Tributylethylammonium ethyl sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An AMES test, an in vitro micronucleus test and a mouse lymphoma test with TBEAES are available. The outcome of all studies is negative.

Link to relevant study records

Reference

Reference 1

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:

December 2007

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

other: "Testing Methods for New Chemical substances"

Version / remarks:

November 2003

GLP compliance:

yes (incl. QA statement)

Remarks:

date certificate 21.07.2004

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1 and 2
Without and with S9-mix: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: Water
- Justification for choice of solvent:
Test compound was stable in water and water has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; 10 µg/plate for TA100 and TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

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

Remarks:

without S9; 10 µg/plate for TA98; 40 µg/plate for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 1 µL/plate for WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, 2-AA

Remarks:

with S9; 2.5 µg/plate in DMSO for TA98, TA100, TA1535 and TA 1537; 10 µg/plate in DMSO for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn and the reduction of the number of revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

Evaluation od Cytotoxicity
Cytotoxicity can be detected by a diminution of the background lawn or a reduction on the number of revertans dow to a mutation factor ≤ 0.5 in relation to the solvent control.

Criteria of validity
a test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100)
- the spontaneous revesion frequencies of control plates without S9 are within the historical control data range.
- corresponding background growth on both the negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plates.

Evaluation of mutagenicity
Mutation factor: mean revertant counts divided by mean values of the solvent control

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertans occurs and/or
- a biologically relevent positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA100 and WP2 uvrA the number of reversions is at least twice as high
- is in tester strains TA1535, TA1537 and TA98 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to the OECD guidelines a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 2500 µg/plate and above and with S9: 5000 µg/plate

Vehicle 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:

cytotoxicity

Remarks:

without S9: 1000 µg/plate and above and with S9: 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 2500 µg/plate and above and with S9: No toxicity observed

Vehicle 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:

cytotoxicity

Remarks:

without S9: 2500 µg/plate and above and with S9: 2500 µg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 2500 µg/plate and above and with S9: No toxicity observed

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Eperiment 1:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Experiment 2:
TA1535: without S9: 2500 µg/plate and above and with S9: 5000 µg/plate
TA1537: without S9: 1000 µg/plate and above and with S9: 5000 µg/plate
TA98: without S9: 2500 µg/plate and above and with S9: No toxicity observed
TA100: without S9: 2500 µg/plate and above and with S9: 2500 µg/plate and above
WP2uvrA: without S9: 2500 µg/plate and above and with S9: No toxicity observed

Conclusions:

In an AMES test, performed according to OECD/EC guideline and GLP principles, TBEAES was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD/EC guideline and in accordance with GLP principles. All bacterial strains showed negative responses up to and including 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No precipitation of the test substance was observed. No toxic efffects of the test substance were noted in any of the five tester strains with and without metabolic activation in experiment 1. Toxic effects of the test substance were noted in all tester strains used in experiment 2 at concentrations of 1000 ug/plate and higher (without metabolic activation) and at concentrations of 2500 ug/plate and higher (with metabolic activation), depending on the tester strain.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that TBEAES is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Feb 2017 - 09 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
Blood samples
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:
Dose-range finding study: age 25, AGT = 13.8 h
First cytogenetic assay: age 22, AGT = 13.2 h
Second cytogenetic assay: age 34, AGT = 13.4 h

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Cytokinesis block (if used):

Cytochalasine B

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without S9-mix, 3 exposure; 27 hr fixation: 125, 250, 500, 1000 and 2000 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 125, 250, 500, 1000 and 2000 µg/mL
With S9-mix, 3 exposure; 27 hr fixation: 125, 250, 500, 1000 and 2000 µg/mL
First cytogenetic test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 500, 1500 and 2000 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 400 and 600 µg/mL

Vehicle / solvent:

- Vehicle used: culture medium
- Justification for choice of vehicle:
Test compound was stable in water and soluble in culture medium. Culture medium has been accepted and approved by authorities and international guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9: 0.25 µg/mL and 0.38 µg/mL for a 3 hour exposure period and 0.15 µg/mL and 0.23 µg/mL for a 24 hour exposure period

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: colchicine

Remarks:

without S9: 0.1 µg/mL for a 3 hour exposure period and 0.05 µg/mL for a 24 hour exposure period

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9: 15 µg/mL and 17.5 µg/mL for a 3 hour exposure period

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 46 ± 2 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 27 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

SPINDLE INHIBITOR: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei.

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the vehicle control using Chi-square statistics.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the second cytogenetic assay only.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No, pH of 2000 µg/mL was 7.94 (solvent control was 7.94)
- Effects of osmolality: No, osmolarity of 2000 µg/mL was 302 mOsm/kg (solvent control 286 mOsm/kg)
- Precipitation: No precipitation was observed up to and including the top dose of 2000 µg/mL


RANGE-FINDING/SCREENING STUDIES: No toxicity was observed up to and including 2000 µg/mL

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See other information on results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (see other information on results).
- Negative (solvent/vehicle) historical control data: The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database (see other information on results).

Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with TBEAES DRY in the Dose-range Finding Test

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	120	320	71	1.90			0
125	128	327	54	1.85			5
250	130	310	66	1.87			3
500	186	259	61	1.75			17
1000	190	276	36	1.69			23
2000	266	238	8	1.50			45

 

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/ml	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	127	315	74	1.90			0
125	173	299	30	1.72			20
250	164	308	39	1.76			16
500	231	249	24	1.59			34
1000	468	37	0	1.07			92
2000	473	28	0	1.06			94

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	120	306	81	1.92			0
125	141	321	62	1.85			8
250	136	317	56	1.84			9
500	183	265	53	1.74			20
1000	194	276	32	1.68			27
2000	227	252	36	1.63			32

Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with TBEAES DRY in the First Cytogenetic Assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	1	0	1	2		2	4
500	10	1	0	1	3		2	5
1500	35	1	1	2	6		1	7
2000	40	0	1	1	4		5	9
0.25 MMC-C	43	1	3	4	33		26	59^***
0.1 Colch	73	20	17	37^***	4		4	8

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	0	0	0	5		3	8
500	11	0	2	2	1		3	4
1500	21	0	2	2	2		2	4
2000	37	2	0	2	6		4	10
15 CP	62	3	5	8^**	14		13	27^***

*)Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾1000 bi- and mononucleated cells were scored for the presence of micronuclei. 
 Duplicate cultures are indicated by A and B.

Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with TBEAES DRY in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	2	1	3	1		0	1
100	14	0	1	1	2		1	3
400	41	1	0	1	2		2	4
600	59	1	2	3	0		0	0
0.15 MMC-C	46	2	1	3	18		14	32^***
0.05 Colch	97	17	24	41^***	0^2)		1^2)	1^*

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾   1000 bi- and mononucleated cells were scored for the presence of micronuclei. 
Duplicate cultures are indicated by A and B.

²⁾  63 and 101 binucleated cells were scored for the presence of micronuclei, respectively. 

Historical Control Data for in vitro Micronucleus Studies of the Solvent Control

	Mononucleated			Binucleated
	+ S9-mix	- S9-mix		+ S9-mix	- S9-mix
	3 hour exposure	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Mean number of micronucleated cells (per 1000 cells)	0.93	1.07	0.87	3.57	3.45	4.14
SD	0.96	1.00	1.11	2.48	2.07	2.67
n	108	110	108	108	110	108
Upper control limit (95% control limits)	3.01	3.59	3.46	9.06	9.18	10.68
Lower control limit (95% control limits)	-1.16	-1.44	-1.71	-1.93	-2.28	-2.41

 

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between January 2014 and November 2016.

Historical Control Data for in vitro Micronucleus Studies of the Positive Control Substances

	Mononucleated		Binucleated
	- S9-mix		+ S9-mix	- S9-mix
	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Mean number of micronucleated cells (per 1000 cells)	16.65	18.27	22.23	26.31	23.36
SD	18.38	20.91	9.15	20.99	19.41
n	224	222	116	224	222
Upper control limit (95% control limits)	60.92	68.40	40.44	63.59	63.09
Lower control limit (95% control limits)	-27.63	-31.87	4.02	-10.97	-16.37

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between January 2014 and November 2016.

Conclusions:

In an in vitro micronucleus test TBEAES was found not to be clastogenic or aneugenic in human lymphocytes.

Executive summary:

An in vitro micronucleus test was conducted according to OECD guideline 487 and in accordance with GLP principles. In the first cytogenetic assay, TBEAES DRY was tested up to a recommended dose level of 2000 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. In the second cytogenetic assay, TBEAES DRY was tested up to 600 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control

database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The

positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and

binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore

concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

TBEAES DRY did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of

S9-mix, in either of the two experiments.

In conclusion, TBEAES DRY is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 March 2017 - 11 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

USED CELLS
- Source of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells. (American Type Culture Collection, (ATCC, Manassas, USA) (2001))

- Suitability of cells: Recommended test system in international guidelines


MEDIA USED:
- Type and identity of media
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM
sodium pyruvate and 2 mM L-glutamin.

Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium: 3 hour exposure basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

- Properly maintained: Stock cultures of the cells were stored in liquid nitrogen (-196°C)
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: not indicated.

Metabolic activation:

with and without

Metabolic activation system:

Rat (male Sprague Dawley) liver S9-homogenate induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 250, 500, 1000, 1500 and 2000 µg/mL
Without S9-mix, 24 hours treatment: 250, 500, 1000, 1500 and 2000 µg/mL

Experiment 1:
Without and with S9-mix, 3 hours treatment: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL

Experiment 2
Without S9-mix, 24 hours treatment: 50, 100, 250, 500, 825, 1000, 1250 and 1500 µg/mL

Vehicle / solvent:

- Vehicle used: RPMI 1640 (exposure medium (R5) Hepes buffered medium (Dutch modification))

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9, in DMSO, 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9, 7.5 µg/mL in Hank's balanced salt solution without calcium and magnesium.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 10^6 cells/mL for 3 hour treatment, 1.25x10^5 cells/mL for 24 hour treatment

CLEANSING
Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine , 2x10^-7 M aminopterine and 1.6x10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were
returned to R10-medium for at least 1 day before starting the experiment.

DURATION
- Exposure duration:
With and without S9-mix: 3 hours
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days, 4 x 10^6 cells were subcultured every day
- Selection time: 11 to 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
STAINING AGENT: 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6x10^5 cells plated/concentration

DETERMINATION OF MUTATION FREQUENCY
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for cloning effiency and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments) (for calculation see "Any other information on materials and methods").

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay is considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).


DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation up to and including a concentration of 2000 μg/ml.

RANGE-FINDING/SCREENING STUDIES:
- After 3 hours of treatment, in the presence and absence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 2000 μg/ml compared to the suspension growth of the solvent control.
- After 24 hours of treatment, The relative suspension growth was 10% at the test item concentration of 2000 μg/ml compared to the relative suspension growth of the solvent control.


FIRST MUTAGENICITY TEST:
- No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with TBEAES DRY either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the TBEAES DRY treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

SECOND MUTAGENICITY TEST:
- The dose levels of 500 to 825 μg/ml showed similar cell growth delay. Therefore, the dose level of 650 µg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 1650 to 2000 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing
- The relative total growth of the highest test item was 8% compared to the total growth of the solvent controls
- No significant increase in the mutation frequency at the TK locus was observed after treatment with TBEAES DRY. The numbers of small and large colonies in the TBEAES DRY treated cultures were comparable to the numbers of small and large colonies of the solvent controls. .

HISTORICAL CONTROL DATA: see table 1 and table 2

ACCEPTABILITY:
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The growth rate over the two-day expression period for the solvent control cultures was between 11 and 13 (3 hour treatment) and 114 and 128 (24 hour treatment).

Table 1: Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutation frequency per 1E^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	86	81	87
SD	23	26	28
n	220	202	273
Upper control limit (95% control limits)	135	135	145
Lower control limit (95% control limits)	37	28	28

SD = Standard deviation n = Number of observations

 Table 2: Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay 

	Mutation frequency per 1E^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	857	688	1710
SD	246	187	815
n	110	102	139
Upper control limit (95% control limits)	1425	1124	4214
Lower control limit (95% control limits)	289	253	-793

SD = Standard deviation n = Number of observations

Conclusions:

Based on the results of a in vitro mammalian cell gene mutation test, performed according to OECD guideline 490 and in accordance with GLP principles, TBEAES DRY is not mutagenic in the mouse lymphoma L5178Y test system.

Executive summary:

A mouse lymphoma assay was conducted with TBEAES DRY according to OECD 490 guideline and in accordance with GLP principles. In the first experiment, TBEAES DRY was tested at concentrations up to and including 2000 μg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. TBEAES DRY did not precipitate in the culture medium at any dose level. In the second experiment, TBEAES DRY was tested up at concentrations up to and including 1500 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The Relative Total Growth (RTG) was 8%. TBEAES DRY did not precipitate in the culture medium at any dose level. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, TBEAES DRY did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in the second experiment with modification in the duration of treatment. In the presence of S9-mix, TBEAES DRY did not induce a significant increase in the mutation frequency. Therefore it is concluded that TBEAES DRY is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

An AMES test was performed according to OECD/EC guidelines and in accordance with GLP principles. All bacterial strains showed negative responses up to and including 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No precipitation of the test substance was observed. No toxic efffects of the test substance were noted in any of the five tester strains with and without metabolic activation in experiment 1. Toxic effects of the test substance were noted in all tester strains used in experiment 2 at concentrations of 1000 ug/plate and higher (without metabolic activation) and at concentrations of 2500 ug/plate and higher (with metabolic activation), depending on the tester strain. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that TBEAES is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

In vitro micronucleus test:

An in vitro micronucleus test was conducted according to OECD guideline 487 and in accordance with GLP principles. In the first cytogenetic assay, TBEAES DRY was tested up to a recommended dose level of 2000 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. In the second cytogenetic assay, TBEAES DRY was tested up to 600 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

TBEAES DRY did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. Based on these results it is concluded that TBEAES DRY is not clastogenic or aneugenic in human lymphocytes.

Mouse lymphoma assay:

A mouse lymphoma assay was conducted with TBEAES DRY according to OECD 490 guideline and in accordance with GLP principles. In the first experiment, TBEAES DRY was tested at concentrations up to and including 2000 μg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. TBEAES DRY did not precipitate in the culture medium at any dose level. In the second experiment, TBEAES DRY was tested at concentrations up to and including 1500 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The Relative Total Growth (RTG) was 8%. TBEAES DRY did not precipitate in the culture medium at any dose level. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, TBEAES DRY did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in the second experiment with modification in the duration of treatment. In the presence of S9-mix, TBEAES DRY did not induce a significant increase in the mutation frequency. Therefore it is concluded that TBEAES DRY is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the available data, TBEAES is not classified for genotoxicity according to CLP Regulation (EC) No. 1272/2008.