Methyl α-D-mannopyranoside

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-08 to 2021-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Methyl α-D-mannopyranoside

EC Number:

210-502-3

EC Name:

Methyl α-D-mannopyranoside

Cas Number:

617-04-9

Molecular formula:

C7H14O6

IUPAC Name:

(2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-methoxyoxane-3,4,5-triol

Test material form:

solid

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line (from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line.
- Normal cell cycle time (negative control): see below

For cell lines:
- Absence of Mycoplasma contamination: Before freezing each batch is screened for mycoplasm contamination.
- Methods for maintenance in cell culture: Thawed stock cultures are propagated at 37 °C in 175 cm2 plastic flasks. About 5 x 105 cells per flask are seeded in 30 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium is supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells are sub-cultured twice a week. All incubations are done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
- Cell cycle length, doubling time or proliferation index: doubling time of V79 cells in stock cultures: approximately 13 hours, determined on May 06, 2011
- Modal number of chromosomes: 22 +/- 2
- Periodically checked for karyotype stability: yes, before freezing

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Please refer to “Methods for maintenance in cell culture” above.

Cytokinesis block (if used):

Cytochalasin B (1.5 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9, prepared in study laboratory
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration of S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures
- quality controls of S9: Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

With regard to the molecular weight of the test item, 1950 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 12.7 to 1950 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment I. The experimental part without S9 mix was repeated in Experiment II since the positive control was invalid (too strong cytotoxicity). The experimental part with S9 mix was repeated in Experiment II as confirmatory experiment since a statistically significant increase and dose-dependency were observed in Experiment I. 1950 µg/mL were chosen as top treatment concentration for Experiment II for both experimental parts (4 hours with and without S9 mix). The continuous treatment without S9 mix was performed in Experiment III with the same top dose (1950 µg/mL).

Vehicle / solvent:

- Solvent used: deionised water

- Justification for choice of solvent: The test item was sufficiently soluble in the solvent.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium did not exceed 10 %.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5.0 – 6.0 x 10^5 cells in 25 cm2 plastic flasks
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment 1: 4 h; Experiment 2: 24 h
- Harvest time after the end of treatment (sampling/recovery times): Experiment 1: 20 h; Experiment 2: none

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinesis block: Cytochalasin B (1.5 μg/mL), Experiment 1: 20 h; Experiment 2: 24 h
- Methods of slide preparation and staining technique used including the stain used: Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvest and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37 °C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) were added to the hypotonic solution and the cells were resuspended carefully. After removal of the supernatant after centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip.
- Number of cells spread and analysed per concentration: duplicate cultures; 500 cells per culture were scored on coded slides for the determination of the CBPI. At least 1000 binucleate cells were scored per culture for micronuclei on coded slides.
- Criteria for scoring micronucleated cells: The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): not applicable


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index

Rationale for test conditions:

The highest treatment concentration in this study, 1950 µg/mL was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval)

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition.
− The results are outside the range of the laboratory historical solvent control data (95 % confidence interval)

Statistics:

Statistical significance will be confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis will be conducted for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.
A linear regression will be performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance will be considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No relevant influence on pH was observed.
- Data on osmolality: No relevant influence on osmolarity was observed.
- Precipitation and time of the determination: No precipitation of the test item in the culture medium was observed at the end of treatment.
- Definition of acceptable cells for analysis: The micronuclei were counted in cells showing a clearly visible cytoplasm area.

STUDY RESULTS
Either Griseofulvin (7.0 µg/mL), MMC (0.2 µg/mL) or CPA (3.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

- Statistical analysis:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control. A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both, biological and statistical significance was considered together.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Cytotoxicity is characterized by the percentages of reduction in the Cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture. In Experiments I, II and III in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

- Genotoxicity results
In Experiment I in the presence of S9 mix, the value of 1.15% micronucleated cells after treatment with the highest applied concentration is statistically significantly increased in comparison to the solvent control but clearly within the 95% control limit of the historical control data (0.01 – 1.99% micronucleated cells). Dose dependency was observed by using a trend test. In the confirmatory Experiment II in the presence of S9 mix none of these findings could be confirmed. Therefore, the findings in Experiment I can be considered as biologically irrelevant. In Experiment II and III in the absence of S9 mix, no relevant increases in micronucleated cells could be observed.

HISTORICAL CONTROL DATA
The historical control data were generated in accordance with the OECD Guideline 487. For the solvent controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) was applied for the evaluation of acceptability and interpretation of the data). For the positive controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The min max range of the positive controls was applied for the evaluation of acceptability

Any other information on results incl. tables

Summary of results

Exp.	interval	concentration	Proliferation index CBPI	Cytostatsis in %*	Micronucleated cells in %**	95 % ctrl limit
Exposure period 4 hrs without S9 mix
II	24 h	Solvent control ^1	1.93		0.8	0-2.45
		Positive control ^2	1.43	53.7	13.20^s
		637	1.92	0.9	0.65
		1114	1.92	1.2	0.65
		1950	1.90	3.2	0.85
Trend test: p-value 0.740
Exposure period 24 hrs without S9 mix
III	24 h	Solvent control ^1	2.21		1.10	0-1.7
		Positive control ^3	2.30	n.c.	10.90^s
		637	2.07	11.8	0.6
		1114	2.11	8.1	0.6
		1950	2.07	11.2	0.8
Trend test: p-value 0.740
Exposure period 4 hrs with S9 mix
I	24 h	Solvent control ^1#	1.96		0.65	0.01-1.99
		Positive control^4	1.54	43.1	6.20^s
		637^#	1.92	3.8	0.7
		1114^#	1.99	n.c.	0.83
		1950^#	1.96	n.c.	1.15^s
Trend test: p-value 0.040^T
II	24 h	Solvent control ^1	1.87		1.25	0.01-1.99
		Positive control^4	1.34	60.5	22.20
		637	1.87	0.1	0.8
		1114	1.87	n.c.	0.75
		1950	1.85	1.6	0.9
Trend test: p-value 0.418

 

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

T Trend analysis via linear regression is significant (p ˂ 0.05)

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1 Deion. water 10.0 % (v/v)

2 MMC 0.2 µg/mL 3

 Griseofulvin 7.0 µg/mL

4 CPA 3.0 µg/m

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.

Executive summary:

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in three independent experiments. Experiment I and II were conducted with and without metabolic activation (S9-mix) with exposure periods of 4 and 24 hours. Experiment III was conducted with out metabolic activation with an exposure period of 24 hours. In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1950 µg/mL of the test item) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. In Experiments I, II and III in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix, the value of 1.15% micronucleated cells after treatment with the highest applied concentration is statistically significantly increased in comparison to the solvent control but clearly within the 95% control limit of the historical control data (0.01 – 1.99% micronucleated cells). Dose dependency was observed using a trend test. In the confirmatory Experiment II in the presence of S9 mix none of these findings could be confirmed. Therefore, the findings in Experiment I can be considered as biologically irrelevant. In Experiment II and III in the absence of S9 mix, no relevant increases in micronucleated cells could be observed. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. The test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.