Sodium N-(hydroxymethyl)glycinate

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study according to an internationally accepted guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium N-(hydroxymethyl)glycinate

EC Number:

274-357-8

EC Name:

Sodium N-(hydroxymethyl)glycinate

Cas Number:

70161-44-3

Molecular formula:

C3H7NO3.Na

IUPAC Name:

sodium N-(hydroxymethyl)glycinate

Test material form:

other: 50% solution in water

Details on test material:

- Name of test material (as cited in study report): Sodium Hydroxymethylglycinate- Received on 11 April 2000- Physical state: clear colourless liquid (solution)- Analytical purity: 51.06%- Lot/batch No.: 04000023467- Storage condition of test material: at room temperature in the dark

Method

Test concentrations with justification for top dose:

28.62, 35.78, 44.72, 55.90, 69.87, 87.34, 109.2, 136.5, 170.6, 213.2, 266.5, 333.2, 416.5, 520.6, 650.8, 813.4, 1017, 1271 µg/mL

Vehicle / solvent:

Test article stock solutions were prepared by dissolving Sodium Hydroxymethylglycinate in purified water to give 12.71 mg/mL. The stock solutions were membrane filter-sterilised (Gelman Acrodisc 32, pore size, 0.2 µm) and subsequent dilutions made using sterile purified water. The test article solutions were protected from light and used within 1.5 hours of initial formulation.

Controls

Untreated negative controls:

yes

Remarks:

Sterile purified water was added to cultures designated as negative controls.

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

The positive control chemicals were dissolved in sterile anhydrous analytical grade dimethyl sulphoxide immediately prior to use.

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumMETABOLIC ACTIVATION SYSTEM:- mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA. - Glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), 150 mM KCl and rat liver S-9 were mixed in the ratio 1 : 1 : 1 :2. An aliquot of the resulting S-9 mix was added to each cell culture designated for treatment in the presence of S-9 to achieve the required final concentration in a total of 10 mL. The final concentration of liver homogenate in the test system was 2%. Cultures treated in the absence ofS-9 received an equal volume of 150 mM KCL BLOOD CULTURES:- Blood from three healthy, non-smoking male volunteers was used for this study- No donor was suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. - An appropriate volume of whole blood was drawn from the peripheral circulation on the same day as culture initiation. Blood was stored refrigerated and pooled prior to use. - Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL heparinised blood into 8.1 mL Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL gentamycin. Phytohaemagglutinin (PHA, reagent grade) was included at a concentration of approximately 2% of culture volume to stimulate the lymphocytes to divide. - Blood cultures were incubated for approximately 48 hours at 37°C and rocked continuously. DURATION- Cultures received pulse treatments (both in the absence and presence of S-9) for 3 hours only.- Cultures were incubated for a further 17 hours before harvesting. HARVESTING:- Approximately 2 hours prior to harvest, colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase. - At the defined sampling time cultures were centrifuged at approximately 300g for 10 minutes- After removal of the supernatant, cells were resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl and incubated at 37°C for 15 minutes to allow cell swelling to occur. - Cells were then fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was removed by repeated centrifuging and resuspension.PREPARATION OF METAPHASE SPREADS:- Lymphocytes were kept in fixative in the refrigerator - Cells were pelleted and resuspended in a minimal amount of fresh fixative (if required) so as to give a milky suspension. - Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides labelled with the appropriate study details. Slides were flamed, as necessary, to improve metaphase spreading. - After the slides had dried, the cells were stained for 5 minutes in 4% (v/v) filtered Giemsa stain in Gurr's pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.SCORING:- Slides were examined, uncoded, for mitotic index (MI) or percentage of cells in mitosis. Slides from enough dose levels from each treatment regime were scored to determine if chemically induced mitotic inhibition had occurred. This is defined as a clear decrease in mitotic index compared with negative controls (based on at least 1000 cells counted), preferably dose-related. - Slides from the selected treatments and from solvent and positive controls were coded using randomly generated letters by a person not connected with the scoring of the slides. Labels bearing only the study reference number, experiment number, the sex of the donor and the code were used to cover treatment details on the slides. Only cells with 44-46 chromosomes were considered acceptable for analysis of structural aberrations.- Any cell with more than 46 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately.

Evaluation criteria:

- a gap was defined as a discontinuity less than the width of the chromatid and no evidence of displacement of the fragment and a deletion is defined as a discontinuity greater than the width of the chromatid and/or evidence of displacement of the fragment.- The aberrant cells in each culture were categorised as follows: 1. cells with structural aberrations including gaps 2. cells with structural aberrations excluding gaps 3. polyploid, endoreduplicated or hyperdiploid cells.The totals for category 2 in negative control cultures were compared with the current laboratory negative control (norm.al) ranges to determine whether the assay was acceptable or not. The proportion of cells in category 2 in test article treated cultures were also compared with normal ranges. - A test article is considered as positive in this assay if: 1. the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicate cultures, and 2. a statistically significant increase in the proportion of cells with structural aberrations ( excluding gaps) occurs at these doses.- Increased incidence of cells with gaps or increased proportions of cells with structural aberrations not exceeding the normal range, or occurring only at very high or very toxic concentrations are likely to be concluded as "equivocal". Full assessment of the biological importance of such increases is likely only to be possible with reference to data from other test systems. - Evidence of a dose-related effect is considered useful but not essential in the evaluation of a positive result. Cells with exchange aberrations or cells with greater than one structural aberration occur very infrequently in negative control cultures. Their appearance is therefore considered to be of particular biological significance.

Statistics:

The statistical significance of any data set was only to be taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentration exceeded the normal range. Under this condition, the statistical method used would be Fisher's exact test. Probability values of p <=0.05 were to be accepted as significant. The proportions of cells in categories 1 and 3 were also examined in relation to historical negative control (normal) ranges and statistical analysis by Fisher's exact test may be used.The proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (12). Probability values of p <=0.05 were to be accepted as significant.

Results and discussion

Test results

Species / strain:

other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: human peripheral blood lymphocytes

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):positive in the presence of S-9: frequencies of structural aberrations significantly elevated for all concentrations analysed as compared to the controls; in the absence of S-9 significantly increased frequencies at the intermediate and high dose.It is concluded that Sodium Hydroxymethylglycinate induced chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).

Executive summary:

Treatments were in the absence and presence of S-9 for 3 hours followed by a 17 hour recovery period prior to harvest (3+ 17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of Sodium Hydroxymethylglycinate on mitotic index. Chromosome aberrations were analysed at three dose levels (see below). The highest concentrations chosen for analysis, 87.34 µg/mL in the absence of S-9 and 170.6 µg/mL in the presence of S-9, induced approximately 59% and 66% mitotic inhibition (reduction in mitotic index) in the absence and presence ofS-9 respectively.

Treatment of cultures with Sodium Hydroxymethylglycinate in the presence of S-9 resulted in frequencies of cells with structural aberrations that were significantly elevated as compared to the concurrent vehicle control for all concentrations analysed. The aberrant cell frequency of all Sodium Hydroxymethylglycinate treated cultures exceeded historical negative control (normal) ranges. Treatment of cultures with Sodium Hydroxymethylglycinate in the absence of S-9 resulted in significantly increased frequencies of cells with structural aberrations at the intermediate and high doses analysed (44.72 and 87.34 µg/mL). However, it was only at the highest concentration analysed (87.34 µg/mL) where large numbers of aberrant cells ( exceeding normal ranges) were observed in both replicate cultures.

It is concluded that Sodium Hydroxymethylglycinate induced chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).