Sodium (S)-lactate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells

Author:

Diaz, D., Scott, A., Carmichael, P., Shi, W., and Costales, C.

Year:

2007

Bibliographic source:

Mutation Research, Vol.630: 1–13

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The evaluation of an automated in vitro micronucleus assay using CHO-K1 cells in 96-well plates was conducted.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

NaCl

IUPAC Name:

sodium chloride

Specific details on test material used for the study:

- Name of test material as cited in study report: Sodium chloride
- Molecular formula (if other than submission substance): NaCl

Method

Target gene:

none

Species / strain

Species / strain / cell type:

other: CHO-K1 cells

Details on mammalian cell type (if applicable):

- Type and identity of media: F12-K Nutrient Mixture (Kaighn’s Modification) (Invitrogen, Carlsbad, CA) containing 10% foetal bovine serum (Invitrogen), 2 mM l-glutamine (Invitrogen), 10 units/mL penicillin/100 g/mL streptomycin.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate

Test concentrations with justification for top dose:

Maximum concentration tested was 200 µM both in the presence and absence of S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in a 96-well plate

DURATION:
- For the S9 treatment, cells were treated with compounds for 3 h, after which the medium was removed, the cells were washed once with warm medium, and fresh medium was added for 18–20 h.
- For the −S9 treatment, cells were treated with compounds continuously for 22–24 h.

SPINDLE INHIBITOR (cytogenetic assays): 6 g/mL of cytochalasin B (Cellomics) was added to the cells for a period of 22–24 h.

STAIN (for cytogenetic assays): Hoechst dye in wash buffer (Cellomics)

NUMBER OF CELLS EVALUATED: A total of 2000 cells per concentration were scored (1000 cells per well); fewer than 2000 cells where scored for wells with high levels of cytotoxicity.

DETERMINATION OF CYTOTOXICITY
Maximum % of cytotoxicity was 31.1 µM with S9 and no cytotoxicity was observed without S9.
For cytotoxicity assessment, a modified version of the cytotoxicity block proliferation index (CBPI) was used. This method takes advantage of the fact that cytotoxicity very often induces cell cycle arrest, which is reflected in a decreased ratio of bi-nucleated to mononucleated cells when using cytochalasin B.
The percentage of cytotoxicity was defined as: 100−100{(CBPIt−1)/(CBPIc−1)}, where CBPI = (number of mononucleated cells + 2×number of binucleated cells)/total number of cells; CBPIt = CBPI of treated cells; CBPIc = CBPI of control cells.

Evaluation criteria:

The automated micronucleus scoring had a sensitivity of 88% and an specificity of 100%, with a predictive value positive of 100% and a predictive value negative of 76% relative to manual scoring methods. A positive result was defined as a statistically significant (t-test, p <0.05) increase in the percentage of micronucleated cells compared to the corresponding control values and a ≥3-fold increase in the percentage of micronucleated cells compared to control values.
A “weak positive” result was defined as a statistically significant (t-test, p < 0.05) increase in the percentage of micronucleated cells compared to the corresponding control values and a ≥2-fold and <3-fold increase in the percentage of micronucleated cells compared to control values.
The assay predictivity terms used throughout the manuscript are defined as follows:
Sensitivity: Proportion of genotoxic compounds (as described in the literature) that tested positive in the automated in vitro micronucleus assay.
Specificity: Proportion of non-genotoxic compounds (as described in the literature) that tested negative in the automated in vitro micronucleus
assay.
Predictive value positive: Proportion of compounds that tested positive in the automated in vitro micronucleus assay and that are truly genotoxic (as described in the literature).
Predictive value negative: Proportion of compounds that tested negative in the automated in vitro micronucleus assay and that are truly non-genotoxic (as described in the literature).

Results and discussion

Test results

Species / strain:

other: CHO-K1 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Maximum % of cytotoxicity was 31.1 µM with S9 and no cytotoxicity was observed without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results, Sodium chloride was negative in an automated in vitro micronucleus assay in CHO-K1 cells.

Executive summary:

Sodium chloride was evaluated in an automated in vitro micronucleus assay using CHO-K1 cells in 96-well plates. For the S9 treatment, cells were treated with Sodium chloride for 3 h, after which the medium was removed, the cells were washed once with warm medium, and fresh medium was added for 18–20 h. For the -S9 treatment, cells were treated with compounds continuously for 22–24 h. At the end of the treatment period, the medium was removed, the cells were washed once with warm medium, and fresh medium containing 6 µg/mL of cytochalasin B was added to the cells for a period of 22–24 h. At the end of the incubation period, the medium was removed, the cells were washed once with warm media, and they were fixed by adding 100 µg/L of a warm solution containing 3.7% formaldehyde and 1 µM of Hoechst dye in wash buffer. The cells were incubated in this solution for 20 min at room temperature, after which they were washed twice with 100 µL washing buffer. Finally, 200 µL washing buffer were added to the cells, the plates were sealed with a plastic cover and were either scanned immediately or stored at 4 °C protected from the light. Plates were stored for no longer than 3 days in order to retain the integrity of the cell dye. Stored plates were allowed to warm-up at room temperature for 30 min before scanning.

Based on the results, Sodium chloride was negative in an automated in vitro micronucleus assay in CHO-K1 cells both in the presence and absence of S9.