Sodium D-glycero-D-gulo-heptonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 02 March 2012 and 07 March 2013.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: 40CFR799.9537

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium glucoheptonate

EC Number:

250-480-2

EC Name:

Sodium glucoheptonate

Cas Number:

31138-65-5

IUPAC Name:

sodium 2,3,4,5,6,7-hexahydroxyheptanoate (non-preferred name)

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Sodium Glucoheptonate
- Physical state: Dark brown liquid
- Analytical purity: 50.5%
- Lot/batch No.: 921000100
- Expiration date of the lot/batch: 09 February 2015
- Storage condition of test material: Room temperature, in the dark
- Other: The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Method

Species / strain

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells 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, at 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test
11.09,22.19,44.38,88.75,177.5, 355, 710, 1420 and 2840 µg/ml.

Experiment 1
4(20)-hour without S9: 0*, 88.75, 177.5, 355, 710*, 1420* and 2840* µg/ml.
4(20)-hour with S9 (2%): 0*,88.75, 177.5, 355, 710*, 1420* and 2840* µg/ml.

Experiment 2
24-hour without S9: 0*, 88.75, 177.5, 355, 710*, 1420* and 2840* µg/ml.
4(20)-hour with S9 (1 %): 0*, 88.75, 177.5, 355, 710*, 1420* and 2840* µg/ml.

* Dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Eagle's minimal essential medium with HEPES buffer (MEM).

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Eagle's minimal essential medium with HEPES buffer (MEM)

Negative solvent / vehicle controls:

yes

Remarks:

Eagle's minimal essential medium with HEPES buffer (MEM)

True negative controls:

not specified

Positive controls:

yes

Remarks:

0.4 and 0.2 µ/ml (Exp. 1 & 2. respectively) (Dissolved in Minimal Essential Medium.)

Positive control substance:

mitomycin C

Remarks:

Without S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Eagle's minimal essential medium with HEPES buffer (MEM)

Negative solvent / vehicle controls:

yes

Remarks:

Eagle's minimal essential medium with HEPES buffer (MEM)

True negative controls:

not specified

Positive controls:

yes

Remarks:

5 µg/ml (both experiments) (Dissolved in dimethyl sulphoxide.)

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix.

Details on test system and experimental conditions:

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

METHOD OF APPLICATION:
The test item was accurately weighed, dissolved in MEM and serial dilutions prepared. The molecular weight of the hydrated form of the test item was calculated to be approximately 284, therefore the maximum recommended dose level was considered to be the 10mM concentration, 2840 µg/ml. It was subsequently discovered that when Sodium Glucoheptonate is in solution in water, as in the sample tested, the water of crystallisation is lost and the molecular weight is therefore reduced to 248.163. Therefore the maximum recommended 10 mM dose concentration should have been 2482 µg/ml. Therefore the test item was consequently over tested at 11.44 mM, beyond the maximum recommended dose level using the molecular weight of the hydrated form of Sodium Glucoheptonate. With no evidence of a genotoxic response in any of the exposure groups it was considered that modest over testing of the test item had no impact on the integrity of the study.
The test item had a water content of 49.5% and an adjustment was made for this when the test item formulations were prepared.
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).
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.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Culture Conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

8.05-9.15 ml MEM, 10% (FBS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.65-0.75 ml heparinised whole blood

With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at 37°C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 ml of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9-mix (i.e. 2% final concentration of S9 in standard cofactors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
In Experiment 2, 1 ml of 10% S9-mix (i.e. 1% final concentration of S9 in standard cofactors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml.
After 4 hours at 37°C, 5% CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37°C in 50/0 CO2 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at 37°C with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 ml of the appropriate vehicle control, test item solution or 0.1 ml of positive control solution. The total volume for each culture was a nominal 10 ml.
After 4 hours at 37°C, 5% CO2 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.0 ml. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 ml of vehicle control, test item dose solution or 0.1 ml of positive control solution. The nominal final volume of each culture was 10 ml. The cultures were then incubated at 37°C, 5% CO2 in humidified air for 24 hours.

The Preliminary Toxicity Test was performed using the methodology of Experiment 1 and Experiment 2 in the absence of 89 except that positive controls were not included.

DURATION
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. 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.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1
i) 4-hour exposure to the test item without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.

Experiment 2
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCI. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCI cell suspension into fresh methanol/glacial acetic acid (3: 1 v/v). The fixative was changed at least three times and the cells stored at approximately 4°C to ensure complete fixation.

Preparation of Metaphase Spreads
The lymphocytes were resuspended in several ml of fresh fixative before centrifugation and resuspension 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 labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

NUMBER OF CELLS EVALUATED:
Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

Evaluation criteria:

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

Statistics:

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

Results and discussion

Test results

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

For tables, please see attachment "Tables 1 -7" below.

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 11.09, 22.19, 44.38, 88.75, 177.5, 355, 710, 1420 and 2840 µg/ml. The maximum dose tested was initially considered to be the maximum recommended 10 mM concentration, calculated from the molecular weight of the dihydrate form of Sodium Glucoheptonate. However, the maximum recommended dose level should have been 2482 µg/ml, equivalent to the 10mM concentration calculated from the molecular weight of the dehydrated form and therefore the maximum recommended dose was exceeded. Sodium Glucoheptonate was tested to a concentration of approximately 11.44 mM.

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 2840 µg/ml in all three exposure groups. The mitotic index data are presented in Table 1. The test item induced some modest evidence of toxicity only in the 24-hour continuous exposure group. The maximum dose level selected for the main experiments was the maximum dose level tested, 2840 µg/ml.

Chromosome Aberration Test - Experiment 1

The qualitative assessment of the slides determined that the toxicity was only marginally greater than that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test item, 2840 µg/ml in the absence and presence of metabolic activation (S9). No precipitate of the test item was observed at the end of exposure in either exposure group.

The mitotic index data are given in Table 2. They confirm the qualitative observations in that only modest inhibition of mitotic index was observed, and that 23% and 18% mitotic inhibition was achieved at 2840 µg/ml in the absence and presence of S9 respectively. Therefore, the maximum dose level selected for metaphase analysis was the maximum dose level tested (2840 µg/ml) for both exposure groups.

The chromosome aberration data are given in Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. 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 not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Tables 4 and 5. 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.

Chromosome Aberration Test - Experiment 2

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 2840 µg/ml in both the absence and presence of S9. No precipitate of the test item was observed at the end of exposure, in either exposure group.

The mitotic index data are given in Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in the absence of S9, and that 38% mitotic inhibition was achieved at 2840 µg/ml. In the presence of S9 no inhibition of mitotic index was observed.

The maximum dose level selected for metaphase analysis was the same as Experiment 1, and was 2840 µg/ml.

The chromosome aberration data are given in Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. 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 not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation. The maximum dose level of the test item exceeded the maximum recommended dose level, however in the absence of any increases in the frequency of cells with aberrations it is considered that this difference is acceptable.

The polyploid cell frequency data are given in Tables 6 and 7. 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.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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 metabolising system in either of two separate experiments. 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 (Scott et al, 1990). The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test", Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, EPA OPPT8 870.5375 and 40CFR799.9537. The study design was also compatible with the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

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

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final Concentration of test item (µg/ml)
4(20)-hour without S9	88.75, 177.5, 355, 710, 1420, 2840
4(20)-hour with S9 (2%)	88.75, 177.5, 355, 710, 1420, 2840
24-hour without S9	88.75, 177.5, 355, 710, 1420, 2840
4(20)-hour with S9 (1%)	88.75, 177.5, 355, 710, 1420, 2840

Results. All vehicle (MEM) 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 indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item was only moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range which exceeded the maximum recommended dose level. The maximum dose level tested was based on the molecular weight of the hydrated form of Sodium Glucoheptonate. It was subsequently discovered that when Sodium Glucoheptonate is in solution, as in the sample tested, the water of crystallisation is lost and the molecular weight is therefore reduced. The test item was consequently over tested at 11.44 mM which was beyond the maximum recommended dose level (10 mM) using the molecular weight of the dihydrate form of Sodium Glucoheptonate. However, since there was no response from the test item at any dose level in any of the exposure groups this was considered to be acceptable and have no impact on the study integrity.

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