Reaction product of D-Glucopyranoside, methyl; esterified with oleic acid, methyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro reverse mutation assay in bacteria (Ames test), OECD 471: Result: negative

in vitro Chromosome Abberation, OECD 473, Result: negative

in vitro TK +/- Mouse Lymphoma Assay, OECD 476: Result. negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-16 - 2015-06-23

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

other: In Vitro Mammalian Chromosome Aberration Test

Specific details on test material used for the study:

The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 μg/mL. However, due to the use of acetone as the solvent, which can be used at a maximum dose concentration of 0.5%, the maximum achievable dose level was 2500 μg/mL.
Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions prepared.
The solubility of the test item was investigated m the Harlan Laboratories Ltd, Mouse Lymphoma Assay, Study number 41403673. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991, Genotoxicity under Extreme Culture Conditions. A report from ICPEMC task Group 9. Mutation Res., 257, 147-204).
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9)

Test concentrations with justification for top dose:

The dose levels selected for the main experiment were based on the results of the preliminary toxicity test and were limited to include the lowest precipitating dose level. The dose levels selected for the main experiment were as follows: 0, 5, 10, 20, 40, 80, 160 (μg/mL).
The dose range for the Preliminary Toxicity Test was 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL. The maximum dose was the maximum achievable dose level, due to formulation difficulties and the necessity of using acetone as the solvent.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 156.25 μg/mL and 78.13 μg/mL in the 4(20)-hour exposure groups in the absence and presence of metabolic activation, respectively and at and above 39.06 μg/mL in the continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2500 μg/mL in all three exposure groups. The test item induced some evidence of toxicity in the 24-hour exposure group only. The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 160 μg/mL for the 4-hour exposure groups and for the continuous exposure group.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range oftest item used was 0, 5, 10, 20, 40, 80 and 160 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range oftest item used was 0, 5, 10, 20, 40, 80 and 160 μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range oftest item used was 0, 5, 10, 20, 40, 80 and 160 μg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Evaluation criteria:

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.

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 induced chromosome 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 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 induced 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 frequency of cells with aberrations compared to the concurrent negative control.
3. The increase observed is considered dose-related

When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploid and endoreduplications.

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.

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.

Key result

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See Remarks

Remarks:

Some toxicity was demonstrated throughout the dose range of the 24-hour continuous exposure group and a 42% reduction in mitotic index was achieved at 160 μg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Some toxicity was demonstrated throughout the dose range of the 24-hour continuous exposure group and a 42% reduction in mitotic index was achieved at 160 μg/mL.
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 up to the maximum dose level of test item, 160 μg/mL in all three exposure groups. Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 80 μg/mL, in the 4(20)-hour exposure group in the absence of S9, and at 160 μg/mL, in the 4(20)-hour exposure group in the presence of S9 and in the 24-hour continuous exposure group. They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in the 4(20)-hour exposure groups in the absence or presence of S9. Some toxicity was demonstrated throughout the dose range of the 24-hour continuous exposure group and a 42% reduction in mitotic index was achieved at 160 μg/mL. The maximum dose level selected for metaphase analysis was the lowest precipitating dose level and was 80 μg/mL for the 4(20)-hour exposure group in the absence of S9, and was 160 μg/mL, for the 4(20)-hour exposure group in the presence of S9 and for the 24-hour continuous exposure group.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the three exposure groups.

Table 2: Mitotic Index - Preliminary Toxicity Test

Dose Level (µg/mL)	4-Hour Without S9		4-Hour With S9		24-Hour Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	2.65	100	1.55	100	4.05	100
9.77	-	-	-	-	3.80	94
19.53	-	-	3.45	223	3.30	81
39.06	3.25	123	2.55	165	3.35 P	83
78.13	3.70	140	1.95 P	126	-P	-
156.25	3.95P	149	3.50 P	226	-P	-
312.5	2.45P	92	-P	-	-P	-
625	-P	-	-P	-	2.40 P	59
1250	-P	-	-P	-	-P	-
2500	4.55P	172	2.45P	158	1.35 P	33

- = Not assessed for mitotic index

NM = No metaphases or insufficient metaphases suitable for scoring

P = Precipitate observed at end of exposure period in blood-free cultures

Table 3: Mitotic Index-Main Experiment (4-hour Exposure Groups)

Dose Level (µg/mL)	4-Hour Without S9				4-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	4.10	7.15	5.63	100	4.05	5.15	4.60	100
5	-	-	-	-	-	-	-	-
10	-	-	-	-	-	-	-	-
20	7.05	8.20	7.63	136	-	-	-	-
40	5.40	5.45	5.43	96	4.65	5.35	5.00	109
80	6.15 P	5.05 P	5.60	100	4.60	4.80	4.70	102
160	-P	-P	-	-	5.35 P	4.95 P	5.15	112
MMC0.4	2.35	2.65	2.50	44	NA	NA	NA	NA
CP5	NA	NA	NA	NA	3.70	2.95	3.33	72

MMC = Mitomycin C

CP = Cyclophosphamide

P = Precipitate

NA =Not applicable

- = Not assessed for mitotic index

Table 4: Mitotic Index - Main Experiment (24-hour Exposure Group)

Dose Level (µg/mL)	24-Hour Without S9
	A	B	Mean	% of Control
0	4.20	2.30	3.25	100
5	1.00	3.20	2.10	65
10	3.90	1.20	2.55	78
20	1.50	2.60	2.05	63
40	2.10	1.50	1.80	55
80	2.60	1.70	2.15	66
160	1.35 P	2.40 P	1.88	58
MMC 0.2	0.80	0.90	0.85	26

MMC = Mitomycin C

CP = Cyclophosphamide

P = Precipitate

NA =Not applicable

- = Not assessed for mitotic index

Table 5: Results of Chromosome Aberration Test - Main Experiment -hour Exposure Without Metabolic Activation (S9)

(attached)

Table 6: Results of Chromosome Aberration Test - Main Experiment 4-hour Exposure With Metabolic Activation (2% S9)

(attached)

Table 7: Results of Chromosome Aberration Test - Main Experiment 24-hour Continuous Exposure Without Metabolic Activation (S9)

(attached)

Table 8: Mean Frequency of Polyploid Cells (%) Main Experiment

Dose Level (µg/mL)	Exposure Group
	4-Hour Without S9	4-Hour With S9	24-Hour Without S9
0	0	0	0
20	0	-	0
40	0	0	0
80	0	0	0
160	-	0	0
MMC 0.4	0	NA	NA
MMC 0.2	NA	NA	0
CP2	NA	0	NA

MMC Mitomycin C

CP Cyclophosphamide

NA Not applicable

- Not determined

Conclusions:

The test item 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 metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro Chromosome Aberration test in human lymphocytes following the OECD Guideline 473 and in compliance with GLP, the potential chromosomal mutagenicity of the test item has been assessed.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, the main experiment was performed using three exposure conditions; 4 hours 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, a 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 selected for the main experiment were based on the results of the preliminary toxicity test and were limited to include the lowest precipitating dose level. The dose levels selected for the main experiment were as follows:

Group	Final concentration of test item (μg/mL)
4(20)-hour without S9	0,5, 10, 20, 40, 80, 160
4(20)-hour with S9 (2%)	0,5, 10, 20, 40, 80, 160
24-hour without S9	0,5, 10, 20, 40, 80, 160

All vehicle (acetone) 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did demonstrate some toxicity in the preliminary toxicity test in the 24-hour exposure group only, however, the dose range for the main experiment was limited to include the lowest precipitating dose level, irrespective of toxicity, as directed in the OECD 473 guideline. The test item 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 lowest precipitating dose level. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-27 - 2015-05-27

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:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MITI/MHW guidelines for testing of new chemical substances

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

other: in vitro mutagenicity in mammalian cells

Specific details on test material used for the study:

Following solubility checks performed in-house, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test item was considered to be a complex mixture (UVCB) and the purity of the test item was treated as 100%. As the test item was formulated in acetone and dosed at 0.5%, the maximum achievable dose level was set at 2500 μg/mL. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991,Genotoxicity under Extreme Culture Conditions. A report from ICPEMC task Group 9. Mutation Res., 257, 147-204).

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9)

Test concentrations with justification for top dose:

Experiment 1:
Concentration (μg/mL) plated for mutant frequency (4-hour):
- S9 mix: 78.13, 156.25, 312.5, 468.74, 546.87, 625
+ S9 mix (2%): 19.53, 39.06, 78.13, 156.25, 312.5, 468.74

Experiment 2:
Concentration (μg/mL) plated for mutant frequency (24-hour):
- S9 mix: 39.06, 78.13, 156.25, 312.5, 390.61
Concentration (μg/mL) plated for mutant frequency (4-hour):
+ S9 mix (1%): 78.13, 156.25, 312.5, 390.61, 468.74, 546.87

Justification: The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle, (Acetone) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

Evaluation criteria:

1. The majority of the plates, for either viability (%V) or TFT resistance are analyzable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120%.
3. The total suspension growth of the solvent control following 4 hour treatment, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24 hour treatment the total suspension growth should be in the range of 32 to 180.
4. The in-house vehicle control mutant frequency is in the range of 50 - 170 x 10-6 cells. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10-6 mutant frequency per survivor are not acceptable and will be repeated.
5. Positive control chemicals (EMS and CP) should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control. The positive controls should ideally yield an absolute increase in total MF, that is an increase above spontaneous background MF (an induced MF [IMF]), of at least 300 x 10-6 cells.
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the relative total growth (RTG) and percentage relative suspension growth (%RSG) should be greater than 10 % of the concurrent selective control group.

Statistics:

The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1
There was no evidence of reductions in viability in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. In the presence of metabolic activation two dose levels 546.87 and 625 μg/mL were not plated out for TFT resistance and viability, due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/-locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell. The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency. It should be noted that all mutant frequency values were within the acceptable range for a vehicle control culture. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation the test item was considered to have been adequately tested. A cloudy precipitate of the test item was observed at and above 156.25 which turned greasy/oily at and above 468.74 μg/mL in both the absence and presence of metabolic activation.

Experiment 2
There was evidence of marked toxicity in both the absence and presence of metabolic activation. There was evidence of a modest reduction in viability in the absence of metabolic activation, therefore indicating that residual toxicity had occurred in this exposure group. In the absence of metabolic activation the dose levels at and above 468.74 μg/mL were not plated out for TFT resistance and viability, due to excessive toxicity. In addition 390.61 μg/mL was plated out, but later excluded from statistical analysis due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances.The 24-hour exposure without metabolic activation treatment, demonstrated that the extended time point had no marked effect on the toxicity of the test item.

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/-locus in L5178Y cells.

Executive summary:

An in vitro TK +/- Mouse Lymphoma Assay according to OECD Guideline 476 and EU Method B17 and in compliance with GLP has been conducted with the test item.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle, (Acetone) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9).

In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the 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 out for viability and expression of mutant colonies were as follows:

Experiment 1

Group	Concentration of test item (μg/mL) plated for mutant frequency
4-hour without S9	78.13 156.25 312.5 468.74 546.87 625
4-hour with S9 (2%)	19.53 39.06 78.13 156.25 312.5 468.74

Experiment 2

Group	Concentration of test item (μg/mL) plated for mutant frequency
24-hour without S9	39.06 78.13 156.25 312.5 390.61
4-hour with S9 (2%)	78.13 156.25 312.5 390.61 468.74 546.87

The maximum dose levels used in the mutagenicity test were limited by test item-induced toxicity. Overall precipitate of the test item was observed around 156.25 μg/mL in the absence and presence of metabolic activation. The vehicle controls (acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating 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, either with or without metabolic activation, in either the first or the second experiment.

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/-locus in L5178Y cells.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-17 - 2017-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine or tryptophan locus in the genome

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl formamide
- Justification for choice of solvent/vehicle: In solubility checks performed in-house the test item was noted to be immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in dimethyl formamide at 50 mg/mL. Dimethyl formamide was, therefore, selected as the vehicle. The test item was originally noted as miscible in acetone at 100 mg/mL but the formulation came out of solution and would not reform as a solution.
The test item was accurately weighed and, on the day of each experiment, approximate halflog dilutions prepared in dimethyl formamide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C. No correction was required for test item purity. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10E-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (direct plate incorporation)
DURATION: Exposure duration: 48 hours
NUMBER OF REPLICATIONS: in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

POCEDURE (Experiment 1 and Experiment 2)
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item or solvent vehicle or 0.1 mL of appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: No reduction in the number of revertants/plate was observed in the range-finding study (Experimant 1) with strains TA98, TA1535, TA1537, TA100 and WP2uvrA.

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (Attachment) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced

marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic

activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2 (see Attachment).

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A test item film (creamy in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

Conclusions:

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

Executive summary:

In a reverse gene mutation assay in bacteria (Ames test) the test substance was evaluated in accordance with OECD Guideline 471, EU Method B13/14, US EPA OPPTS 870.5100 and Japans Guidelines for Screening Mutagenicity Testing Of Chemicals and in compliance with GLP.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations.

The dose range was amended, following the results of Experiment 1, and was 15 to 5000 μg/plate. Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A test item film (creamy in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

The tets material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria (Ames test)

In a key study (Thompson, 2017) a reverse gene mutation assay in bacteria (Ames test) has been performed to evaluate the test substance in accordance with OECD Guideline 471, EU Method B13/14, USEPA OPPTS 870.5100 and Japans Guidelines for Screening Mutagenicity Testing Of Chemicals and in compliance with GLP.

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia colistrain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations.

The dose range was amended, following the results of Experiment 1, and was 15 to 5000 μg/plate. Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A test item film (creamy in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

The tets material was considered to be non-mutagenic under the conditions of this test.

A supporting QSAR prediction with QSAR Toolbox 4.0 was performed for D-glucopyranoside methyl 2,6 -dioleate. The method to predict the gene mutation potential was determined to be suitable for the substance. According to Protein binding by OASIS v1.4 (subcategorization) and in vitro mutagenicity (Ames test) alerts by ISS (subcategorization) no alerts have been found. The substance is therefore predicted to be negative for gene mutation in bacteria (Ames test).

Chromosome aberration data (in vitro)

In the key study an in vitro Chromosome Aberration test in human lymphocytes (Bowles, 2015) following the OECD Guideline 473 and in compliance with GLP, the potential chromosomal mutagenicity of the test item has been assessed.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, the main experiment was performed using three exposure conditions; 4 hours 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, a 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 selected for the main experiment were based on the results of the preliminary toxicity test and were limited to include the lowest precipitating dose level. The dose levels selected for the main experiment were as follows:

Group	Final concentration of test item (μg/mL)
4 (20)-hour without S9	0,5, 10, 20, 40, 80, 160
4 (20)-hour with S9 (2%)	0,5, 10, 20, 40, 80, 160
24-hour without S9	0,5, 10, 20, 40, 80, 160

All vehicle (acetone) 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did demonstrate some toxicity in the preliminary toxicity test in the 24-hour exposure group only, however, the dose range for the main experiment was limited to include the lowest precipitating dose level, irrespective of toxicity, as directed in the OECD 473 guideline. The test item 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 lowest precipitating dose level. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Mammalian cell gene mutation data (in vitro)

In the key study an in vitro TK +/- Mouse Lymphoma Assay (Brown, 2015) according to OECD Guideline 476 and EU Method B17 and in compliance with GLP has been conducted with the test item.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle, (Acetone) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9).

In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the 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 out for viability and expression of mutant colonies were as follows:

Experiment 1

Group	Concentration of test item (μg/mL) plated for mutant frequency
4-hour without S9	78.13 56.25 312.5 468.74 546.87 625
4-hour with S9 (2%)	19.53 39.06 78.13 156.25 312.5 468.74

Experiment 2

Group	Concentration of test item (μg/mL) plated for mutant frequency
24-hour without S9	39.06 78.13 156.25 312.5 390.61
4-hour with S9 (2%)	78.13 156.25 312.5 390.61 468.74 546.87

The maximum dose levels used in the mutagenicity test were limited by test item-induced toxicity. Overall precipitate of the test item was observed around 156.25 μg/mL in the absence and presence of metabolic activation. The vehicle controls (acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating 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, either with or without metabolic activation, in either the first or the second experiment.

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/-locus in L5178Y cells.

Justification for classification or non-classification

Based on the all negative results of recent in vitro studies and modelled reverse-bacteria data available for the test item, it is considered to be neither mutagenic nor genotoxic. According to the Regulation (EC) No 1272/2008 (CLP Regulation) the test item does not meet the criteria for classification and will not require labelling as a mutagen.