Bis(2-hydroxyethyl) sulphone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro reverse gene mutation in bacteria (Ames):

BS-1 was tested for mutagenicity using Salmonella typhimurium (TA100 and TA98) as indicator strains and the liver microsome fraction of rats for metabolic activation system (S9 Mix).

The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety and Health Law" and "GLP Standards Applied to Industrial Chemicals" of "Law Concerning the Examination and Regulation of Manufacture, etc. , of Chemical Substance".

The number of revertant colonies of BS-1 did not show any tendency to increase twice or more over that in negative control

for all strains. It was confirmed that the test was appropriately performed because the negative control and the

positive control induced the reasonable increase in the number of revertant colonies.

Based on the above results, it is concluded that BS-1 was non-mutagenic in this test system.

BS-1 was tested for mutagenicity using Salmonella typhimurium (TA1535 and TA1537), Escherichia coli (WP2uvrA/pKM101) as indicator strains and the liver microsome

fraction of rats for metabolic activation system (S9 Mix). The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety

and Health Law" and "GLP Standards Applied to Industrial Chemicals" of "Law Concerning the Examination and Regulation of Manufacture, etc. , of Chemical Substance".

The number of revertant colonies of BS-1 increased twice or more over that in negative control in TA1535+S9Mix at main test. But this response is not reproducible because the number of revertant colonies did not increase more than double of spontaneous reversion at dose range finding test and confirmatory test-1 and -2. Thus, it is thought that the number of revertant colonies in TA1535+S9Mix at main test increased because of variability. For the other strains, the number of revertant colonies did not show any tendency to increase twice or more over that in negative control. It was confirmed that the test was appropriately performed because the negative control and the positive control induced the reasonable increase in the number of revertant colonies.

Based on the above results, it is concluded that BS-1 was non-mutagenic in this test system.

In-vitro cytogenicity in mammalian cells (chromose aberration)

 Introduction

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 (Scottet al., 1990). 

 

 Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the in the absence of any marked toxicity the maximum concentration should be the maximum recommended dose level. The dose levels selected for the Main Test were as follows:

Exposure Group

Final concentration of test item BHES (µg/mL)

4(20)-hour without S9

48.13, 96.25, 192.5, 385, 770, 1540

4(20)-hour with S9 (2%)

48.13, 96.25, 192.5, 385, 770, 1540

24-hour without S9

48.13, 96.25, 192.5, 385, 770, 1540

Results

All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended dose level.

 

Conclusion

The test item,BHES was considered to be non-clastogenic to human lymphocytes in vitro.

In-vitro gene mutation in mammalian cells (Mouse Lymphoma Assay):

 Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group	Concentration of BHES (µg/mL) plated for mutant frequency
4-hour without S9	48.13, 96.25, 192.5, 385, 770, 1540
4-hour with S9 (2%)
24-hour without S9

 Results……..

With no evidence of precipitate or test item-induced toxicity, the maximum dose level used in the Mutagenicity Test was the 10mM limit dose of 1540 µg/mL. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

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:

The study was conducted between 2 September 1997 and 16 September 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety and Health Law"

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test substance: BS-1
Sample nurnber: 97-180
Concentration: 65 percent aqueous solution

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The highest dose of the test solution was 10μl because the concentration of test substance was 65%.
Solutions of five different concentration, 0. 391, 0. 156, 0. 6 25, 2. 50 and 10. 0 μl per per plate were used for dose range finding test.
For main test, concentration of the test solution was prepared to be 0. 6 25, 1.25, 2.50, 5.00 and 10.0 μl per plate.

Vehicle / solvent:

sterilized pure water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterilized pure water

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO. 0.01 and 0.1µg/plate for TA100 and TA98

Positive control substance:

other: 2- ( 2-Furyl)-3- (5-nitro- 2-furyl)acrylamide (AF2)

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterilized pure water

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO. 5 µg/plate for TA100 and TA98

Positive control substance:

other: Benzo[a]pyrene (BP)

Remarks:

with S9 mix

Details on test system and experimental conditions:

Control test
For the solvent control test, three plates were used.
Meanwhile, two plates were used for positive control test.
Every positive control was dissolved in DMSO and stored in a freezer at - 20 °C.

For the test substance two plates of each dose were used in every test.

Test method
Test solution (0.1 ml) was mixed with 0.1 M sodium phosphate buffer (pH 7.4, 0.5 ml) and the overnight culture of tester strain (0.1 ml) and then the mixture was pre-incubated at
37 °C for 20 minutes with gentle shaking. For metabolic activation, S9 Mix (0.5 ml) was mixed instead of 0.1 M sodium phosphate buffer. After pre-incubation, melted soft agar ( 2 ml)
was added and the resulting mixture was poured onto minimal glucose agar plate. The soft agar contained 0.5 mM D-biotin and 0.5 mM L-histidine.
After incubation for 2 days, revertant colonies were counted and average number of colonies in each dose and control were obtained.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding test:
The number of revertant colonies did not increase twice or more over that in negative control in test strains both with and without S9Mix.

Main test:
The number of revertant colonies did not increase twice or more over that in negative control in test strains both with and without S9Mix.

Results from the main test are presented in the table below

Test period		97/9/11~97/9/16
With (+) or without (-) S9 mix	Dose µg/plate	Number of revertant colonies /plate
		Base-pair substitution type		Frameshift type
		TA100		TA98
S9 Mix (-)	Solvent control	127		18
		167		28
		140	(145)	23	(23)
	1      0.625	136		25
		145	(141)	14	(20)
	2        1.25	155		19
		127	(141)	23	(21)
	3          2.5	140		18
		140	(140)	26	(22)
	4              5	144		14
		128	(136)	23	(19)
	5           10	137		21
		142	(140)	23	(22)
S9 Mix (+)	Solvent control	139		38
		116		48
		127	(127)	40	(42)
	1      0.625	112		39
		161	(137)	43	(41)
	2        1.25	122		40
		145	(134)	40	(40)
	3          2.5	155		37
		130	(143)	40	(39)
	4              5	136		28
		148	(142)	44	(36)
	5           10	133		27
		155	(144)	40	(34)
Positive control without S9 mix	Chemical	AF2		AF2
	Dose µg/plate	0.01		0.1
	Revertants /plate	1166 1240	(1203)	164 140	(152)
Positive control with S9 mix	Chemical	BP		BP
	Dose µg/plate	5		5
	Revertants /plate	1122 1036	(1079)	293 367	(330)

The figures in parentheses show the mean of each plate.

Positive controls

AF2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

BP: Benzo[a]pyrene

Conclusions:

The number of revertant colonies of BS-1 did not show any tendency to increase twice or more over that in negative control
for all strains. It was confirmed that the test was appropriately performed because the negative control and the
positive control induced the reasonable increase in the number of revertant colonies.
Based on the above results, it is concluded that BS-1 was non-mutagenic in this test system.

Executive summary:

BS-1 was tested for mutagenicity using Salmonella typhimurium (TA100 and TA98) as indicator strains and the liver microsome fraction of rats for metabolic activation system (S9 Mix).

The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety and Health Law" and "GLP Standards Applied to Industrial Chemicals" of "Law Concerning the Examination and Regulation of Manufacture, etc. , of Chemical Substance".

The test substance was judged non-mutagenic in this test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 17 February 1998 and 12 March 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety and Health Law"

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test Substance: BS-1
Sample number: 97-180
Concentration: 65 percent aqueous solution

Target gene:

histidine or tryptophan

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The highest dose of the test solution was 10μl because the concentration of test the substance was 65%. Solutions of five
different concentration, 0.391, 0.156 , 0.625, 2.50 and 10.0 μl per plate were used for dose range finding test.
For main test and confirmatory test for TA1535 + S9 Mix, concentration of the test solution was prepared to be 0. 625, 1.25, 2.50, 5.00 and 10.0 μl
per plate. Solutions of five different concentration, 0.156, 0.313, 0.625, 1.25 and 2.50 μl per plate were used for confirmatory test for TA1537 - S9 Mix.

Vehicle / solvent:

sterilized pure water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterilized pure water

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO. 0.01 µg/plate for strain WP2uvrA/pKM101

Positive control substance:

other: 2-(2-Furyl)-3- (5-nitro-2-furyl)acrylamide (AF2)

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterilized pure water

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO. 0.5 µg/plate for strain TA1535

Positive control substance:

sodium azide

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterlized pure water

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO. 80 µg/plate for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterilized pure water

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO. Dissolved in DMSO. 2 µg/plate for strain TA1535 and 1 µg/plate for strain WP2urvA/pKM101

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterilized pure water

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO. 5 µg/plate for strain TA1537

Positive control substance:

benzo(a)pyrene

Remarks:

with S9

Details on test system and experimental conditions:

Control test
To confirm the activity of strains and 89 Mix, solvent control tests and positive control tests were carried out with
and without S9 Mix for each strain.
For the solvent control test, three plates were used. Meanwhile, two plates were used for positive control test.

For the test substance two plates of each dose were used in every test.

Test method
Test solution (0.1ml) was mixed with 0. 1 M sodium phosphate buffer (pH 7.4, 0.5 ml) and the overnight culture of
tester strain (0.1 ml) and then the mixture was pre-incubated at 37 °C for 20 minutes with gentle shaking. For metabolic
activation, S9 Mix (0.5 ml) was mixed instead of 0.1 M sodium phosphate buffer. After pre-incubation, melted soft agar (2 ml)
was added and the resulting mixture was poured onto minimal glucose agar plate. The soft agar contained 0. 5 mM D-biotin and
0. 5 mM L-histidine for Salmonella typhimurium strains, and 0. 5mM L-tryptophan for Escherichia coli strain.
After incubation for 2 days, revertant colonies were counted and average number of colonies in each dose and control
were obtained.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In the initial test (+ S) the number of revertent colonies increased twice or more over that in the solvent control. However, two confirmatory tests were carried out where the number of revertent colonies did not increase twice or more over the control.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

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:

Dose range finding test:
Although a slight increase in the number of revertant colonies was observed in TA1535+S9Mix and TA1537-S9Mix, the
twofold increase beyond spontaneous reversion was not shown in all test strains.

Main test:
The number of revertant colonies increased twice or more over that in negative control in TA1535+S9Mix. In other strains,
the number of revertant colonies did not show any tendency to increase though a slight increase in the number of revertant
colonies was observed in TA1537-S9Mix. Therefore, confirmatory test-1 and -2 were performed for TA1535+S9Mix and TA1537-S9Mix

Confirmatory test-1 and -2:
The number of revertant colonies did not increase twice or more over that in negative control in test strains.

The results of the main test are presented in the table below

Test period		98/02/25~98/02/27
With (+) or without (-) S9 mix	Dose µg/plate	Number of revertant colonies /plate
		Base-pair substitution type				Frameshift type
		TA1535		WP2urvA/pKM101		TA1537
S9 Mix (-)	Solvent control	8		66		5
		8		80		5
		16	(11)	62	(69)	5	(5)
	1      0.625	12		77		7
		17	(15)	85	(81)	11	(9)
	2        1.25	7		70		10
		14	(11)	92	(81)	8	(9)
	3          2.5	13		55		4
		16	(15)	71	(63)	9	(7)
	4              5	18		68		4
		16	(17)	70	(69)	10	(7)
	5           10	11		48		7
		12	(12)	77	(63)	9	(8)
S9 Mix (+)	Solvent control	8		87		12
		7		92		8
		9	(8)	97	(92)	15	(12)
	1      0.625	12		72		16
		9	(11)	92	(82)	10	(13)
	2        1.25	13		62		13
		17	(15)	82	(72)	10	(12)
	3          2.5	15		71		8
		18	(17)	89	(80)	8	(8)
	4              5	10		94		15
		19	(15)	80	(87)	9	(12)
	5           10	15		90		8
		12	(14)	86	(88)	12	(10)
Positive control without S9 mix	Chemical	NaN3		AF2		9AA
	Dose µg/plate	0.5		0.01		80
	Revertants /plate	241 244	(243)	2042 1956	(1999)	402 338	(370)
Positive control with S9 mix	Chemical	2AA		2AA		BP
	Dose µg/plate	2		1		5
	Revertants /plate	203 216	(210)	132 144	(138)	161 157	(159)

The figures in parentheses show the mean of each plate

Positive controls

AF2: 2-(2-F uryJ)-3-(5-nitro-2-furyl)acrylamide

NaN₃: Sodium azide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo[a]pyrene

Results from Confirmatory test 1 are displayed in the following table

Test period		98/02/25~98/02/27
With (+) or without (-) S9 mix	Dose µg/plate	Number of revertant colonies /plate
		Base-pair substitution type		Frameshift type
		TA1535		TA1537
S9 Mix (-)	Solvent control			3
				4
				9	(5)
	1      0.625			4
				4	(4)
	2        1.25			5
				6	(6)
	3          2.5			5
				5	(5)
	4              5			2
				7	(5)
	5           10			3
				2	(3)
S9 Mix (+)	Solvent control	17
		22
		18	(19)
	1      0.625	15
		28	(22)
	2        1.25	17
		23	(20)
	3          2.5	21
		20	(21)
	4              5	14
		31	(23)
	5           10	33
		22	(23)
Positive control without S9 mix	Chemical			9AA
	Dose µg/plate			80
	Revertants /plate			714 512	(613)
Positive control with S9 mix	Chemical	2AA
	Dose µg/plate	2
	Revertants /plate	350 374	(362)

Results from the Confirmatory test 2 are displayed in the table below

Test period		98/02/25~98/02/27
With (+) or without (-) S9 mix	Dose µg/plate	Number of revertant colonies /plate
		Base-pair substitution type		Frameshift type
		TA1535		TA1537
S9 Mix (-)	Solvent control			12
				14
				13	(13)
	1      0.625			11
				14	(13)
	2        1.25			6
				10	(8)
	3          2.5			9
				15	(12)
	4              5			2
				9	(6)
	5           10			10
				19	(15)
S9 Mix (+)	Solvent control	13
		12
		11	(12)
	1      0.625	8
		13	(11)
	2        1.25	7
		10	(9)
	3          2.5	8
		12	(10)
	4              5	11
		11	(11)
	5           10	8
		13	(11)
Positive control without S9 mix	Chemical			9AA
	Dose µg/plate			80
	Revertants /plate			473 466	(470)
Positive control with S9 mix	Chemical	2AA
	Dose µg/plate	2
	Revertants /plate	354 404	(379)

Conclusions:

The number of revertant colonies of BS-1 increased twice or more over that in negative control in TA1535+S9Mix at main test. But this response is not reproducible because the number of revertant colonies did not increase more than double of spontaneous reversion at dose range finding test and confirmatory test-1 and -2. Thus, it is thought that the number of revertant colonies in TA1535+S9Mix at main test increased because of variability. For the other strains, the number of revertant colonies did not show any tendency to increase twice or more over that in negative control. It was confirmed that the test was appropriately performed because the negative control and the positive control induced the reasonable increase in the number of
revertant colonies.
Based on the above results, it is concluded that BS-1 was non-mutagenic in this test system.

Executive summary:

BS-1 was tested for mutagenicity using Salmonella typhimurium (TA1535 and TA1537), Escherichia coli (WP2uvrA/pKM101) as indicator strains and the liver microsome

fraction of rats for metabolic activation system (S9 Mix). The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety

and Health Law" and "GLP Standards Applied to Industrial Chemicals" of "Law Concerning the Examination and Regulation of

Manufacture, etc. , of Chemical Substance". The test substance was judged non-mutagenic in this test system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 14 July 2016 and 08 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro Mammalian Chromosome Aberration Test

Specific details on test material used for the study:

Identification: BHES
Physical state/Appearance: White solid block
Batch: 160226
Purity: 99.64%
Expiry Date: 01 March 2017
Storage Conditions: Room temperature in the dark

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability
- Suitability of cells: The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Cell cycle length, doubling time or proliferation index: Average generation time for human lymphocytes is approx. 16 hrs
- Sex, age and number of blood donors if applicable:
Preliminary toxicity test: male, aged 25 years
Main experiment: male, aged 26 years
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Modal number of chromosomes: 44-48

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver homogenate metabolizing system (S9), at a 2% final concentration

Test concentrations with justification for top dose:

Preliminary test:
The dose range for the Preliminary Toxicity Test was 6.02, 12.03, 24.06, 48.13, 96.25, 192.5, 385, 770 and 1540 µg/mL.
The maximum dose was the maximum recommended dose level, the 10 mM concentration.

Main experiment:
In the absence of any marked toxicity in the preliminary test, the maximum dose level selected for the Main Experiment was the maximum recommended dose level, and was 1540µg/mL for the 4(20)-hour exposure groups and for the continuous exposure group.
The test concentration were as follows:
4(20)-hour without S9 - 48.13, 96.25, 192.5, 385, 770 and 1540 µg/mL
4(20)-hour with S9 - 48.13, 96.25, 192.5, 385, 770 and 1540µg/mL
24-hour without S9 - 48.13, 96.25, 192.5, 385, 770 and 1540 µg/mL

Vehicle / solvent:

The test item was soluble in culture medium (Minimal Essential Medium (MEM)) at 15.4 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/mL for 4-hour exposure 0.05 µg/mL for the 24-hour exposure

Positive control substance:

mitomycin C

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Details on test system and experimental conditions:

Cells
Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/BNF S9 10/04/16 was used in this study.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 48hrs
- Exposure duration: 4 and 24 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24hrs

SPINDLE INHIBITOR: demecolcine (Colcemid 0.1 µg/mL)

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: 2000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

The following criteria were used to determine a valid assay:
- The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
- All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
- The required number of cells and concentrations were analyzed.

Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

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. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was not dose related

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mitotic index data for the Main Experiment confirm the qualitative observations of the preliminary test in that no dose-related inhibition of mitotic index was observed in any of the three exposure groups. In the 4(20)-hour exposure group in the absence of S9, no toxicity was observed at the maximum recommended dose level, 1540 µg/mL. In the presence of S9, there was very modest toxicity demonstrated at 770 µg/mL and 1540 µg/mL with 17% and 10% mitotic inhibition, respectively. The 24-hour exposure group did demonstrate marked toxicity with 53% and 50% mitotic inhibition at 192.5 µg/mL and 770 µg/mL, respectively. However, it was considered that these reductions were due to a discrepancy in the mitotic index between the ‘A’ and ‘B’ replicates, with the ‘B’ replicates being much lower than the ‘A’ replicates. The toxicity demonstrated in the 24-hour exposure group was not dose related and the reductions in mitotic index at 192.5 µg/mL and 770 µg/mL were considered not to be a true representation of the toxicity in this exposure group. An inhibition of mitotic index of 39% was noted at 1540 µg/mL in the 24-hour continuous exposure group.

Preliminary Toxicity Test:

No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1540 µg/mL in all three exposure groups. The test item induced no marked evidence of toxicity in any of the exposure groups.  

In the absence of any marked toxicity the maximum dose level selected for the Main Experiment was the maximum recommended dose level, and was 1540µg/mL for the 4(20)-hour exposure groups and for the continuous exposure group.

Chromosome Aberration Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of BHES (µg/mL)
4(20)-hour without S9	0*, 48.13, 96.25, 192.5*, 385*, 770*, 1540*, MMC0.2*
4(20)-hour with S9 (2%)	0*, 48.13, 96.25, 192.5*, 385*, 770*, 1540*, CP2*
24-hour without S9	0*, 48.13, 96.25, 192.5*, 385*, 770*, 1540*, MMC 0.05*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 1540 µg/mL in all three exposure groups.

No precipitate of the test item was observed in the blood cultures at the end of the exposure in any of the three exposure groups.

The maximum dose level selected for metaphase analysis was the maximum recommended dose level, the 10 mM concentration dose level (1540 µg/mL).

The assay was considered valid as it met all of the following criteria:

- The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

- All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

- The required number of cells and concentrations were analyzed.

- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure Without Metabolic Activation (S9)

Treatment Group	Replicate	Mitotic Index (%)	Number of Cells Scored	Number of Aberrations						Total Number of Aberrations		Frequency of Aberrant Cells (%)
				Gaps	Chromatid		Chromosome		Others
					Breaks	Exchanges	Breaks	Exchanges	X	(+ Gaps)	(-Gaps)	(+Gaps)	(-Gaps)
Vehicle Control (MEM)	A	5.45	150	0	0	0	0	0	0	0	0	0	0
	B	5.35	150	0	0	0	0	0	0	0	0	0	0
	Total	10.80	300	0	0	0	0	0	0	0	0	0	0
		(100)										(0.0)	(0.0)
	A	4.65	150	1	0	0	0	0	0	1	0	1	0
192.5	B	5.85	150	2	0	0	1	0	0	3	1	3	1
mg/mL	Total	10.50	300	3	0	0	1	0	0	4	1	4	1
		(97)										(1.3)	(0.3)
	A	6.50	150	0	0	0	0	0	0	0	0	0	0
385	B	4.85	150	0	1	0	1	0	0	2	2	2	2
mg/mL	Total	11.35	300	0	1	0	1	0	0	2	2	2	2
		(105)										(0.7)	(0.7)
	A	5.40	150	1	0	0	2	0	0	3	2	2	1
770	B	6.80	150	2	0	0	0	0	0	2	0	2	0
mg/mL	Total	12.20	300	3	0	0	2	0	0	5	2	4	1
		(113)										(1.3)	(0.3)
	A	4.60	150	0	0	0	0	0	0	0	0	0	0
1540	B	6.85	150	0	0	0	0	0	0	0	0	0	0
mg/mL	Total	11.45	300	0	0	0	0	0	0	0	0	0	0
		(106)										(0.0)	(0.0)
Positive Control	A	3.70	86^a	0	5	8	2	0	0	15	15	15	15
MMC 0.2	B	4.25	133^a	2	16	4	0	0	0	22	20	17	17
mg/mL	Total	7.95	199	2	21	12	2	0	0	37	35	32	32***
		(74)										(16.1)	(15.6)

MMC     Mitomycin C

a             Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

***          P < 0.001

MEM Minimal Essential Medium

Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure With Metabolic Activation (2% S9)

Treatment Group	Replicate	Mitotic Index (%)	Number of Cells Scored	Number of Aberrations						Total Number of Aberrations		Frequency of Aberrant Cells (%)
				Gaps	Chromatid		Chromosome		Others
					Breaks	Exchanges	Breaks	Exchanges	X	(+ Gaps)	(-Gaps)	(+Gaps)	(-Gaps)
Vehicle Control (MEM)	A	5.25	150	3	0	0	0	0	0	3	0	3	0
	B	5.50	150	0	2	0	0	0	0	2	2	1	1
	Total	10.80	300	3	2	0	0	0	0	5	2	4	1
		(100)										(1.3)	(0.3)
	A	5.00	150	1	2	0	0	0	0	3	2	3	2
192.5	B	5.30	150	0	0	0	0	0	0	0	0	0	0
mg/mL	Total	10.50	300	1	2	0	0	0	0	3	2	3	2
		(96)										(1.0)	(0.7)
	A	5.95	150	1	0	0	0	0	0	1	0	1	0
385	B	5.75	150	0	0	0	0	0	0	0	0	0	0
mg/mL	Total	11.35	300	1	0	0	0	0	0	1	0	0	0
		(109)										(0.3)	(0.0)
	A	4.10	150	0	1	0	0	0	0	1	1	1	1
770	B	4.85	150	0	0	0	0	0	0	0	0	0	0
mg/mL	Total	12.20	300	0	1	0	0	0	0	1	1	1	1
		(83)										(0.3)	(0.3)
	A	4.45	150	0	0	0	0	0	0	0	0	0	0
1540	B	5.20	150	1	0	0	0	0	0	1	0	1	0
mg/mL	Total	11.45	300	1	0	0	0	0	0	1	0	1	0
		(90)										(0.3)	(0.0)
Positive Control	A	2.40	40^a	1	18	2	1	0	0	22	21	16	15
MMC 0.2	B	2.65	61^a	4	19	1	2	0	0	26	22	19	16
mg/mL	Total	7.95	101	5	37	3	3	0	0	48	43	35	31***
		(47)										(34.7)	(30.7)

CP         Cyclophosphamide

a            Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

***        P < 0.001

MEM Minimal Essential Medium

Results of Chromosome Aberration Test – Main Experiment 24-hour Continuous Exposure
Without Metabolic Activation (S9)

Treatment Group	Replicate	Mitotic Index (%)	Number of Cells Scored	Number of Aberrations						Total Number of Aberrations		Frequency of Aberrant Cells (%)
				Gaps	Chromatid		Chromosome		Others
					Breaks	Exchanges	Breaks	Exchanges	X	(+ Gaps)	(-Gaps)	(+Gaps)	(-Gaps)
Vehicle Control (MEM)	A	3.15	150	1	0	0	0	0	0	1	0	1	0
	B	3.90	150	0	1	0	0	0	0	1	1	1	1
	Total	7.05	300	1	1	0	0	0	0	2	1	2	1
		(100)										(0.7)	(0.3)
	A	2.05	150	2	0	0	2	0	0	4	2	3	2
192.5	B	1.25	150	0	0	0	0	0	0	0	0	0	0
µg/mL	Total	3.30	300	2	0	0	2	0	0	4	2	3	2
		(47)										(1.0)	(0.7)
	A	3.05	150	1	4	0	0	0	0	5	4	5	4
385	B	2.85	150	0	0	0	0	0	0	0	0	0	0
µg/mL	Total	5.90	300	1	4	0	0	0	0	5	4	5	4
		(84)										(1.7)	(1.3)
	A	2.40	150	0	2	0	0	0	0	2	2	2	2
770	B	1.11	150	1	2	0	0	0	0	3	2	3	2
µg/mL	Total	3.51	300	1	4	0	0	0	0	5	4	5	4
		(50)										(1.7)	(1.3)
	A	1.95	150	0	0	0	0	0	0	0	0	0	0
1540	B	2.35	150	0	0	0	0	0	0	0	0	0	0
µg/mL	Total	4.30	300	0	0	0	0	0	0	0	0	0	0
		(61)										(0.0)	(0.0)
Positive Control	A	1.20	105^a	5	13	0	4	0	0	22	17	19	15
MMC 0.05	B	1.10	95^a	2	19	4	3	0	0	28	26	16	15
µg/mL	Total	2.30	200	7	32	4	7	0	0	50	43	35	30***
		(33)										(17.5)	(15.0)

MMC     Mitomycin C

a            Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

***        P < 0.001

MEM Minimal Essential Medium

Conclusions:

BHES did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

 Introduction

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 (Scottet al., 1990). 

 Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the in the absence of any marked toxicity the maximum concentration should be the maximum recommended dose level. The dose levels selected for the Main Test were as follows:

Exposure Group	Final concentration of test item BHES (µg/mL)
4(20)-hour without S9	48.13, 96.25, 192.5, 385, 770, 1540
4(20)-hour with S9 (2%)	48.13, 96.25, 192.5, 385, 770, 1540
24-hour without S9	48.13, 96.25, 192.5, 385, 770, 1540

Results

All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended dose level.

Conclusion

The test item,BHES was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 01 July 2016 and 26 July 2016

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)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian gene mutaation study

Specific details on test material used for the study:

Identification: BHES
Physical state/Appearance: White solid block
Batch: 160226
Purity: 99.64%
Expiry Date: 01 March 2017
Storage Conditions: Room temperature, in the dark

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Suitability of cells: The thymidine kinase heterozygote system, TK +/- to TK -/- has been extensively validated
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Methods for maintenance in cell culture if applicable: The stocks of cells are stored in liquid nitrogen at approximately -196 °C.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The molecular weight of the test item was 154.18 therefore the maximum proposed dose level in the solubility test was set at 1540 µg/mL, the 10 mM limit dose. The purity of the test item was 99.64% and was therefore not accounted for during formulation of the test item.

Preliminary Cytoxicity test:
0, 6.02, 12.03, 24.06, 48.13, 96.25, 192.5, 385, 770, 1540 µg/mL

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Main experiment:
4 hr exposure (with and without S9) and 24 hr exposure - 0, 48.13, 96.25, 192.5, 385, 770, 1540 µg/mL

Vehicle / solvent:

The test item was accurately weighed and formulated in R0 medium prior to serial dilutions being prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

400µg/mL and 150 µg/mL respectively, was used in the 4-hour and 24-hour exposure groups

Positive control substance:

ethylmethanesulphonate

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1.5 µg/mL was used in the 4-hour exposure group

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium, in suspension
- Cell density at seeding (if applicable):
preliminary toxicty tests 5 x 10^5 cells/ml for 4-hour exposure and 1.5 x 10^5 cells/ml for 24-hour exposure. All groups were serially diluted to 2 x 10^5 calls/ml after the exposure period, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
Main test: 1 x 10^-6 cells/ml for 4-hour exposure and 0.3 x 10^6 cells/ml for 24-hour exposure. All groups were serially diluted to 2 x 10^5 cells.ml after the exposure period. Before plating cells were diluted to 1 x 10^4 cells/ml (or 10 cells/ml for viability assessment)

DURATION
- Preincubation period: Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment.
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): 4 µg/mL 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Data evaluation:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
To define positive and negative responses the Global Evaluation Factor (GEF) of 126 x 10^-6 is used. This is the increase in mutation frequency (MF) above the concurrent control.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

Calculation of Percentage Relative Suspension Growth (%RSG):
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Day 2 Viability (%V):
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells / total wells plated
%V = (-ln P(0) x 100) / number of cells per well

Calculation of Relative Total Growth (RTG):
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = %V / mean solvent control %V
Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%

Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

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:
There was no evidence of any marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in any of the three exposure groups. No precipitate of the test item was observed at any of the dose levels.

Mutagenicity Test:
As was seen in the Preliminary Cytotoxicity Test, there was no evidence of any marked toxicity following exposure to the test item in any of the three exposure groups, as indicated by the %RSG and RTG values. There was also no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had also not occurred. Acceptable levels of toxicity were seen with the positive control substances.
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, including the 10 mM limit dose, in any of the three exposure groups. No precipitate of the test item was observed at any of the dose levels.

Preliminary Cytotoxicity Test

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
6.02	96	93	97
12.03	111	83	113
24.06	94	84	110
48.13	96	92	98
96.25	99	80	83
192.5	96	100	106
385	94	85	89
770	97	82	81
1540	107	96	90

A summary of the results of the main experiment is presented in the tables below:

Concentration (µg/mL)			4-Hours-S9					Concentration (µg/mL)			4-Hours+S9
			%RSG	RTG	MF§						%RSG	RTG	MF§
0			100	1.00	128.30			0			100	1.00	145.30
48.13			92	0.94	123.27			48.13			88	0.93	157.37
96.25			94	1.04	118.10			96.25			97	1.05	137.25
192.5			101	1.22	88.25			192.5			92	0.98	137.43
385			101	1.12	113.45			385			91	1.03	138.74
770			96	1.14	114.29			770			96	0.96	157.34
1540			94	1.12	110.85			1540			88	0.99	128.05
MF threshold for a positive response = 254.30								MF threshold for a positive response = 271.30
Positive control								Positive control
EMS								CP
400			74	0.62	1030.03			1.5			73	0.48	1065.80

Concentration (µg/mL)		24-Hours-S9
		%RSG	RTG	MF§
0		100	1.00	158.20
48.13		109	1.15	123.61
96.25		124	1.21	157.76
192.5		96	0.96	160.56
385		85	0.86	163.92
770		110	1.03	169.01
1540		107	1.02	150.35
MF threshold for a positive response = 284.20
Positive control
EMS
150		49	0.39	1482.67

A summary of analysis for all test groups is displayed in the following tables:

4-Hour test group (-S9)

Concentration (µg/mL)			SG	%RSG	%V	RTG	MF§
0			12.76	100	78.43	1.00	128.30
48.13			10.83	92	80.34	0.94	123.27
96.25			11.49	94	86.56	1.04	118.10
192.5			12.23	101	94.51	1.22	88.25
385			12.60	101	87.30	1.12	113.45
770			11.77	96	93.66	1.14	114.29
1540			11.95	94	93.66	1.12	110.85
Positive control EMS
Concentration (µg/mL)			SG	%RSG	%V	RTG	MF§
400			9.47	74	65.31	0.62	1030.03

GEF =126, therefore MF threshold for a positive response = 254.30

4-Hour test group (+S9)

Concentration (µg/mL)			SG	%RSG	%V	RTG	MF§
0			10.82	100	73.67	1.00	145.30
48.13			9.59	88	78.43	0.93	157.37
96.25			11.32	97	80.34	1.05	137.25
192.5			9.92	92	79.06	0.98	137.43
385			10.06	91	83.01	1.03	138.74
770			10.66	96	74.24	0.96	157.34
1540			10.52	88	82.33	0.99	128.05
Positive control CP
Concentration (µg/mL)			SG	%RSG	%V	RTG	MF§
1.5			8.24	73	48.70	0.48	1065.80

GEF =126, therefore MF threshold for a positive response = 271.30

24-Hour test group (-S9)

Concentration (µg/mL)			SG	%RSG	%V	RTG	MF§
0			63.22	100	90.38	1.00	158.20
48.13			67.90	109	97.17	1.15	123.61
96.25			75.58	124	91.18	1.21	157.76
192.5			61.38	96	89.59	0.96	160.56
385			55.50	85	88.81	0.86	163.92
770			69.24	110	85.11	1.03	169.01
1540			65.84	107	88.81	1.02	150.35
Positive control EMS
Concentration (µg/mL)			SG	%RSG	%V	RTG	MF§
150			39.70	49	56.92	0.39	1482.67

GEF =126, therefore MF threshold for a positive response = 284.20

A summary of mutation frequencies for all test groups is displayed in the following tables:

4-Hour test group (-S9)

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		160	768	712	768	48.3	684	768	73.8	0.40
48.13		77	384	359	384	41.9	340	384	75.7	0.36
96.25		68	384	356	384	43.7	341	384	68.6	0.39
192.5		58	384	370	384	19.6	339	384	65.9	0.24
385		67	384	361	384	35.4	338	384	73.1	0.33
770		59	384	355	384	41.9	339	384	66.5	0.39
1540		59	384	362	384	31.5	334	384	74.5	0.31
400 EMS		104	384	252	384	322.5	232	384	385.8	0.46

4-Hour test group (+S9)

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		176	768	703	768	60.0	685	768	77.6	0.44
48.13		80	384	351	384	57.3	333	384	90.8	0.39
96.25		77	384	359	384	41.9	333	384	88.7	0.33
192.5		79	384	352	384	55.0	341	384	75.1	0.43
385		73	384	356	384	45.6	333	384	85.8	0.35
770		87	384	348	384	66.3	340	384	82.0	0.45
1540		74	384	358	384	42.6	337	384	79.3	0.36
1.5 CP		145	384	227	384	539.8	293	384	277.7	0.63

24-Hour test group (-S9)

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		126	768	695	768	55.3	650	768	92.3	0.38
48.13		55	384	356	384	39.0	330	384	78.0	0.34
96.25		62	384	349	384	52.4	323	384	94.9	0.36
192.5		64	384	358	384	39.1	314	384	112.3	0.27
385		65	384	353	384	47.4	318	384	106.2	0.32
770		70	384	352	384	51.1	320	384	107.1	0.33
1540		65	384	347	384	57.0	331	384	83.6	0.41
150 EMS		123	384	244	384	398.3	211	384	526.0	0.45

KEY TO TABLES 1 TO 10

$       =       Cell counts (x10^5 cells/mL).  Set up on previous day to 2 x 10^5 cells/mL unless otherwise stated in parenthesis.

%RSG       =       Relative Suspension Growth

RTG       =       Relative Total Growth

%V       =       Viability Day 2

A,B       =       Replicate cultures

CP       =       Cyclophosphamide

EMS       =       Ethylmethanesulphonate

MF§       =       5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Nv       =       Number of wells scored, viability plates

Yv       =       Number of wells without colonies, viability plates

Ym       =       Number of wells without colonies, mutation plates

Nm       =       Number of wells scored, mutation plates

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

 Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group	Concentration of BHES (µg/mL) plated for mutant frequency
4-hour without S9	48.13, 96.25, 192.5, 385, 770, 1540
4-hour with S9 (2%)
24-hour without S9

 Results……..

With no evidence of precipitate or test item-induced toxicity, the maximum dose level used in the Mutagenicity Test was the 10mM limit dose of 1540 µg/mL. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on negative results in in-vitro studies conducted on the substance, the substance does not need to be classified for germ cell mutagenicity.