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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

946-364-2

Molecular formula:

C43H78O8

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- State of aggregation: yellow liquid
-Others: storage at room temperature, in the dark

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.

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.