(18) 3.2	12 8 16	(12) 4.0
+	500	112 P 82 P 85 P	(93) 16.5	8 P 9 P 10 P	(9) 1.0	26 P 23 P 23 P		(24) 1.7	14 P 20 P 18 P	(17) 3.1	11 P 8 P 8 P	(9) 1.7
+	1500	108 P 98 P 81 P	(96) 13.7	8 P 10 P 16 P	(11) 4.2	21 P 25 P 21 P		(22) 2.3	21 P 18 P 22 P	(20) 2.1	14 P 12 P 11 P	(12) 1.5
+	5000	108 P 102 P 89 P	(100) 9.7	8 P 10 P 9 P	(9) 1.0	21 P 19 P 20 P		(20) 1.0	18 P 16 P 20 P	(18) 2.0	11 P 12 P 10 P	(11) 1.0
Positive controls S9-Mix +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		820 705 786	(770) 59.1	254 202 213	(223) 27.4	98 165 151		(138) 35.3	167 129 152	(149) 19.1	258 338 269	(288) 43.4

 

Table4              Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 30 December 2008						To: 02 January 2009
Without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
-	0	123 126 112	(120) 7.4#	19 29 27	(25) 5.3	26 20 15	(20) 5.5		16 13 12	(14) 2.1	15 15 13	(14) 1.2
-	50	80 95 74	(83) 10.8	19 25 24	(23) 3.2	25 23 16	(21) 4.7		13 15 13	(14) 1.2	12 12 13	(12) 0.6
-	150	97 81 101	(93) 10.6	19 21 19	(20) 1.2	25 13 20	(19) 6.0		12 11 16	(13) 2.6	14 13 11	(13) 1.5
-	500	89 P 82 P 69 P	(80) 10.1	24 P 29 P 22 P	(25) 3.6	16 P 20 P 21 P	(19) 2.6		9 P 15 P 15 P	(13) 3.5	12 P 13 P 13 P	(13) 0.6
-	1500	80 P 78 P 91 P	(83) 7.0	27 P 21 P 20 P	(23) 3.8	22 P 21 P 24 P	(22) 1.5		16 P 14 P 11 P	(14) 2.5	11 P 14 P 13 P	(13) 1.5
-	5000	80 P 72 P 93 P	(82) 10.6	27 P 23 P 26 P	(25) 2.1	17 P 24 P 21 P	(21) 3.5		16 P 13 P 16 P	(15) 1.7	14 P 10 P 13 P	(12) 2.1
Positive controls S9-Mix -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		387 448 501	(445) 57.0	85 106 70	(87) 18.1	92 102 110	(101) 9.0		136 174 163	(158) 19.6	132 104 315	(184) 114.6

Table5              Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 30 December 2008						To: 02 January 2009
With S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
+	0	85 91 86	(87) 3.2#	15 18 13	(15) 2.5	33 24 27	(28) 4.6		29 29 24	(27) 2.9	16 14 13	(14) 1.5
+	50	92 84 91	(89) 4.4	15 19 16	(17) 2.1	26 25 29	(27) 2.1		29 25 27	(27) 2.0	16 10 12	(13) 3.1
+	150	92 92 91	(92) 0.6	9 16 11	(12) 3.6	27 26 26	(26) 0.6		25 24 25	(25) 0.6	13 11 11	(12) 1.2
+	500	92 P 82 P 80 P	(85) 6.4	8 P 14 P 16 P	(13) 4.2	29 P 23 P 24 P	(25) 3.2		24 P 26 P 26 P	(25) 1.2	12 P 18 P 11 P	(14) 3.8
+	1500	84 P 73 P 70 P	(76) 7.4	14 P 16 P 15 P	(15) 1.0	22 P 22 P 23 P	(22) 0.6		21 P 23 P 27 P	(24) 3.1	10 P 13 P 15 P	(13) 2.5
+	5000	85 P 69 P 86 P	(80) 9.5	13 P 16 P 17 P	(15) 2.1	24 P 28 P 24 P	(25) 2.3		28 P 26 P 22 P	(25) 3.1	10 P 15 P 15 P	(13) 2.9
Positive controls S9-Mix +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		599 743 616	(653) 78.7	102 103 103	(103) 0.6	505 448 183	(379) 171.8		144 181 223	(183) 39.5	103 81 93	(92) 11.0

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

#        Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. 

Results. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A powdery precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion. The test material was considered to be non-mutagenic under the conditions of this test.