Tribenzylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test (reference 7.6.1-1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-29 to 2020-10-15

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:

21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: the strains used contain mutations in the histidine operon
Escherichia coli: the strain used carries a defect in one of the genes for tryptophan biosynthesis

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver S9 mix from rats pretreated with ß-Naphthoflavone/Phenobarbital
- method of preparation of S9 mix: The complete S9 Mix was prepared with components: sodium phosphate-buffer 0.5 mL + Rat liver homogenate (S9) 0.1 mL (1st series) or 0.2 mL (2nd series) + Magnesium Chloride/ potassium Chloride Solution (0.4 M/1.64 M) 0.02 mL + Glucose-6-phosphate x 1 H2O, disodium salt 1.61 mg + Nicotinamide adenine dinucleotide phosphate, disodium salt 3.15 mg + ultra-pure water 0.38 mL (1st series) or 0.28 mL (2nd series)
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % and 20 % S9 in the S9 mix were used. 0.5 mL S9 mix were added per plate with metabolic activation.
- quality controls of S9: metabolic capability

Test concentrations with justification for top dose:

1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate
2nd series: 15.8, 50, 158, 500, 889 µg/plate
The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system. Therefore, 5000 µg/plate was chosen as the appropriate maximum test material concentration.

Vehicle / solvent:

- Solvent used: DMSO

- Justification for choice of solvent: The selection of the solvent for this assay was based on the available information from a prelimi-nary solubility test. DMSO showed best Performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

- Justification for percentage of solvent in the final culture medium: Analysis of the historical data and independent experiments of the laboratory, and experience of other research groups showed that the chosen amounts of the selected solvents have no influence on the spontaneous mutation rate of the bacteria used. For this reason, no untreated controls were used.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: 4-Nitro-o-phenylenediamine (4-NOPD), 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

The assay is considered valid if the following criteria were met:
• Regular background growth in the negative control
• The mean number of revertants in the negative (solvent) controls are within the normal range for this test system taking published ranges and the current historical data of the laboratory into account, and
• The positive control chemicals should induce an increase the mean number of revertants above a threshold of 2-fold (TA98, TA100, WP2 uvrA) or 3-fold (TA1535, TA1537) as compared to the concurrent negative controls
• A minimum of five analyzable concentrations, at least three of them without showing signs of toxic effects, evident as a reduction in the number of revertants below a factor of 0.5 compared to the concurrent negative control, should be present.

A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA98, TA 100, WP2 uvrA) or 3-fold (TA1535, TA 1537) as compared to the concurrent negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met
Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response.

Statistics:

Tables of individual and mean values were generated by use of a validated, automated data processing program ("Ames Study Manager" and modules by Instem, Stone, Staffordshire, UK, TOXID8004).

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate.
- Other confounding effects: No other effects observed.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Please refer to "Any other information on results".

Ames test:
- Signs of toxicity : No toxicity to the bacteria was observed with the exception of one occasion in TAI 537 at 5000 ug/plate (without S9 mix).
- Please refer to "Any other information on results" for values.


HISTORICAL CONTROL DATA
- Please refer to "Any other information on results" for values.

1st series:

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA98	TA100	TA1535	TA1537	WP2 uvrA
	Results without S9
DMSO	36 ± 9	125 ± 13	33 ± 1	14 ± 6	31 ± 5
5	39 ± 3	146 ± 20	46 ± 14	8 ± 4	24 ± 6
15.8	36 ± 12	147 ± 4	68 ± 8	13 ± 6	26 ± 4
50	46 ± 8	133 ± 23	75 ± 7	14 ± 1	22 ± 8
158	39 ± 4	108 ± 4	82 ± 22	11 ± 4	25 ± 4
500	35 ± 6 ME	95 ± 9 ME	75 ± 13 ME	8 ± 3 ME	24 ± 2 ME
1580	38 ± 14ME	101 ± 8 ME	77 ± 11 ME	8 ± 3 ME	19 ± 1 ME
5000	30 ± 2ME	98 ± 6 ME	56 ± 4 ME	6 ± 1 ME	22 ± 3 ME
4-NOPD (20)	225 ± 25
4-NOPD (60)				82 ± 4
NaN3 (2)		1105 ± 27	920 ± 11
NQO (2)					1521 ± 119
	Results with S9
DMSO	45 ± 4	119 ± 24	12 ± 3	9 ± 4	36 ± 5
5	38 ± 1	111 ± 5	9 ± 3	13 ± 4	30 ± 7
15.8	44 ± 10	115 ± 6	7 ± 3	11 ± 4	23 ± 4
50	41 ± 5	116 ± 8	11 ± 2	11 ± 4	27 ± 2
158	39 ± 8	104 ± 7	9 ± 3	10 ± 3	26 ± 4
500	36 ± 6 ME	94 ± 4 ME	7 ± 2 ME	9 ± 2 ME	20 ± 10 ME
1580	35 ± 5 ME	81 ± 4 ME	8 ± 3 ME	7 ± 4 ME	20 ± 8 ME
5000	29 ± 8 ME	74 ± 9 ME	9 ± 3 ME	6 ± 1 ME	24 ± 7 ME
2-AA (2)	902 ± 83	1531 ± 51
2-AA (5)			256 ± 23	488 ± 105
2-AA (10.0)					320 ± 32

2nd series:

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA98	TA100	TA1535	TA1537	WP2 uvrA
	Results without S9
DMSO	42 ± 2	108 ± 7	13 ± 3	16 ± 3	34 ± 4
15.8	35 ± 6	114 ± 29	21 ± 0	20 ± 3	21 ± 4
50	44 ± 7	113 ± 13	22 ± 5	13 ± 4	32 ± 5
158	33 ± 3	90 ± 4	23 ± 1	15 ± 2	35 ± 1
500	30 ± 8 ME	84 ± 8 ME	18 ± 3 ME	12 ± 3 ME	24 ± 6 ME
889	33 ± 8 ME	88 ± 2 ME	16 ± 3 ME	12 ± 3 ME	26 ± 7 ME
4-NOPD (20)	505 ± 23
4-NOPD (60)				108 ± 4
NaN3 (2)		1955 ± 92	1193 ± 76
NQO (2)					2007 ± 88
	Results with S9
DMSO	45 ± 6	131 ± 15	16 ± 3	16 ± 3	40 ± 3
15.8	54 ± 16	120 ± 5	18 ± 5	25 ± 2	34 ± 6
50	42 ± 1	144 ± 15	13 ± 5	18 ± 4	36 ± 4
158	53 ± 10	122 ± 25	15 ± 7	18 ± 2	32 ± 7
500	36 ± 2 ME	100 ± 13 ME	10 ± 2 ME	11 ± 2 ME	27 ± 6 ME
889	36 ± 6 ME	105 ± 17 ME	12 ± 1 ME	9 ± 1 ME	25 ± 1 ME
2-AA (2)	579 ± 45	1057 ± 38
2-AA (5)			225 ± 16	191 ± 83
2-AA (10.0)					297 ± 8

E= Precipitation until end of experiment

M= Manual count

Historical Data

The historical data have been obtained in experiments between 01/2019 and 12/2019.

Negative Controls

Strain	TA 98		TA 100
S9 Mix	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent
Total Plates	424	424	420	416
Number of Values	87	87	86	85
Minimum	8	10	85	93
Maximum	49	52	141	172
Mean	33	39	112	128
Standard Deviation	6.7	7.7	12.5	15.3

Strain	TA 1535*		T A 1537		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent
Total Plates	268	268	272	272	416	416
Number of Values	48	48	49	49	85	85
Minimum	22	9	5	6	24	25
Maximum	45	53	19	16	45	55
Mean	31	28	10	11	32	37
Standard Deviation	5.0	7.2	2.7	2.6	4.5	6.5

 

 

Positive Controls

Strain	TA 98			TA 100
S9 Mix	Without		With	Without	With
Compound	DAUN	4-NOPD	2-AA	NaN3	2-AA
Total Plates	90	127	217	215	213
Number of Values	35	52	87	86	85
Minimum	86	68	73	871	354
Maximum	1414	726	5113	2224	5269
Mean	524	446	801	1654	1816
Standard Deviation	337.2	115.1	665.7	281.1	1037.5

 

Strain	T A 1535		TA 1537			WP2 uvrA
S9 Mix	Without	With	Without		With	Without	With
Compound	NaN3	2-AA	9-AA	4-NOPD	2-AA	NQO	2-AA
Total Plates	139	139	62	79	141	213	213
Number of Values	48	48	21	28	49	85	85
Minimum	620	79	104	70	69	386	99
Maximum	1427	273	3715	123	799	2661	729
Mean	947	184	897	99	374	1611	298
Standard Deviation	160.4	49.5	930.4	12.6	198.1	415.9	107.4

 

*ln the present study, TA1535 of a different origin was used which has a generally lower spontaneous mutation frequency as com-pared to the TA1535 strain used to generate the historical control data.

Conclusions:

It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Executive summary:

A study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse mutation test in the absence and presence of a rat liver metabolizing system (S9 mix) according to OECD 471. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pre-treated with ß-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10 % S9 in the 1st and 20 % S9 in the 2nd series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate. No toxicity to the bacteria was observed with the exception of one occasion in TAI 537 at 5000 µg/plate (without S9 mix). Under the experimental conditions reported, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse mutation test in the absence and presence of a rat liver metabolizing system (S9 mix) according to OECD 471. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pre-treated with ß-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10 % S9 in the 1st and 20 % S9 in the 2nd series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate. No toxicity to the bacteria was observed with the exception of one occasion in TAI 537 at 5000 µg/plate (without S9 mix). Under the experimental conditions reported, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Justification for classification or non-classification

The available data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.