9-(2‘-Bromo-biphenyl-2-y)-9H-fluoren-9-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 471. The test material was not mutagenic under the conditions of this assay.

Link to relevant study records

Reference

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:

12.11.2014-11.02.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

without S9 mix: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate
with S9 mix: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate

Vehicle / solvent:

Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 60 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as 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 of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above 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.

Key result

Species / strain:

S. typhimurium TA 98

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

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

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

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 1537 at the highest applied concentration of 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

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

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS

Effects of pH: none

Water solubility: not tested

Precipitation: Precipitation was observed on the test plates at concentrations of 800 μg/plate and
above in the absence and presence of S-9.

Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Slight toxic effects occurred in strain TA 1537 at the highest applied concentration of 5000 μg/plate (with and without S9 mix in experiment I).

Summary of Experiment I

 

Metabolic activation	Test group	Dose Level (per plate)	Revertant Colony Counts
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	DMSO		21, 20, 21, 19, 15	4, 5, 9, 15, 11	14, 20, 26, 21, 39	117, 158, 130, 129, 134	228, 252, 296, 278, 243
	Test item	5	26, 21, 23	10, 19, 14	28, 30, 38	147, 117, 143	288, 257, 279
		16	24, 29, 26	10, 5, 10	29, 18, 29	137, 130, 119	221, 228, 273
		50	20, 19, 24	10, 14, 8	28, 20, 23	135, 128, 132	259, 316, 251
		160	26, 25, 24	9, 8, 14	24, 30, 31	147, 112, 130	224, 232, 244
		500	24, 30, 28	11, 13, 14	38, 30, 34	130, 144, 142	238, 259, 273
		1600	23 M P, 19 M P, 25 M P	7 M P, 10 M P, 8 M P	26 M P, 33 M P, 21 M P	109 M P, 106 M P, 115 M P	234 M P, 241 M P, 214 M P
		5000	20 M P, 22 M P, 17 M P	2 M P, 3 M P, 4 M P	16 M P, 22 M P, 25 M P	105 M P, 96 M P, 95 M P	234 M P, 239 M P, 241 M P
	2NF	5			630, 563, 520
	NaN3	2	822, 717, 702			980, 1085, 994
	AAC	50		168, 153, 109
	MMC	0,2					710, 644, 738
With activation	DMSO		21, 19, 20, 19, 14	21, 20, 15, 16, 13	33, 44, 33, 33, 30	104, 101, 94, 107, 114	226, 253, 252, 224, 237
	Test item	5	15, 14, 16	14, 14, 21	49, 44, 48	110, 110, 94	205, 233, 209
		16	13, 11, 19	16, 14, 15	34, 38, 38	88, 85, 93	223, 222, 264
		50	9, 18, 20	25, 24, 13	43, 26, 30	74, 89, 145	253, 153, 192
		160	14, 23, 10	15, 11, 21	41, 46, 43	105, 104, 108	291, 271, 233
		500	18, 19, 10	16, 8, 15	40, 40, 38	115, 100, 108	237, 173, 203
		1600	9 M P, 18 M P, 9 M P	17 M P, 15 M P, 14 M P	28 M P, 28 M P, 30 M P	94 M P, 87 M P, 112 M P	189 M P, 212 M P, 199 M P
		5000	14 M P, 16 M P, 20 M P	10 M P, 11 M P, 9 M P	37 M P, 24 M P, 22 M P	98 M P, 98 M P, 106 M P	172 M P, 223 M P, 205 M P
	B[a]P	10			413, 398, 34
	AAN	5	199, 232, 264	98, 100, 81		952, 1090, 855	794, 802, 733

 Summary of Experiment II

Metabolic activation	Test group	Dose Level (per plate)	Revertant Colony Counts
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	DMSO		16, 20, 6, 16, 19	6, 15, 5, 12, 9	24, 30, 26, 30, 27	115, 105, 96, 117, 118	299, 307, 203, 199, 207
	Test item	20,48	12, 22, 15	12 M B, 14, 9	25, 30, 35	100, 115, 107	259, 249, 232
		51,2	16, 17, 16	16, 12, 11	26, 40, 40	135, 116, 110	264, 243, 248
		128	26, 20, 10	6, 7, 7	34, 25, 34	133, 112, 118	223, 214, 267
		320	10, 11, 24	6, 6, 9	21, 26, 22	105, 117, 130	268, 273, 234
		800	19 P, 11 M P, 20 P	7 P, 7 P, 11 P	32 P, 46 P, 27 P	108 P, 133 P, 137 P	279 P, 280 P, 216 P
		2000	20 M P, 14 M P, 19 M P	8 M P, 9 P, 6 M P	20 M P, 22 M P, 25 M P	96 M P, 104 M P, 98 M P	273 P, 267 P, 238 P
		5000	13 M P, 19 M P, 18 M P	7 M P, 7 M P, 6 M P	24 M P, 29 M P, 25 M P	101 M P, 98 M P, 102 M P	243 P, 253 P, 234 P
	2NF	5			1091, 1101, 816
	NaN3	2	573, 585, 536			891, 860, 889
	AAC	50		203, 168, 194
	MMC	0,2					543, 647, 612
With activation	DMSO		21, 20, 20, 14, 20	16, 15, 11, 19, 20	44, 31, 35, 35, 37	105, 113, 80, 95, 105	299, 224, 196, 197, 233
	Test item	20,48	11, 6, 12	19, 21, 20	29, 30, 36	102, 121, 103	229, 228, 204
		51,2	14, 15, 12	21, 21, 19	30, 27, 46	96, 131, 95	201, 204, 199
		128	12, 17, 5	21, 16, 12	29, 36, 35	97, 95, 126	277, 191, 216
		320	10, 11, 17	20, 26, 14	42, 25, 51	84, 90, 105	206, 212, 188
		800	11 P, 27 P, 21 P	19 P, 12 P, 21 M P	39 P, 32 P, 44 P	100 P, 98 P, 121 P	202 P, 158 P, 264 P
		2000	10 M P, 10 M P, 16 M P	18 M P, 17 M P, 16 M P	31 M P, 27 M P, 18 M P	95 M P, 74 M P, 83 M P	187 M P, 198 M P, 200 M P
		5000	11 M P, 16 M P, 12 M P	21 M P, 18 M P, 18 M P	30 M P, 24 M P, 36 M P	86 M P, 74 M P, 89 M P	201 M P, 216 M P, 214 M P
	B[a]P	10			440, 383, 315
	AAN	5		49, 112, 61		568, 606, 690	861, 990, 985

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 AAC 2-NF MMC B[a]P AAN	sodium azide 9-Aminoacridine 2-Nitrofluorene Mitomycin C Benzo[a]pyrene 2-Aminoanthracene	P M	Precipitate Manual count

Conclusions:

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 or frameshifts in the genome of the strains used.
Therefore, the test material is considered to be non-mutagenic in this reverse mutation assay.

Executive summary:

Purpose

This study was performed to investigate the potential of the test material to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102:

Methods

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 471. In detail 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:

Experiment I:       5; 16; 50; 160; 500; 1600; and 5000 µg/plate

Experiment II:     20.48;51.2; 128; 320; 800; 2000; and 5000 µg/plate

Each series was performed with and without S9 mix.

Results

The test item precipitated in the overlay agar in the test tubes from 1600 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed starting at 800 µg/plate without S9 mix and at 1600 µg/plate and above with S9 mix in experiment I. In experiment II precipitation on the plates was found at concentrations ranging from 800 µg/plate to 5000 µg/plate independently from the S9 mix supplementation.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 1537 at 5000 µg/plate

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material 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 and showed a distinct increase of induced revertant colonies.

Conclusion

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 or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.