Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

B-TEGME was not mutagenic in several bacterial and mammalian test systems in vitro covering different genetic endpoints:

Negative in two Ames tests: OECD 471, 2007 and 1989

Negative in the HPRT test: OECD 476, 2010

Negative in the chromosome aberration assay: OECD 473, 2010 and 1991

In conclusion there were no indications of genotoxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-10-12 to 2007-11-09

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:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Substance type: Borated Glycol Ether
- Physical state:Clear colourless liquid
- Analytical purity: 100%
- Isomers composition: Not applicable
- Purity test date: Not provided
- Lot/batch No.: DEG4131078
- Expiration date of the lot/batch: 2009-06-05
- Stability under test conditions: Room temperature (20°C)

Target gene:

Defective excision repair gene in Salmonella thyphymirium strains (uvrB) and Escherichia coli strain (uvrA).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
TA 98: University of California, Berkeley Divison of Biochemistry & Molecular Biology, CA 94720 USA
E. coli WP2 (uvrA), TA 1537, TA 1535 and TA 100: TRINOVA BIOCHEM GMBH, Kerkrader Stra e 10, D-35394 Giessen
- Suitability of cells: cells selected according to guidelines

Metabolic activation:

with and without

Metabolic activation system:

S9 (CCR; batch Nos. 250507 and 200707)

Test concentrations with justification for top dose:

1st study (plate incorporation method): 5.0, 1.6, 0.5, 0.16 and 0.05 mg/plate (factor root 10)
2nd study (preincubation method): 5.0, 2.3, 1.1, 0.5 and 0.23 mg/plate (factor root 5)

Vehicle / solvent:

distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

benzo(a)pyrene

other: 2-amino-anthracen, ICR 191, 4-nitro-o-phenylendiamine, nitrofurantoine

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- plate incorporation:
2.0 ml top agar with 0.5 mM Histidin/Biotin (or L-Tryptophan for E.coli) for plates without S9
1.5 ml top agar with 0.5 mM Histidin/Biotin (or L-Tryptophan for E.coli) for plates with S9
0.5 ml S9 mix for plates with metabolic activation
0.1 ml bacteria (overnight culture)

- preincubation:
0.35 ml test/reference item solution and double distilled water
1.75 ml buffer solution or S9-mix
0.35 ml bacteria (overnight culture)
After preincubation for 20 min. At 33-37°C: 0.7 ml of the precincubated solution and 1.5 ml top agar with 0.5 nM Histidin/Biotin or L-Tryptophan (for E.coli) solution

DURATION
- Preincubation period: 20 minutes (33-37°c)
- Exposure duration: 48 hours (36.2-37.7°C)

NUMBER OF REPLICATIONS: 3 per concentration level and control (Titer control replicates were prepared 2-fold)

Evaluation criteria:

The rate of induced revertant His+ colonies was derermined.
In addition, genotypes were evaluated. Plates of the mutagenicity test were inspected for present and reduced background lawn after an incubation time of 48h. Plates with colonies which correspond not with the typical shape and colour of Salmonella thyphimuium or Escherichia coli were regarded as contaminated and were not included in calculations.

Statistics:

Arithmetic mean values and standard deviations were calculated based on colonies per plate of three replicates. For evaluation of the results, the induction rate of the mean values was calculated (revertant colonies test item/revertant colonies control). If there is a concentration effect relationship over at least 3 concentrations and/or induction rate equal to or greater than 2, then the test item is classified as mutagenic.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- There were no confounding factors

RANGE-FINDING/SCREENING STUDIES: No

COMPARISON WITH HISTORICAL CONTROL DATA: No

ADDITIONAL INFORMATION ON CYTOTOXICITY: No

Remarks on result:

other: plate incorporation & preincubation

Table 1: Mutagenic and cytogenic effects of the test item in the Ames test

Strain	S9	Tested concentration range (mg/plate)		Lowest mutagenic concentration	Lowest cytotoxic concentration (mg/plate)
		1st study	2nd study	(mg/plate)	1st study	2nd study
TA 1537	-	0.05 – 0.5	0.23 -5.0	none	not cytotoxic up to 5.0	not cytotoxic  up to 5.0
	+
TA 98	-
	+
TA 100	-
	+
TA 1535	-
	+
E coli WP2 (uvrA)	-
	+

Conclusions:

The test item is regarded to be not mutagenic under the test conditions.

Executive summary:

The mutagenic effect of B-TEGME was determined in a bacterial reverse mutation test according to OECD No. 471 in two independent study parts (plate incorporation and preincubation method). Test systems were the Salmonnella thyphimurium strains TA 1537, TA 98, TA100 and TA 1535 (uvrB) and Escherichia coli WP2 (uvrA) strains with (+) and without (-) the metabolic system S9 (from male Wistar rats), respectively. Positive and negative conrols were included in each study. B-TEGME was dissolved in double distilled water and applied once at the start of the experiments with the concentration ranges: 0.05 – 0.5 mg/plate in the 1st experiment and 0.23 -5.0 mg/plate in the second experiment. The exposure duration was 48h.

Mutagenic and cytotoxic effects were absent up to the limit dose of 5 mg/plate.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010/01/14 - 2010/05/28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

With the exception noted below the work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards.

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: DEG4129165
- Purity : 89.8 %
- Date received: 03 December 2009
- Expiration date of the lot/batch: 31 October 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: specific gravity was determined and used to calculate the appropriate dose volume for the required dose level

FORM AS APPLIED IN THE TEST: test material was used as supplied

Target gene:

To assess the potential mutagenicity of the test material the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells was addressed within the assay.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Cell Line
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.

Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal bovine serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

PB/betaNF S9 was prepared from the livers of male Sprague Dawley CD strain rats. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 prepreparation on the fourth day.

Test concentrations with justification for top dose:

The molecular weight of the test material was calculated to be approximately 500.4, therefore the maximum dose level was 5000 µg/ml. The purity of the test material was 89.8% and was accounted for in the formulations. There was no significant change in pH when the test material was dosed into media. However the osmolality did increase by 50 mOsm at the top dose and was considered to be at the limit of acceptability therefore the maximum dose was limited to 4000 µg/ml. Further details for osmalality and pH are given in table 1.

Preliminary Cytotoxicity Test
Doses of 15.63 to 4000 µg/ml were used in the preliminary cytotoxicity test.

Mutagenicity Test - Experiment 1
The dose levels of the controls and the test material are given table below:
Group Final concentration of B-TEGME (µg/ml)
4(20)-hour without S9 0*, 125*, 250*, 500*, 1000*, 2000*, 4000*, EMS 500* and 750*
4(20)-hour with S9 0*, 125*, 250*, 500*, 1000*, 2000*, 4000*, DMBA 0.5* and 1.0*

Mutagenicity Test - Experiment 2
The dose levels of the controls and the test material are given below:
Group Final concentration of B-TEGME (µg/ml)
4(20)-hour without S9 0*, 125*, 250*, 500*, 1000*, 2000*, 4000*, EMS 200*
4(20)-hour with S9 0*, 125*, 250*, 500*, 1000*, 2000*, 4000*, DMBA 0.5* and 1.0*

EMS = Ethylmethanesulphonate
* = Dose levels plated for mutant frequency
DMBA = Dimethyl benzanthracene

No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Hams F12 cell culture media
- Justification for choice of solvent/vehicle: The test material formed a solution with the solvent suitable for dosing.
No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hams F12

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hams F12

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Preliminary Cytotoxicity Test
A preliminary cytotoxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation and a continuous exposure of 24 hours without metabolic activation. The cells were plated out at 3 x 10^6 cells/75 cm2 flask for the 4-hour exposure groups and at 1.5 x 10^6 cells/75 cm2 flask for the 24-hour exposure group approximately 20 hours before dosing. On dosing, the growth media was removed and replaced with serum free media (Ham's F12) for the 4-hour exposure group and with Ham’s F12 with 1% serum for the 24-hour exposure group. One flask per dose level was treated with and without S9 metabolic activation, 9 dose levels using halving dilutions and vehicle controls were dosed. The dose range of test material used was 15.63 to 4000 µg/ml. Exposure was for 4 hours or 24 hours at 37°C, after which the cultures were washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in growth medium; a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 ml of growth medium and incubated for 6 to 7 days at 37°C ± 2°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies.
Results from the preliminary cytotoxicity test were used to select the test material dose levels for the mutagenicity experiments.

Mutagenicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 3 x 10^6/75 cm2 flask for the 4 hour exposure groups or 1.5 x 10^6/75 cm2 flask for the 24 hour exposure group and allowed to attach overnight before being exposed to the test or control materials. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with six dose levels of test material, and vehicle and positive controls. Treatment was either for 4 hours in serum free media (Ham's F12) or for 24 hours in Ham’s F12 with 1% serum at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The dose range of test material was 125 to 4000 µg/ml both in the presence and absence of S9 metabolic activation and in both Experiment 1 and Experiment 2.
At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in growth medium. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10^6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in growth media and incubated at 37°C ± 1.5°C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

During the 7 Day expression period the cultures were sub-cultured and maintained at 2 x 10^6 cells/225 cm2 flask on days 3 or 4 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 ml of growth medium to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10^5 cells/75 cm2 flask (5 replicates per group) in Ham's F12 growth media (5% serum), supplemented with 10 µg/ml 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with giemsa. Mutant colonies were manually counted and recorded for each flask.
The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.

i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10^-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10^-6 will not be used for the evaluation of a test material.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test material clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test materials with little or no mutagenic activity, should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test material in culture medium. Where a test material is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test material.

Evaluation criteria:

Extended information are given above in the section 'Details on test system and conditions'.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
Doses of 15.63 to 4000 µg/ml were used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in Table 1. It can be seen that there was no consistent dose-related reduction in the cloning efficiency (CE) either in the presence or absence of S9.

Mutagenicity Test - Experiment 1
The Day 0 and Day 7 cloning efficiencies are presented in Tables 2 and 3. All of the vehicle controls had acceptable levels for cloning efficiencies. It can be seen that there was no marked toxicity with the test material when compared to the vehicle controls. The toxicity observed was similar to that seen in the preliminary cytotoxicity test.
All of the vehicle control cultures had mutant frequencies within the expected range. The positive control materials induced significant increases in mutant frequency. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The mutation frequency counts and mean mutation frequency per survivor values are presented in Tables 2 and 3. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10^-6 with or without the presence of S9.

Mutagenicity Test - Experiment 2
The Day 0 and Day 7 cloning efficiencies are presented in Tables 4 and 5. All of the vehicle controls had acceptable levels for cloning efficiencies. It can be seen that, as in Experiment 1, there was no marked toxicity with the test material when compared to the vehicle controls, and the toxicity observed was similar to that seen in the preliminary cytotoxicity test. All of the vehicle control cultures had mutant frequencies within the expected range. The positive control materials induced significant increases in mutant frequency. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The mutation frequency counts and mean mutation frequency per survivor per values are presented in Tables 4 and 5. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10^-6 with or without the presence of S9.

Table 1: Preliminary Cytotoxicity Results

	4-hour -S9						4-hour +S9						24-hour -S9
Dose (µg/ml)	Count			Mean	% CE	% Control	Count			Mean	% CE	% Control	Count			Mean	% CE	% Control
0	142	146	156	148.0	74.0	100	142	146	118	135.3	67.7	100	168	170	164	167.3	83.7	100
15.63	132	134	140	135.3	67.7	91	110	111	126	115.7	57.8	85	167	161	160	162.7	81.3	97
31.25	130	126	132	129.3	64.7	87	104	102	116	107.3	53.7	79	169	170	166	168.3	84.2	101
62.5	136	138	125	133.0	66.5	90	113	120	135	122.7	61.3	91	155	157	141	151.0	75.5	90
125	148	135	119	134.0	67.0	91	117	134	128	126.3	63.2	93	166	151	154	157.0	78.5	94
250	139	151	132	140.7	70.3	95	100	112	125	112.3	56.2	83	127	157	128	137.3	68.7	82
500	109	130	148	129.0	64.5	87	101	108	103	104.0	52.0	77	136	142	142	140.0	70.0	84
1000	138	120	130	129.3	64.7	87	117	123	120	120.0	60.0	89	168	155	151	158.0	79.0	94
2000	121	123	128	124.0	62.0	84	121	118	118	119.0	59.5	88	144	155	151	150.0	75.0	90
4000	145	138	135	139.3	69.7	94	140	150	129	139.7	69.8	103	156	170	156	160.7	80.3	96

CE = cloning efficiency

Table 2: Experiment 1 - Without Metabolic Activation (S9)

		Day 0 Viability						Day 7 Viability						Day 7 Mutant
Dose (µg/ml)		Colonies/flask (200 cells plated/flask)			% CE	% Control	Mean % Control	Colonies/flask (200cells plated /flask)			% CE	% Control	Mean % Control	Colonies /flask (2x10^5cells plated/flask )					MF	MFS  10^-6	Group SD	Group MFS 10^-6
0	A	160	162	166	81.3	100.0	100	156	173	192	86.8	100.0	100	3	4	3	2	5	17	19.6	1.33	16
	B	143	155	161	76.5	100.0		208	185	207	100.0	100.0		5	2	3	2	1	13	13.0
125	A	141	144	161	74.3	91.4	93	192	182	202	96.0	110.6	103	3	5	4	2	3	17	17.7	0.92	17
	B	150	149	139	73.0	95.4		187	198	189	95.7	95.7		3	4	3	3	2	15	15.7
250	A	131	169	162	77.0	94.7	102	166	182	172	86.7	99.8	96	1	3	0	3	5	12	13.8	1.78	15
	B	164	166	171	83.5	109.2		160	198	191	91.5	91.5		5	4	3	1	1	14	15.3
500	A	132	137	141	68.3	84.0	92	187	178	194	93.2	107.3	88	3	6	8	8	5	30	32.2	2.41	25
	B	149	159	150	76.3	99.8		142	142	132	69.3	69.3		4	3	1	3	2	13	18.8
1000	A	162	144	149	75.8	93.2	105	127	121	131	63.2	72.7	72	1	1	1	0	1	4	6.3	0.63	9
	B	181	183	175	89.8	117.4		154	152	121	71.2	71.2		1	2	2	2	1	8	11.2
2000	A	161	122	163	74.3	91.4	99	157	154	136	74.5	85.8	84	3	3	2	4	2	14	18.8	1.32	14
	B	169	155	163	81.2	106.1		186	181	122	81.5	81.5		1	1	0	4	2	8	9.8
4000	A	135	133	132	66.7	82.0	91	196	176	199	95.2	109.6	101	6	2	2	4	5	19	20.0	1.57	17
	B	164	150	143	76.2	99.6		195	193	172	93.3	93.3		3	4	2	4	1	14	15.0
EMS 500	A	71	54	60	30.8	37.9	42	139	137	161	72.8	83.9	77	26	30	28	23	27	134	184.0	5.76	156
	B	57	78	79	35.7	46.6		160	117	139	69.3	69.3		14	19	18	14	24	89	128.4
EMS 750	A	39	33	36	18.0	22.1	29	107	115	112	55.7	64.1	62	29	40	34	37	38	178	319.8	7.93	253
	B	47	61	55	27.2	35.5		112	116	126	59.0	59.0		24	18	21	22	25	110	186.4

EMS = Ethylmethanesulphonate

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Table 3: Experiment 1 - With Metabolic Activation (S9) 

		Day 0 Viability						Day 7 Viability						Day 7 Mutant
Dose (µg/ml)		Colonies/flask (200 cells plated/flask)			% CE	% Control	Mean % Control	Colonies/flask (200 cells plated /flask)			% CE	% Control	Mean % Control	Colonies /flask (2x10^5cells plated/flask )					MF	MFS   10^-6	Group SD	Group MFS 10^-6
0	A	173	175	171	86.5	100.0	100	194	192	180	94.3	100.0	100	3	3	2	1	4	13	13.8	1.23	13
	B	162	188	199	91.5	100.0		159	156	151	77.7	100.0		0	1	2	3	3	9	11.6
125	A	150	160	164	79.0	91.3	96	152	154	160	77.7	82.3	93	2	3	4	4	3	16	20.6	1.10	18
	B	185	185	184	92.3	100.9		167	162	157	81.0	104.3		2	4	4	1	2	13	16.0
250	A	170	163	178	85.2	98.5	99	144	163	146	75.5	80.0	93	2	3	4	2	3	14	18.5	0.97	17
	B	172	180	195	91.2	99.6		167	166	157	81.7	105.2		3	2	1	4	2	12	14.7
500	A	191	154	178	87.2	100.8	98	156	157	166	79.8	84.6	93	4	2	5	3	4	18	22.5	1.10	20
	B	177	151	191	86.5	94.5		164	150	158	78.7	101.3		4	2	2	3	2	13	16.5
1000	A	186	197	198	96.8	111.9	105	148	141	153	73.7	78.1	85	0	2	3	4	4	13	17.6	1.49	14
	B	180	181	177	89.7	98.0		158	131	141	71.7	92.3		0	2	3	1	1	7	9.8
2000	A	186	138	147	78.5	90.8	89	196	187	164	91.2	96.6	96	2	2	3	3	2	12	13.2	1.57	17
	B	179	148	150	79.5	86.9		153	140	153	74.3	95.7		2	6	3	4	0	15	20.2
4000	A	185	151	168	84.0	97.1	97	164	169	191	87.3	92.6	92	5	3	5	2	3	18	20.6	1.81	13
	B	158	174	195	87.8	96.0		135	151	141	71.2	91.6		0	1	2	0	1	4	5.6
DMBA 0.5	A	153	159	163	79.2	91.5	87	151	156	140	74.5	79.0	76	45	35	43	36	38	197	264.4	5.07	321
	B	145	151	155	75.2	82.1		115	112	114	56.8	73.2		51	41	37	40	46	215	378.3
DMBA 1	A	113	105	94	52.0	60.1	54	89	91	90	45.0	47.7	49	45	45	40	36	44	210	466.7	8.06	583
	B	78	90	92	43.3	47.4		77	74	86	39.5	50.9		58	62	53	52	51	276	698.7

DMBA = Dimethyl benzanthracene

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Table 4: Experiment 2 - Without Metabolic Activation (S9) 

		Day 0 Viability						Day 7 Viability						Day 7 Mutant
Dose (µg/ml)		Colonies/flask (200 cells plated/flask)			%CE	% Control	Mean % Control	Colonies/flask (200 cells plated /flask)			% CE	% Control	Mean % Control	Colonies /flask (2x10^5cells plated/flask)					MF	MFS  10 ^-6	GroupSD	Group MFS 10^-6
0	A	149	159	168	79.3	100.0	100	149	171	174	82.3	100.0	100	0	0	0	0	1	1	1.2	0.32	1
	B	149	153	161	77.2	100.0		148	169	144	76.8	100.0		0	0	0	0	0	0	0.0
125	A	146	152	171	78.2	98.5	98	171	179	146	82.7	100.4	100	0	0	0	0	0	0	0.0	0.00	0
	B	155	144	148	74.5	96.5		143	145	174	77.0	100.2		0	0	0	0	0	0	0.0
250	A	163	149	140	75.3	95.0	96	139	147	142	71.3	86.6	90	0	0	0	1	0	1	1.4	0.32	1
	B	143	150	152	74.2	96.1		139	161	132	72.0	93.7		0	0	0	0	0	0	0.0
500	A	142	161	158	76.8	96.8	96	152	151	141	74.0	89.9	90	2	0	0	0	0	2	2.7	0.67	2
	B	148	156	137	73.5	95.2		129	147	139	69.2	90.0		1	0	0	0	0	1	1.4
1000	A	143	159	155	76.2	96.0	93	159	160	174	82.2	99.8	100	0	0	1	0	0	1	1.2	0.84	4
	B	138	143	139	70.0	90.7		159	162	139	76.7	99.8		0	2	1	0	2	5	6.5
2000	A	137	159	141	72.8	91.8	90	168	172	147	81.2	98.6	94	0	0	0	0	1	1	1.2	0.53	4
	B	127	133	150	68.3	88.6		141	121	149	68.5	89.2		1	1	1	1	0	4	5.8
4000	A	147	132	121	66.7	84.0	89	158	172	164	82.3	100.0	102	0	0	1	1	1	3	3.6	0.48	2
	B	153	144	136	72.2	93.5		171	159	148	79.7	103.7		0	0	0	0	0	0	0.0
EMS 200	A	59	56	40	25.8	32.6	25	98	87	96	46.8	56.9	60	18	12	9	12	14	65	138.8	3.69	130
	B	35	26	23	14.0	18.1		102	96	92	48.3	62.9		18	11	9	14	7	59	122.1
EMS 300	A	26	34	23	13.8	17.4	14	86	88	79	42.2	51.2	53	19	28	30	18	25	120	284.6	4.67	279
	B	12	21	15	8.0	10.4		79	82	94	42.5	55.3		27	20	27	25	17	116	272.9

EMS =Ethylmethanesulphonate

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Table 5: Experiment 2 - With Metabolic Activation (S9)

		Day 0 Viability						Day 7 Viability						Day 7 Mutant
Dose (µg/ml)		Colonies/flask (200 cells plated/flask)			% CE	% Control	Mean % Control	Colonies/flask (200cells plated/flask)			% CE	% Control	Mean % Control	Colonies /flask (2x10^5cells plated/flask)					MF	MFS  10^-6	Group SD	Group MFS 10^-6
0	A	161	154	151	77.7	100.0	100	170	168	161	83.2	100.0	100	0	0	2	1	1	4	4.8	0.79	5
	B	195	190	188	95.5	100.0		149	159	166	79.0	100.0		1	2	0	0	1	4	5.1
125	A	152	144	143	73.2	94.2	85	170	181	163	85.7	103.0	98	0	0	2	1	2	5	5.8	0.82	6
	B	122	164	147	72.2	75.6		141	149	155	74.2	93.9		1	2	1	0	1	5	6.7
250	A	116	143	138	66.2	85.2	84	176	149	178	83.8	100.8	97	0	0	1	0	0	1	1.2	0.32	1
	B	149	164	166	79.8	83.6		143	154	149	74.3	94.1		0	0	0	0	0	0	0.0
500	A	132	135	146	68.8	88.6	80	148	174	139	76.8	92.4	93	0	0	1	1	1	3	3.9	0.70	4
	B	137	139	135	68.5	71.7		145	142	161	74.7	94.5		0	2	0	1	0	3	4.0
1000	A	184	151	149	80.7	103.9	90	176	178	156	85.0	102.2	101	0	0	0	3	1	4	4.7	1.06	4
	B	150	130	156	72.7	76.1		141	163	165	78.2	98.9		1	0	2	0	0	3	3.8
2000	A	155	162	153	78.3	100.9	89	171	178	196	90.8	109.2	98	2	1	2	0	0	5	5.5	0.82	6
	B	149	132	162	73.8	77.3		151	132	129	68.7	86.9		0	2	1	1	1	5	7.3
4000	A	134	152	156	73.7	94.8	91	145	165	178	81.3	97.8	99	0	0	1	0	0	1	1.2	0.84	4
	B	160	168	167	82.5	86.4		141	171	162	79.0	100.0		2	1	2	0	0	5	6.3
DMBA 0.5	A	110	111	129	58.3	75.1	67	115	116	111	57.0	68.5	69	25	18	17	24	22	106	186.0	2.77	188
	B	127	107	106	56.7	59.3		104	101	121	54.3	68.8		18	23	19	21	22	103	189.6
DMBA 1	A	118	112	112	57.0	73.4	69	108	101	119	54.7	65.7	72	24	24	20	17	24	109	199.4	12.29	267
	B	109	142	116	61.2	64.0		116	122	129	61.2	77.4		32	34	56	46	37	205	335.1

DMBA = Dimethyl benzanthracene

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Conclusions:

The test material did not induce significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Executive summary:

The study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The protocol used was designed to comply with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used was the plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Chinese hamster ovary (CHO) CHO-Kl cells were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose range of test material was selected based on the results of a preliminary cytotoxicity test and was 125 to 4000 µg/ml in both the presence and absence of metabolic activation. The maximum dose was limited to 4000 µg/ml as a result of an increase in osmolality of 50 mOsm at the maximum recommended dose of 5000 µg/ml, which was considered to be at the limit of acceptability.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

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

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010/01/28 - 2010/04/28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: is acceptable to the Japanese New Chemical Substance Law (METI)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice.

Type of assay:

other: in vitro mammalian chromosome aberration assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Substance type: Borated Glycol Ether
- Lot/batch No.of test material: DEG4129165
- Purity : 89.8 %
- Date received: 03 December 2009
- Expiration date of the lot/batch: 31 October 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: specific gravity was determined and used to calculate the appropriate dose volume for the required dose level

FORM AS APPLIED IN THE TEST: test material was used as supplied

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Cell Growth Inhibition test (µg/mL)
4h exposure (after 20 hour cell harvest) without S9: 0, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000
4(20)-hour with S9 0, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000
24-hour without S9 0, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000

Experiment 1: (µg/mL)
4(20)-hour without S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, MMC 0.4*
4(20)-hour with S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, CP 5*
(* Dose levels selected for metaphase analysis)


Experiment 2: (µg/mL)
24-hour without S9 0*, 125, 250*, 500*, 1000*, 2000*, 4000, MMC 0.4*
4(20)-hour with S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, CP 5*
(* Dose levels selected for metaphase analysis)

CP: cyclophosphoamide
MMC: mitomycin C

Vehicle / solvent:

MEM tissue culture medium was selected as the solvent because the test material was soluble in it at the required concentrations. A correction for the purity of the test material was made when the test material formulations were prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM tissue culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM tissue culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period: Approximately 48 hrs
- Exposure duration:
Experiment 1: 4 hrs with and without S9
Experiment 2: 24 hrs without S9, 4 hrs with S9

- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SPINDLE INHIBITOR: Demecolcine

STAIN: When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: 100/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy: Frequency of polyploid cells

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Result tables are given below. Please refer to tables 1 to 4.

Preliminary Toxicity Test (Cell Growth Inhibition Test)
The dose range of the test material for the Preliminary Toxicity Test was 15.63 to 4000 µg/ml. The maximum dose was limited by osmolality issues observed at the maximum recommended 5000 µg/ml concentration. It can be seen that the test material showed some evidence of toxicity in all three exposure groups. No precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure period, in any of the exposure groups. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 4000 µg/ml in the 4(20)-hour treatment, both in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 4000 µg/ml.
Dose selection for Experiment 1 and 2 was based on mild toxicity and the maximum practical dose level (4000 µg/ml).

Chromosome Aberration Test – Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of B-TEGME (µg/ml)
4(20)-hour without S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, MMC 0.4*
4(20)-hour with S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, CP 5*
(* Dose levels selected for metaphase analysis)
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 4000 µg/ml in the presence and absence of metabolic activation (S9). The data show that no growth inhibition was achieved in either the presence or absence of S9.
No precipitate of the test material was observed at the end of the treatment period in the presence or absence of S9.
The maximum dose level selected for metaphase analysis was the maximum practical dose level (4000 µg/ml).
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation (S9).
The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of B-TEGME (µg/ml)
24-hour without S9 0*, 125, 250*, 500*, 1000*, 2000*, 4000, MMC 0.4*
4(20)-hour with S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, CP 5*
(* Dose levels selected for metaphase analysis)
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 4000 µg/ml in the absence and presence of S9.
These data show 50% growth inhibition was achieved at 2000 µg/ml in the absence of S9. However, only 7% growth inhibition was achieved at 4000 µg/ml in the presence of S9.
No precipitate of the test material was observed at the end of the treatment period in the presence or absence of S9.
The maximum dose level selected for metaphase analysis in the presence of S9 was the same as Experiment 1, and was the maximum practical dose level of 4000 µg/ml. However the maximum dose level selected in the absence of S9 was limited by toxicity to 2000 µg/ml.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Table 1: Short Term Treatment Test - Experiment 1 Without Metabolic Activation (S9)

Treatment Period (hours)	S9 mix	Concentration ȝg/ml		Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)							g	Cell Growth Index	Number and Percentages of Cells Showing Numerical Aberrations (%)
				Observed	ctb	cte	csb	cse	Others	Total		Mitotic Index (%)	Observed	Polyploids	Others	Total
4	-	Negative Control (MEM) 0	A	100	2	0	0	0	0	2	0	4.80	100	0	0	0
			B	100	0	0	0	0	0	0	0	3.50	100	0	0	0
			Total %	200 (100)	2 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (1.0)	0 (0.0)	(100)	200	0	0	0 (0.0)
	-	1000	A	100	0	0	0	0	0	0	2	5.00	100	0	0	0
			B	100	1	0	1	0	0	2	0	6.35	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	2 (1.0)	2 (1.0)	(137)	200	0	0	0 (0.0)
	-	2000	A	100	0	0	0	0	0	0	1	6.70	100	0	0	0
			B	100	0	0	0	0	0	0	2	4.75	100	0	0	0
			Total %	200 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	3 (1.5)	(138)	200	0	0	0 (0.0)
	-	4000	A	100	0	0	0	0	0	0	1	5.30	100	0	0	0
			B	100	0	0	0	0	0	0	0	4.90	100	0	0	0
			Total %	200 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	(123)	200	0	0	0 (0.0)
	-	Positive Control (MMC) 0.4	A	50^a	17	16	2	1	0	29	3	2.05	50	0	0	0
			B	50^a	16	21	3	0	0	31	5	2.05	50	0	0	0
			Total %	100 (100)	33 (33.0)	37 (37.0)	5 (5.0)	1 (1.0)	0 (0.0)	60*** (60.0)	8 (8.0)	(49)	100	0	0	0 (0.0)

MMC = Mitomycin C

*** = p < 0.001

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.

 

Table 2: Short Term Treatment Test - Experiment 1 With Metabolic Activation (S9)

Treatment Period (hours)	S9 mix	Concentration ȝg/ml		Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)							g	Cell Growth Index	Number and Percentages of Cells Showing Numerical Aberrations (%)
				Observed	ctb	cte	csb	cse	Others	Total		Mitotic Index (%)	Observed	Polyploids	Others	Total
4	+	Negative Control(MEM) 0	A	100	0	0	0	0	0	0	0	5.90	100	0	0	0
			B	100	1	0	1	0	0	2	1	5.30	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	2 (1.0)	1 (0.5)	(100)	200	0	0	0 (0.0)
	+	1000	A	100	0	0	1	0	0	1	0	7.40	100	0	0	0
			B	100	0	0	0	0	0	0	0	7.00	100	0	0	0
			Total %	200 (100)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	(129)	200	0	0	0 (0.0)
	+	2000	A	100	0	0	0	0	0	0	0	6.90	100	0	0	0
			B	100	1	0	1	0	0	2	0	6.30	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	2 (1.0)	0 (0.0)	(118)	200	0	0	0 (0.0)
	+	4000	A	100	0	0	0	0	0	0	0	6.25	100	0	0	0
			B	100	1	0	0	0	0	1	0	6.55	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	(114)	200	0	0	0 (0.0)
	+	Positive Control(CP) 5	A	100	11	4	1	0	0	15	9	2.00	100	0	0	0
			B	50^a	14	6	4	0	0	20	9	1.15	50	0	0	0
			Total %	150 (100)	25 (16.7)	10 (6.7)	5 (3.3)	0 (0.0)	0 (0.0)	35*** (23.3)	18 (12.0)	(28)	150	0	0	0 (0.0)

CP = Cyclophosphamide

*** = p < 0.001

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.

Table 3: Continuous Treatment Test - Experiment 2

Treatment Period (hours)	S9 mix	Concentration mg/ml		Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)							g	Cell Growth Index	Number and Percentages of Cells Showing Numerical Aberrations (%)
				Observed	ctb	cte	csb	cse	Others	Total		Mitotic Index (%)	Observed	Polyploids	Others	Total
24	-	Negative Control (MEM) 0	A	100	1	0	0	0	0	1	0	6.60	102	2	0	2
			B	100	0	0	0	0	0	0	1	6.25	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	1 (0.5)	(100)	202	2	0	2 (1.0)
	-	250	A	100	1	0	1	0	0	2	0	5.00	100	0	0	0
			B	100	0	0	0	0	0	0	0	5.20	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	2 (1.0)	0 (0.0)	(79)	200	0	0	0 (0.0)
	-	500	A	100	1	0	0	0	0	1	0	4.55	100	0	0	0
			B	100	0	0	0	0	0	0	0	5.95	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	(82)	200	0	0	0 (0.0)
	-	1000	A	100	0	0	0	0	0	0	0	4.20	100	0	0	0
			B	100	0	0	0	0	0	0	0	3.70	100	0	0	0
			Total %	200 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	(61)	200	0	0	0 (0.0)
	-	2000	A	100	0	0	0	0	0	0	0	3.40	100	0	0	0
			B	100	0	0	1	0	0	1	1	3.00	100	0	0	0
			Total %	200 (100)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	1 (0.5)	1 (0.5)	(50)	200	0	0	0 (0.0)
	-	Positive Control (MMC) 0.2	A	50^a	10	12	0	1	0	16	2	0.85	50	0	0	0
			B	50^a	6	12	8	1	0	23	5	0.80	50	0	0	0
			Total %	100 (100)	16 (16.0)	24 (24.0)	8 (8.0)	2 (2.0)	0 (0.0)	39*** (39.0)	7 (7.0)	(13)	100	0	0	0 (0.0)

MMC = Mitomycin C

*** = p < 0.001

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.

Table 4: Short Term Treatment Test - Experiment 2 With Metabolic Activation (S9)

Treatment Period (hours)	S9 mix	Concentration mg/ml		Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)							g	Cell Growth Index	Number and Percentages of Cells Showing Numerical Aberrations (%)
				Observed	ctb	cte	csb	cse	Others	Total		Mitotic Index (%)	Observed	Polyploids	Others	Total
4	+	Negative Control (MEM) 0	A	100	0	0	0	0	0	0	1	8.75	100	0	0	0
			B	100	0	0	0	0	0	0	0	5.55	100	0	0	0
			Total %	200 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	(100)	200	0	0	0 (0.0)
	+	1000	A	100	0	0	1	0	0	1	0	6.70	100	0	0	0
			B	100	1	0	0	0	0	1	0	6.60	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	2 (1.0)	0 (0.0)	(93)	200	0	0	0 (0.0)
	+	2000	A	100	0	0	0	0	0	0	0	6.95	100	0	0	0
			B	100	0	0	0	0	0	0	0	7.20	100	0	0	0
			Total %	200 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	(99)	200	0	0	0 (0.0)
	+	4000	A	100	0	0	0	0	0	0	1	5.55	100	0	0	0
			B	100	1	0	0	0	0	1	0	7.80	100	0	0	0
			Total %	200 (100)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	1 (0.5)	(93)	200	0	0	0 (0.0)
	+	Positive Control (CP) 5	A	50^a	11	3	1	0	0	15	6	2.25	50	0	0	0
			B	100	6	8	9	0	0	21	10	1.80	100	0	0	0
			Total %	150 (100)	17 (11.3)	11 (7.3)	10 (6.7)	0 (0.0)	0 (0.0)	36*** (24.0)	16 (10.7)	(28)	150	0	0	0 (0.0)

CP = Cyclophosphamide

*** = p < 0.001

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.

Conclusions:

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and is acceptable to the Japanese New Chemical Substance Law (METI).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition in one of the exposure groups.

In conclusion, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential mutagenic effect of B-TEGME was determined in two reverse mutation assays according to OECD test guideline No. 471, of which the most recent study according to GLP and current standards was considered as the key study (Clariant, USO117742, 2007), and a previous study (non-GLP) was considered as supporting (BASF, 40M0575/884367, 1989). In both studies, there was no mutagenicity up to the limit dose of 5000 µg/plate and two independent study parts took place (plate incorporation and preincubation method). Test systems were the Salmonnella thyphimurium strains TA 1537, TA 98, TA100 and TA 1535 (uvrB) and Escherichia coli WP2 (uvrA) strains with (+) and without (-) the metabolic system S9 (from male Wistar rats), respectively. Positive and negative controls were included in each study. The test item was dissolved in distilled water and applied once at experimental starting with the concentration ranges given. The duration was 48h. Mutagenic and cytotoxic effects were absent.

A key study for gene mutation potential in mammalian cells of B-TEGME was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells (Shell, 2259-0067, 2010). The protocol used was designed to comply with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent. Chinese hamster ovary (CHO) CHO-Kl cells were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose range of test material was selected based on the results of a preliminary cytotoxicity test and was 125 to 4000 µg/ml in both the presence and absence of metabolic activation. The maximum dose was limited to 4000 µg/ml as a result of an increase in osmolality of 50 mOsm at the maximum recommended dose of 5000 µg/ml, which was considered to be at the limit of acceptability.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test material was considered to be non-mutagenic to CHO cells under the conditions of the test.

A key study for chromosome aberration potential of B-TEGME was studied in vitro in human peripheral lymphocytes (Shell, 2259-0066, 2010). This study was conducted according to OECD 473 and GLP standards, and was therefore considered to be relevant, adequate and reliable. The study supplements microbial systems insofar as it identifies potential genotoxins that produce chromosomal aberrations rather than gene mutations. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of metabolising system S9, at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition in one of the exposure groups. In conclusion, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Chromosome aberration potential was also studied with a brake fluid containing 17% B-TEGME in cultured Chinese Hamster Ovary (CHO) cells (Shell, 92.090, 1991). Although the study was also conducted under GLP and according to OECD test 473, this was considered as a supporting study sine the test material contained less B-TEGME. Cells were treated for either 3 hours in the presence of S9 mix or for 24 hours in the absence of S9 mix. Metaphase cells were analysed, demonstrating that the brake fluid and hence also B-TEGME did not induce structural chromosome aberrations in cultured human lymphocytes both in the presence and in the absence of S9 mix, under the experimental conditions described.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.