N-{[1,1'-Biphenyl]-2-yl}-N-(9,9-dimethyl-9H-fluoren-2-yl)-7'-phenyl-9,9'-spirobi[fluoren]-7-amine

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:

2018-11-23 till 2018-11-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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 E. coli WP2

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:

Pre-Experiment/Experiment I & Experiment II: 3; 10; 33; 100; 333; 1000; 2500; 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:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

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:

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:

valid

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strain TA 1535 in exp.1 at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not tested
Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in strain TA1535 at the highest tested concentration in the presence of metabolic activation in experiment I.

Remarks on result:

other: reverse mutation assay migrated from the field Test System

Summary of Experiment I

Study Name: 1929000	Study Code: Envigo 1929000
Experiment: 1929000 VV Plate	Date Plated: 23.11.2018
Assay Conditions:	Date Counted: 26.11.2018

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without Activation	DMSO		12 ± 3	11 ± 1	32 ± 3	115 ± 8	47 ± 8
	Untreated		13 ± 2	13 ± 2	34 ± 10	123 ± 11	45 ± 7
	test item	3 µg	12 ± 3	8 ± 2	32 ± 5	127 ± 9	34 ± 1
		10 µg	11 ± 4	13 ± 3	28 ± 9	131 ± 11	40 ± 7
		33 µg	13 ± 3	13 ± 3	34 ± 8	121 ± 13	40 ± 6
		100 µg	11 ± 4	14 ± 3	39 ± 6	111 ± 20	46 ± 7
		333 µg	11 ± 1^P	12 ± 5^P	29 ± 2^P	114 ± 11^P	41 ± 1^P
		1000 µg	12 ± 2^P M	8 ± 2^P M	20 ± 3^P M	112 ± 9^P M	37 ± 8^P
		2500 µg	9 ± 1^P M	8 ± 2^P M	23 ± 6^P M	92 ± 4^P M	26 ± 2^P M
		5000 µg	9 ± 3^P M	8 ± 2^P M	26 ± 8^P M	86 ± 3^P M	23 ± 6^P M
	NaN3	10 µg	1158 ± 62			1912 ± 57
	4-NOPD	10 µg			494 ± 18
	4-NOPD	50 µg		86 ± 20
	MMS	2.0 µL					953 ± 36
With Activation	DMSO		15 ± 3	10 ± 3	38 ± 5	149 ± 17	51 ± 9
	Untreated		17 ± 2	10 ± 2	53 ± 4	144 ± 10	51 ± 8
	test item	3 µg	12 ± 6	14 ± 2	51 ± 5	140 ± 6	54 ± 7
		10 µg	13 ± 1	11 ± 1	51 ± 3	143 ± 8	50 ± 10
		33 µg	13 ± 1	9 ± 3	40 ± 7	138 ± 8	47 ± 8
		100 µg	11 ± 2	12 ± 3	54 ± 8	142 ± 13	53 ± 8
		333 µg	12 ± 1^P	9 ± 3^P	45 ± 4^P	168 ± 14^P	42 ± 1^P
		1000 µg	8 ± 2^P M	11 ± 4^P M	35 ± 3^P M	154 ± 10^P	45 ± 7^P
		2500 µg	10 ± 3^P M	10 ± 2^P M	30 ± 10^P M	138 ± 3^P M	36 ± 7^P M
		5000 µg	5 ± 1^P M	9 ± 1^P M	24 ± 2^P M	122 ± 5^P M	29 ± 1^P M
	2-AA	2.5 µg	445 ± 26	226 ± 12	3707 ± 160	4228 ± 130
	2-AA	10.0 µg					342 ± 16

Summary of Experiment II

Study Name: 1929000	Study Code: Envigo 1929000
Experiment: 1929000 HV2 Pre	Date Plated: 27.11.2018
Assay Conditions:	Date Counted: 30.11.2018

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without Activation	DMSO		13 ± 4	12 ± 2	26 ± 3	128 ± 13	49 ± 9
	Untreated		13 ± 3	9 ± 2	29 ± 6	147 ± 8	54 ± 16
	Test material	3 µg	12 ± 3	10 ± 2	30 ± 9	118 ± 11	44 ± 3
		10 µg	14 ± 3	11 ± 5	31 ± 2	131 ± 4	48 ± 2
		33 µg	10 ± 2	12 ± 3	29 ± 3	125 ± 11	52 ± 8
		100 µg	13 ± 5	8 ± 2	33 ± 3	122 ± 11	46 ± 10
		333 µg	10 ± 1^P	8 ± 2^P	29 ± 7^P	123 ± 13^P	47 ± 6^P
		1000 µg	10 ± 4^P M	10 ± 1^P	22 ± 8^P	127 ± 2^P	51 ± 3^P
		2500 µg	8 ± 2^P M	12 ± 4^P M	22 ± 5^P M	103 ± 3^P M	40 ± 3^P M
		5000 µg	9 ± 3^P M	7 ± 2^P M	16 ± 4^P M	78 ± 14^P M	24 ± 4^P M
	NaN3	10 µg	1073 ± 22			1725 ± 14
	4-NOPD	10 µg			502 ± 18
	4-NOPD	50 µg		80 ± 12
	MMS	2.0 µL					799 ± 50
With Activation	DMSO		12 ± 2	11 ± 5	40 ± 7	145 ± 18	50 ± 11
	Untreated		14 ± 4	12 ± 2	52 ± 6	151 ± 14	52 ± 5
	Test material	3 µg	9 ± 1	12 ± 5	45 ± 3	139 ± 16	54 ± 1
		10 µg	9 ± 4	10 ± 3	47 ± 14	134 ± 5	55 ± 8
		33 µg	9 ± 2	10 ± 2	39 ± 4	162 ± 19	55 ± 8
		100 µg	13 ± 2	13 ± 2	41 ± 6	151 ± 20	49 ± 3
		333 µg	11 ± 1^P	14 ± 3^P	52 ± 10^P	138 ± 8^P	54 ± 2^P
		1000 µg	11 ± 4^P M	11 ± 3^P M	39 ± 7^P	133 ± 9^P M	58 ± 5^P
		2500 µg	11 ± 4^P M	9 ± 2^P M	25 ± 3^P M	141 ± 7^P M	43 ± 1^P M
		5000 µg	7 ± 3^P M	7 ± 1^P M	27 ± 7^P M	139 ± 7^P M	43 ± 6^P M
	2-AA	2.5 µg	408 ± 55	192 ± 13	4371 ± 397	4082 ± 130
	2-AA	10.0 µg					329 ± 9

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

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 Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test material was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were 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:                                3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate.

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.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in strain TA1535 at the highest tested concentration in the presence of metabolic activation in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any concentration 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 in induced revertant colonies.

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.