reaction mass of Calcium chloride and Sodium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data are available on the reaction mass itself. However, several studies are available for the two components (calcium chloride and sodium chloride). Furthermore, calcium chloride is naturally present in cells and the structure does not indicate a genotoxic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: publication

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: sufficient information is available for the interpretation of results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

none

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histedine locus

Species / strain / cell type:

other: Ames reversion test with his - S. typhimurium strains TA1535, TA1537, TA1538, TA98, TA100 and, in part, TA97, and in a DNA-repair test with trp- E. coli strains WP2 (repair-proficient), WP67 (uvrA polA ) and CM871 (uvrA- recAlexA -).

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix, prepared according to Ames et al. (1975), contained 10% liver $9 fractions from Aroclor- treated Sprague-Dawley rats, whose protein concentration had been adjusted to 30 mg/ml.

Test concentrations with justification for top dose:

Each compound was tested with each strain, both with and without S9 mix, at various dilutions (in duplicate or triplicate plates) performed by a
geometric ratio of 2, starting from its solubility or toxicity limit.
Specific test concentrations not mentioned in the report.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

other: not specified in the report but out of 135 chemicals tested few were the historical positive control.

Positive control substance:

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and spot test

NUMBER OF REPLICATIONS: duplicate


Determination OF CYTOTOXICITY:
- Method
other: by back ground lawn

Evaluation criteria:

Criteria for positivity of results included rate of increase of induced versus spontaneous revertants, dose dependence and reproducibility in separate
experiments (De Serres and Shelby, 1979).
The indications concerning the effect of S9 mix on mutagenicity, are intended to specify the metabolic trends of positive compounds in the
presence of liver S9 fractions, as inferred from dose-response data obtained by varying the concentrations either of test compound or of S9
fraction.
In the Ames test the mutagenic potency was expressed by dividing the number of revertants in excess of controls - as determined at the top level
of the linear part of dose-response curves with the most sensitive strain (identified with a C) symbol + by the corresponding amount of compound (in nmoles). Potency data were in the absence of S9 mix for direct-acting mutagens and in the presence of S9 mix for mutagens requiring
metabolic activation or undergoing an increase in activity. For negative compounds, 'less than' figures were caluclated by dividing an arbitrary
value of 100 revertants by the nmoles corresponding to the maximum dose of compound tested.

Statistics:

The plate count means were presented.

Species / strain:

other: Salmonella typhimurium strains TA97, TA98, TA100. TA1535, TA 1537 and, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not specified in the report

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: yes

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: A 2-M concentration of NaCI reduces survival.

It is known also that 2 M NaCI enhances the mutation frequency, somatic recombination andgene conversion in diploid yeast (Magni and Sora, unpublished data; Parker et al., 1986). Therefore,NaCI may induce chromosome breakage as well asmutations in yeast.

none

Conclusions:

Interpretation of results: negative with and without metabolic activation.

Based on the results of the experiment NaCl was negative in the Ames reverse mutation assay.

Executive summary:

All the compounds were assayed in the Ames test with 5 his- S. typhimurium strains (TA1535, TA1537, TA1538, TA98 and TA100), whose genetic features were described by Ames et al. (1975). Salmonella strains were a kind gift from Prof. Bruce N. Ames (Department of Biochemistry, University of California, Berkeley, CA, U.S.A.).

The plate-incorporation test was performed according to the standard procedure described by Ames et al. (1975), with some revisions suggested by Bruce N. Ames (personal communication) and now published by Maron and Ames (1983).

Each compound was tested with each strain, both with and without S9 mix, at various dilutions (in duplicate or triplicate plates) performed by a geometric ratio of 2, starting from its solubility or toxicity limit. Throughout the period of experiments the spontaneous reversion rate of Salmonella tester strains was within the ranges indicated by Ames et al. (1975) and Maron and Ames (1983).

All the compounds were assayed in the Ames test with 5 his- S. typhimurium strains (TA1535, TA1537, TA1538, TA98 and TA100), whose genetic features were described by Ames et al. (1975). Salmonella strains were a kind gift from Prof. Bruce N. Ames (Department of Biochemistry, University of California, Berkeley, CA, U.S.A.).

3 isogenic E. coli strains were used in the DNA-repair test, i.e. WP2 (wild-type, repair-profi164 cient) (Witkin, 1956), WP67 (uvrA-polA ) (Witkin, 1967) and CM871 (uvrA- recA lexA ) (Tweats et al., 1981). In some preliminary assays with the spot test strains WP2uvrA (uurA) (Hill,1958) and TM1080 (polA- lexA- plasmid R391) (Venturini and Monti-Bragadin, 1978) were also used. All these bacteria carry an ochre nonsense mutation blocking an intermediate process in the synthesis of tryptophan (Bridges et al., 1967). E. coli strains were a kind gift from Prof. C. Monti- Bragadin (Institute of Microbiology, University of Trieste, Italy).

The plate-incorporation test was performed according to the standard procedure described by Ames et al. (1975), with some revisions suggested by Bruce N. Ames (personal communication) and now published by Maron and Ames (1983).

Each compound was tested with each strain, both with and without S9 mix, at various dilutions (in duplicate or triplicate plates) performed by a geometric ratio of 2, starting from its solubility or toxicity limit. Throughout the period of experiments the spontaneous reversion rate of Salmonella tester strains was within the ranges indicated by Ames et al. (1975) and Maron and Ames (1983).

Criteria for positivity of results included rate of increase of induced versus spontaneous revertants, dose dependence and reproducibility in separate experiments (De Serres and Shelby, 1979).

The indications concerning the effect of S9 mix on mutagenicity, are intended to specify the metabolic trends of positive compounds in the

presence of liver S9 fractions, as inferred from dose-response data obtained by varying the concentrations either of test compound or of S9

fraction.

In the Ames test the mutagenic potency was expressed by dividing the number of revertants in excess of controls - as determined at the top level

of the linear part of dose-response curves with the most sensitive strain (identified with a C) symbol + by the corresponding amount of compound (in nmoles). Potency data were in the absence of S9 mix for direct-acting mutagens and in the presence of S9 mix for mutagens requiring

metabolic activation or undergoing an increase in activity. For negative compounds, 'less than' figures were caluclated by dividing an arbitrary value of 100 revertants by the nmoles corresponding to the maximum dose of compound tested.

Based on the results of the experiment NaCl was negative in the Ames reverse mutation assay.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

other: publication

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: sufficient information is available for the interpretation of results.

Qualifier:

no guideline available

Principles of method if other than guideline:

none

GLP compliance:

not specified

Type of assay:

other: bacterial DNA-repair test

Target gene:

Trytophane locus

Species / strain / cell type:

other: in a DNA-repair test with trp- E. coli strains WP2 (repair-proficient), WP67 (uvrA polA ) and CM871 (uvrA- recAlexA -).

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix, prepared according to Ames et al. (1975), contained 10% liver $9 fractions from Aroclor- treated Sprague-Dawley rats, whose protein concentration had been adjusted to 30 mg/ml.

Test concentrations with justification for top dose:

Each compound was tested with each strain, both with and without S9 mix, at various dilutions (in duplicate or triplicate plates) performed by a
geometric ratio of 2, starting from its solubility or toxicity limit.
Specific test concentrations not mentioned in the report.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

other: not specified in the report but out of 135 chmicals tested few were the historical positive control.

Positive control substance:

not specified

Details on test system and experimental conditions:

not specified in the report

Evaluation criteria:

A ratio between the MICs in repair-proficient and -deficient strains greater than 2 (this minimum difference was accepted only if confirmed
in 5 experiments) was considered to be significant in this test.
In the liquid DNA-repair micromethod, the genotoxic potency was calculated by relating the differences of MICs in repair-deficient (rep-) and -proficient (rep +) strains to the corresponding nmoles of compound. For negative compounds, according to the above formula, potency is 0 when the MICs are overlapping in repair-deficient and -proficient bacteria. Half of negative compounds in this study had 'greater than' values of MICs, because they were devoid of toxicity even at the highest concentration tested. In these cases, 'less than' figures of potency were calculated by arbitrarily ascribing MICs 2- and 4-fold higher than the maximum concentration tested in repair-deficient and -proficient strains, respectively. In fact, using 2-fold dilutions, these would have been the theoretical minimum levels of toxicity, if compounds could have been tested at higher concentrations per well.

Statistics:

The plate count means were presented.

Species / strain:

other: trp- E. coli strains WP2 (repair-proficient), WP67 (uvrA polA ) and CM871 (uvrA- recAlexA -).

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not specified in the report

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: yes

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: A 2-M concentration of NaCI reduces survival.

It is known also that 2 M NaCI enhances the mutation frequency, somatic recombination andgene conversion in diploid yeast (Magni and Sora, unpublished data; Parker et al., 1986). Therefore,NaCI may induce chromosome breakage as well asmutations in yeast.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

none

Conclusions:

Interpretation of results: negative

Based on the results of the experiment NaCl was negative in the DNA-repair test.

Executive summary:

Compounds of various chemical classes were comparatively assayed in the DNA-repair test with trp- E. coli strains WP2 (repair-proficient), WP67 (uvrA polA ) and CM871 (uvrA- recAlexA -). A liquid micromethod procedure for the assessment of the minimal inhibitory concentration (MIC)

of test compounds, was used.

This procedure is similar to those described by Kada et al. (1980) in the rec-assay system with B. subtilis and by McCarroll et al. (1981a) with various strains of E. coil. All the reagents needed were dispensed and diluted with the aid of a multichannel

pipette in disposable, sterile Microtiter plates containing 8 rows of 12 350 micro liter wells. 50 micro liter of liquid compounds or of water solutions were distributed in the first wells of six 8-well rows. DMSO-soluble compounds were distributed in 10 micro liter amounts, in order

to avoid toxic effects due to this solvent, and diluted 1 : 5 by adding 40 micro liter of nutrient broth No. 2 (Oxoid Ltd., Basingstoke, Hants, Great Britain).

The initial concentration of each compound was governed either by its solubility or by its toxicity

for bacteria, as inferred from preliminary assays. Starting from this concentration, compounds were further diluted in nutrient broth for a total of 8 2-fold dilutions (50 micro lilter/well, 6 wells/dilution). In order to save reagents, in confirmatory assays the exact inhibitory concentration of compounds could be generally centered by preparing only 4 instead of 8 dilutions, thus assaying 4 instead of 2 compounds per Microtiter plate.

Of the above six 8-well rows per compound, 3 were filled with 50 micro liter 0.2 M phosphate-buffered

saline (PBS), and 3 with 50 btl S9 mix. Finally, each row of wells was filled with 100 micro liter of one of the 3 bacterial tester strains. Bacterial inocula had been obtained from 16-h cultures in nutrient broth, supplemented with 10% DMSO, distributed into

sterile tubes (0.5 ml/tube) and stored at -80°C.

A sample of each strain had been preliminarily thawed and titrated in nutrient agar (nutrient broth supplemented with 1.8% Difco Bacto agar). Immediately before use, cultures were thawed and suitably diluted in fresh nutrient broth in order to yield approximately 103 bacteria per well. In each experiment, controls of bacterial density were prepared by seeding 100 micro liter (i.e. the same amount

dispensed in eagh well) in nutrient agar pour plates. The exact number of bacteria used was scored after 16 h at 37°C.

The plates were sealed with self-adhesive acetate tape in order to prevent spreading of volatile compounds from well to well, and the reagents were

thoroughly mixed for 5 min by means of a multiple microshaker apparatus (Tecnolabo A.S.S.I.,

Milan, Italy). Bacterial growth in each well was visually evaluated after 16 h at 37°C, by observing the increase in turbidity of the medium and/or formation of a pellet of settled cells on the bottom of wells. Since in some cases the visual examination was complicated by turbidity due to high concentration of compounds (especially in the presence of S9 mix) or to chemical precipitation, microdrops were subcultured by means of a wire

loop from the suspected well on the surface of nutrient agar plates. In general, 1 row of 8 wells could be subcultured on a single agar plate divided

into 8 sectors, but some multiple device might be easily designed in order to accelerate this step. Growth of bacterial colonies on agar plates was

checked after an additional 8-24-h incubation at 37°C.

Therefore, the liquid micromethod procedure led to the comparative assessment, within 16-40 h, of the MIC of each compound towards each of the

3 bacterial strains, with or without S9 mix. Each compound was assayed in at least 5 separate experiments in order to check the reproducibility

of results. A ratio between the MICs in repair-proficient and -deficient strains greater than 2 (this minimum difference was accepted only if confirmed

in 5 experiments) was considered to be significant in this test.

Based on the study results minimal inhibitory concentration (MIC) value for the NaCl was < 0.00001.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: Publication

Adequacy of study:

weight of evidence

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The publication has sufficient information for interpretation of results.

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

Target gene:

none

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), 2mM 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 microM both in the presence and absence of S9.

Vehicle / solvent:

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

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 microM 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 compound was classified as “positive” (+), if it induced at least a 3-fold increase in
the percentage of micronucleated cells compared to untreated controls (1% DMSO vehicle control for the −S9 condition and 1% DMSO+ S9
control for the +S9 condition). A compound was classified as “weak positive” (±) if it induced an increase in micronucleated cells between 2–3-fold compared to controls, and it was classified as “negative” if the percentage of micronucleated cells was less than 2-fold compared to controls. For the predictivity calculations, “weak positive” compounds were considered “positive”

Statistics:

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).

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 microM with S9 and no cytotoxicity was observed without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified in the report
- Effects of osmolality:not specified in the report
- Evaporation from medium:not specified in the report

RANGE-FINDING/SCREENING STUDIES: not specified in the report

COMPARISON WITH HISTORICAL CONTROL DATA: not specified in the report

ADDITIONAL INFORMATION ON CYTOTOXICITY: Maximum % of cytotoxicity was 31.1 microM with S9 and no cytotoxicity was observed without S9.

See the attched word doc. "tables_figures" for Results

Conclusions:

Interpretation of results: negative with and without metabolic activation.

Based on the experimental conditions NaCl 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.

CHO-K1 cells were pre-loaded with a cell dye that stains the cytoplasm, after which the cells were treated with the test compounds for either 3 h (for the +S9 condition) or 24 h (for the −S9 condition). A total of 10 concentrations were tested, of which the top five concentrations were scored (limited by either cytotoxicity or solubility). At the end of the incubation period the cells were fixed and their DNA was stained with Hoechst. The visualization and scoring of the cells was done using an automated fluorescent microscope coupled with proprietary automated image analysis software provided by Cellomics (Pittsburg, PA). A total of 46 compounds were used in this evaluation, including 8 aneugens and 25 clastogens with varied mechanisms of action. Thirteen non-genotoxic compounds were also included.

CHO-K1 (Chinese Hamster Ovary cells, ATCC Item Number CCL-61) were maintained in F12-K Nutrient Mixture (Kaighn’s Modification) (Invitrogen, Carlsbad, CA) containing 10% foetal bovine serum (Invitrogen), 2mM l-glutamine (Invitrogen), 10 units/ml penicillin/100 micro gram/ml streptomycin. Cells were plated at 4000 cells/well (−S9) or 5000 cells/well (+S9) into the wells of a 96-well plate, in a volume of 100 micro liter per well and cultured overnight (18–22 h) prior to the start of the assay.

The following day, the media was removed and replaced with 75 micro liter per well of cell dye solution (Cellomics), prepared by adding 5.5 micro liter of cell dye to 11 ml of medium. The cells were incubated with the cell dye for 1 h. In the meantime the test compounds and controls were prepared. The S9 metabolic regeneration system was prepared in incomplete medium (ICM, serum-free McCoy’s medium), by adding0.8 mg/ml of NADP, 1.5 mg/ml of isocitric acid and 0.6 mg/ml of arochlor-induced rat liver S9 homogenate (Moltox, Boone, NC). The final concentration of rat liver S9 was 1.5% (w/v).

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. At the end of the treatment period, the medium was removed, the cells were washed once with warm medium, and fresh medium containing 6 micro gram/ml of cytochalasin B (Cellomics) was added to the cells for a period of 22–24 h. Although these treatment regimes include recovery times, it has been shown that different treatment-recovery schedules (including and excluding recovery times) are suitable for the detection of micronuclei in CHO cells.

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 micro liter of a warm solution containing 3.7% formaldehyde and 1microM of Hoechst dye in wash buffer (Cellomics). The cells were incubated in this solution for 20 min at room temperature, after which they were washed twice with 100 microl ofwash buffer. Finally, 200 mictro liter of wash 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 towarm-up at room temperature for 30 min before scanning.

Based on the experimental conditions NaCl was negative in an automated in vitro micronucleus assay in CHO-K1 cells both in the presence and absence of S9.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

other: publication

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to GLP/guidelines.This study contains sufficient amount of information for interpretation of the results.

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of the present investigation was to evaluate the utility of the alkaline unwinding assay applied to a mammalian cell line, for predicting
the genotoxic potential of chemical agents and to establish criteria for positive and negative results. By using mouse lymphoma cells and similar
exposure conditions to those used in the mouse lymphoma thymidine kinase (TK) locus forward mutation assay (Clive et al., 1979), the sensitivity
of the end-point measured in the alkaline unwinding method can be compared with mutations at the TK locus for a number of compounds with a
known mutagenic activity.

GLP compliance:

not specified

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

DNA damage

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium containing 10% horse serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

not applicable

Test concentrations with justification for top dose:

All compounds, with a few exceptions, were tested to concentrations that were clearly toxic to the cells.
NaCl Concentration: 0, 5.06 x 10- 2, 7.59 x 10- 2, 10.1x 10- 2, 15.2 x 10- 2 (mole/l)

Vehicle / solvent:

no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

other: Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension

DURATION
- Exposure duration: for 1-2 h in medium without radioactive isotope. S9 mix or Fop without $9 mix was then added, after which exposure was initiated by adding the compound to be tested.

NUMBER OF REPLICATIONS: not reported

NUMBER OF CELLS EVALUATED: not reported

DETERMINATION OF CYTOTOXICITY
- Method: Cell samples were mixed with an equal volume of trypan blue (0.5% in 0.9% NaC1) and the proportion of cells that excluded the dye was determined. To minimise the underestimation of viability, due to prolonged exposure to the test substance during counting, cells were counted in the
order of decreasing test compound concentration. Control cells were counted last.

Evaluation criteria:

The criteria used for classification were the following.
(1) An increase in the relative fraction of ssDNA of 6.5% at a relative toxicity of less than 5% is considered positive.
(2) An increase in the relative fraction of ssDNA that is greater than the increase in the relative toxicity at the corresponding concentrations of the
test compound at relative toxicities of 5% to 50% is considered positive if this occurs at 2 or more concentrations. If such an increase is seen at one concentration the result is classified as equivocal.
(3) The classifications under 1 and 2 are true if the increase in relative fraction of ssDNA is dose-related.
(4) A result is considered negative if a toxic response is obtained and no increase in the fraction of ssDNA is seen or if the increase is smaller than
the corresponding increase in relative toxicity. If toxicity is not evident the result cannot be adequately evaluated.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: not specified in the report

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In addition to these qualitative differences between the sensitivity of the 2 assays, the alkaline unwinding assay appears to possess a somewhat
lower sensitivity for some classes of compound that are detected by both tests.

None

Conclusions:

Interpretation of results: positive without metabolic activation

Based on the study results NaCl was positive in the Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution.

Executive summary:

A rapid genotoxicity test, based on the measurement of the proportion of single- to double-stranded DNA by alkaline unwinding and hydroxyapatite elution in mouse lymphoma cells treated in vitro with various chemicals, was evaluated.

Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens, toxic compounds not usually tested for genotoxicity and non-toxic compounds not thought to be genotoxic were tested.

Mouse lymphoma L5178Y/TK +/ (-3.7.2 C) cells, obtained from Dr. D. Clive, were used throughout this work. Culture media and conditions were according to the procedures for the mouse lymphoma mutagenicity assay (Clive and Spector, 1975). The cells were determined to be free from mycoplasma by the uridine/uracil incorporation technique (Stanbridge and Schneider, 1976).

Exponentially growing cells were labelled overnight with 0.2-0.3 micro Ci [methyl-3H]thymidine per ml culture. After 16-18 h the culture was centrifuged, the radioactive medium was discarded and the cells washed with Hanks' balanced salt solution

(HBSS), resuspended and incubated at 37 °C for 1-2 h in medium without radioactive isotope. S9 mix or Fop without S9 mix was then added, after which exposure was initiated by adding the compound to be tested. The final cell concentration

was approximately 2 x 105 cells/ml and the serum concentration 3% during exposure. Chemicals

were dissolved in Fop, water, ethanol or dimethyl sulphoxide at 100 times the greatest concentration to be tested, and appropriate dilutions made. If the solubility was too low to make a 100 times concentrated solution, a concentration 5

times that to be tested was made in Fop, with appropriate modifications of the test procedure to obtain the same cell density during the incubation.

After 3 h exposure, 0.5-ml samples were set aside for viability estimation and the remaining 2.0 ml of cells were centrifuged and washed with HBSS to terminate the exposure. Strand breaks were detected by alkaline unwinding and hydroxyapatite elution, according to

Ahnstrom and Erixon (1981) with modifications as follows. The relative DNA content was determined by mixing 2-4-ml samples from the ssDNA and dsDNA fractions with Insta Gel scintillation cocktail and measuring the radioactivity in a liquid scintillation counter.

The results obtained were compared with those from the mouse lymphoma TK locus forward-mutation assay, providing a basis for assessing the relative sensitivity of the 2 assays using the same cells exposed to chemicals under similar conditions. Clear evidence of DNA-damaging activity was obtained with 43 of the compounds, while 4 gave equivocal results. Of the remaining 31 compounds, 14 were toxic without inducing DNA damage while the rest were non-toxic and did not induce any DNA damage.

Based on the study results NaCl was Positive in the Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only with metabolic activation

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, TA98

Metabolic activation:

with

Metabolic activation system:

S9

Test concentrations with justification for top dose:

max 5.0 mg/plate

Details on test system and experimental conditions:

Ames test

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, TA98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Conclusions:

Interpretation of results: negative with metabolic activation

Negative in the test conditions.

Executive summary:

in a Ames test (similar to OECD 471 guideline), performed in the following TA strains: TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98 calcium chloride did not induce mutation in bacteria in presence of metabolic activation.

 

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

no test with metabolic activation

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster fibroblast cell line CHL

Metabolic activation:

without

Test concentrations with justification for top dose:

max. 4 mg/plate

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Conclusions:

The results are negative without metabolic activation.

Negative in the test conditions.

Executive summary:

A chromosome aberration test similar to the OECD 473 guideline was performed on Chinese hamster fibroblast cell line CHL with Calcium chloride. The results are negative without metabolic activation.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: publication

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The publication contains sufficient information for the interpretation of study results.

Qualifier:

equivalent or similar to guideline

Guideline:

other: mouse lymphoma assay [Clive et al., 1979; Jotz and Mitchell, 1981; Oberly et al., 1984]

Principles of method if other than guideline:

none

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (tk) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were maintained in culture according to the procedures of Turner et al. [1984].
Cells were maintained in log-phase growth for a 2-day expression period and then cloned with TFT for selection (1 lig/m1) and without TFT for determination of viability in Fischer's medium containing 0.22% Baltimore Biological Laboratory (BBL) agar.
- Properly maintained: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

Experiment 1
0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.5, 4.5, 5.5 mg/ml

Experiment 2
4.75, 4.86, 4.97, 5.08, 5.19, 5.30, 5.49, 5.52, 5.63, 5.74

Experiment 3
4.75, 4.86, 4.97, 5.08, 5.19, 5.30, 5.49, 5.52, 5.63, 5.74, 5.85

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

not specified in the report

DURATION

- Exposure duration: 4 hours
- Expression time (cells in growth medium): After 9-11 days of incubation at 37°C, colonies were counted

NUMBER OF REPLICATIONS: not specified

NUMBER OF CELLS EVALUATED: not specified

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

This assay measures forward mutation at the functionally heterozygous thymidine kinase (tk) locus based on resistance to trifluorothymidine (TFT). We have been investigating factors that may impact test design and also influence the interpretation of test results. Factors that effect the magnitude of the quantitated mutant frequency include expression time [Moore and Clive, 1982] and both the growth medium [Moore and Howard, 1982] and the agar used in the cloning medium [Meyer et al., 1986].

Statistics:

no data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality:
- Evaporation from medium: no data
- Water solubility: yes

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment, there was insufficient cytotoxicity to reach a 10-20% survival range, and the mutant frequency (and number of mutants) showed only a very small dose-related increase. It was judged a "no test"—that is, there was insufficient information to make a decision. The second and third* experiments included slightly higher concentrations. Both experiments showed a dose-related increase in both mutant frequency and mutant number.

In the first experiment, there was insufficient cytotoxicity to reach a 10-20% survival range, andthe mutant frequency (and number of mutants) showed only a very small dose-relatedincrease. It was judged a "no test"—that is, there was insufficient information to make a decision. The second and third experiments included slightly higher concentrations. Both experiments showed a dose-related increase in both mutant frequency and mutant number.

Colony sizing was performed for selected doses.

Colony sizing for weakresponses is some what difficult to interpret. The majority of induced mutant colonieswere small colonies (data not shown), which is consistent with the report by Gallowayet al. [1987] and Ashby and Ishidate [1986] that high-salt concentrations induce chromosome aberrations.

Conclusions:

Interpretation of results : in this test, high concentrations of sodium chloride are positive without metabolic activation.

This experiment with sodium chloride demonstrates the importance of carefully evaluating weak mutagenic responses observed with high concentrations of test compounds. The positive mutagenicity probably is due not to a direct interaction with DNA but to some indirect mechanism resulting from the extremely nonphysiological condition of the test.

Executive summary:

High Concentrations of Sodium Chloride was evaluated for a "Positive" Response at the TK Locus of L5178Y/TK+/- Mouse Lymphoma Cells.

Cells were maintained in culture according to the procedures of Turner et al.[1984]. L5178Y/TIC^H--3.7.2C cells were treated with sodium chloride for 4 hr inthe absence of exogenous metabolic activation according to procedures described byTurner et al., 1984. Cells were maintained in log-phase growth for a2-day expression period and then cloned with TFT for selection (1 lig/m1) and without TFT for determination of viability in Fischer's medium containing 0.22% BaltimoreBiological Laboratory (BBL)agar. After 9-11 days of incubation at 37°C, colonie swere counted.

Test results generated for the mouse lymphoma assay, the magnitude of the induced mutant frequency is relatively low, and the concentration of test agent required to obtain the response is enormous.

This experiment with sodium chloride demonstrates the importance of carefully evaluating weak mutagenic responses observed with high concentrations of test compounds. The positive mutagenicity probably is due not to a direct interaction with DNA but to some indirect mechanism resulting from the extremely nonphysiological condition of the test.the colony-size distribution determined by an Artek model 880automatic colony counter modified with a 10-turn potentiometer [Moore et al., 1985].

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: publication

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to GLP/guidelines.This study contains sufficient amount of information for interpretation of the results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

None

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium (Seromed), supplemented with 10% (v/v) fetal calf serum (Seromed), 100 units penicillin (Hoechst), and 0.128 mg/ml dihydrostreptomycin sulfate (Heyl) in 10-cm petri dishes .
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

not applicable

Test concentrations with justification for top dose:

NaC1 conc. (mOsm/kg H20): 200,1 50, 125, 100, 80, 300 (control)
sodium chloride (NaCI) was dissolved in distilled water; the stock solution was further diluted until the appropriate osmolality was reached.

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: monolayer

DURATION
- Exposure duration: 30 min at 37°C

SPINDLE INHIBITOR (cytogenetic assays): colcemide
STAIN (for cytogenetic assays): BrdUrd

NUMBER OF REPLICATIONS: not reported

NUMBER OF CELLS EVALUATED: 100-200/dose

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in
evaluating the test results. Statistical significance should not be the only determining factor for a positive response.
An increase in the number of polyploid cells may indicate that the test substance has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has
the potential to inhibit cell cycle progression.
A test substance for which the results do not meet the above criteria is considered nonmutagenic in this system.
Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
Positive results from the in vitro chromosome aberration test indicate that the test substance induces structural chromosome aberrations in
cultured mammalian somatic cells. Negative results indicate that, under the test conditions, the test substance does not induce chromosome
aberrations in cultured mammalian somatic cells.

Statistics:

not reported

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

not specified

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: not specified in the report

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Effects of osmolality: yes


RANGE-FINDING/SCREENING STUDIES: no data
COMPARISON WITH HISTORICAL CONTROL DATA: nodata
ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

See the attached word Doc. "tables_figures":

Details of the attchment:

TABLE L Chromosomal Aberrations in V 79 Hamster Cells Induced by DilutedMcCoy's Medium (the Cells Were Treated for 30 min Under

Hypotonic Conditions; Fixation was Done 15 hr Later)2

TABLE III. Chromosomal Aberrations in V 79 Hamster Cells Induced by NaCI (the Cells Were Treated for 30 min Under Hypotonic Conditions,Fixation was Done15 hr Later)2

TABLE IV.Chromosomal Aberrations in BrdUrd-labelled V 79Hamster Cells Inducedby NaCI (theCells Were Treated for 30min Under Hypotonic Conditions, Fixation was Done 15 hr Later)

TABLEV.Chromosomal Aberrations in V 79 Hamster Cells Induced by AS(the Cells Were Treated for 30 min Under Hypotonic Conditions;Fixationwas Done 15 hr Later)

TABLE VI.Chromosomal Aberrations in BrdUrd-labelledV 79 Hamster Cells Induced by AS (the Cells Were Treated for 30 min UnderHypotonicConditions; Fixation wasDone 15hr Later)

TABLE VII. Chromosomal Aberrations in V 79 Hamster Cells Induced by Tris (the Cells Were Treated for 30 min Under Hypotonic Conditions;Fixation was Done 15 hr Later)"

TABLE VIII. Chromosomal Aberrations in BrdUrd-labelled V 79 Hamster Cells Inducedby Tris (the Cells Were Treated for 30 min Under Hypotonic Conditions; Fixation was Done 15 hr Later)"

Conclusions:

Interpretation of results: positive without metabolic activation.

Hypotonic NaCl solutions of less than 150 mOsm/kg H10 clearly elevated the frequency of chromosomal aberrations. Treatment with a NaCl solution of 80-100 mOsm/kg 1-120 induced a maximum frequency of chromosomal aberrations.

Executive summary:

V 79 hamster cells were grown at 37°C in a humidified atmosphere with 5% CO2 in McCoy's 5A medium (Seromed), supplemented

with 10% (v/v) fetal calf serum (Seromed), 100 units penicillin (Hoechst), and 0.128 mg/ml dihydrostrepto­mycin sulfate (Heyl) in 10-cm petridishes (Greiner); 48 hr before the treatment 105 cells from the stock culture were seeded in 6-cm petridishes with 5 ml of complete medium.

Sodium chloride (NaCI), and trishydroxymethylami­nomethane (Tris), were dissolved in distilled water; the stock solution was further diluted until the appropriate osmolality was reached. Undiluted McCoy's medium had 300 mOsm/kg H20. A dilution of 1:9 (medium:water) reduced the osmolality to 30 mOsm/kg H20. For AS, NaCI, and Tris there is a clear correlation of molarity and osmolality. The molarity (mM) of AS solutions has to be multiplied by a factor of three to give the osmolality (mOsm/kg H20); for NaCI it is a factor of two and forTris it is 1.5.

The osmolality of all solutions was measured with a microosmometer; all chemicals were purchased from Merck and had a purity of 99.5%. The pH of the AS and NaCI solutions was 5.5 independent of the concentration. The pH of all Tris solutions was adjusted with HCI to 7.2 to equal that of McCoy's medium. The pH of McCoy's medium changed with dilution. Undiluted medium had a pH of 7.2; a dilution to 200 mOsm/kg H20 had no influence on pH. Further dilution led to a pH decrease and medium of 55 mOsm/kg H20 had a pH of 5.9.

Hypotonic treatment was carried out on monolayer cells in petri dishes. The culture medium was discarded; then the cells were washed with 5 ml of prewarmed phosphate-buffered saline (PBS). Immediately afterward 5 ml of prewarmed salt solution or diluted McCoy's medium was added. After 30 min at 37°C the salt solution or the hypotonic medium was discarded; the cells were washed once with

PBS, and then freshly prepared culture medium with BrdUrd was added. After a culture time of 16 hr, including a 2-hr colcemide treatment, the cells were fixed in methanol:acetic acid and stained with 5% Giemsa or according to the fluorescenceplus Giemsa (FPG) technique.

All experiments were done twice or three times without BrdUrd and once with BrdUrd labelled cells.

Hypotonic NaCl solutions of less than 150 mOsm/kg H2O clearly elevated the frequency of chromosomal aberrations. Treatment with a NaCl solution of 80-100 mOsm/kg 1-120 induced a maximum frequency of chromosomal aberrations. A further reduction of the osmolality led to less aberrations, but the number of totally damaged cells, bearing complex aberrations which were not scora­ble, was increased. In the case of NaCl a reduction of the osmolality to one-half led to an increased aberration frequency. the aberrations were mostly of the chromosome type. Treatment with less than 60 mOsm/kg H₂0 reduced the mitotic index to nearly zero, which may be due to osmotic shock.

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: other: Thymidine Kinase locus assay

Type of information:

other: publication

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to GLP/guidelines.This study contains sufficient amount of information for interpretation of the results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

no data

GLP compliance:

not specified

Type of assay:

other: Mouse Lymphoma L5178Y thymidine kinase locus assay

Target gene:

TK+/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium containing 10% horse serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

20mmol/l

Vehicle / solvent:

no data

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
DURATION
- Exposure duration:4 hour
- Expression time (cells in growth medium): 48 h

Evaluation criteria:

Total survival was calculated from the suspension growth and cloning efficiency data and the mutation frequency expressed as the number of mutant cells/106 viable cells. Test compounds were generally tested up to a concentration that reduced total growth to 10-20% of the solvent control except in those cases when low solubility precluded the achievement of toxic concentrations. Assays which gave irregular dose response or an absence of dose response were retested.

Statistics:

The number of colonies formed on the six replicate control agar plates and the three replicate plates from each treated culture was tested for normal distribution according to Shapiro and Wilk (1965) and found to be normally distributed in 92% of the cases (n = 383). Further, the replicates were subjected to analysis of variance, which showed that the variance of the control and treated replicates was equal in 95% of the comparisons (n = 317). Therefore, a pairwise two-tailed Student's t-test was performed on each set of treated replicates versus the corresponding solvent control replicates.

The statistical analyses were performed using the SAS computer programming package.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

other: not specified in the report

Cytotoxicity / choice of top concentrations:

other: not specified in the report

Additional information on results:

no data

none

Conclusions:

Interpretation of results : positive without metabolic activation

The compound Sodium Chloride was positive in a gene mutation test in mammalian celles at concentrations in the range 0.02-1 mol/l.

Executive summary:

Mutagenicity testing was conducted on the mouse lymphoma TK+/- - TK-/- forward mutation assay. Test compound was obtained from the E.Merck (FRG) with highest available purity.

Heterozygous L5178Y TK+/- 3.7.2.C cells were obtained from Dr Donald Clive. Stock cultures were grown in Fischer’s medium containing 10% horse serum with additions. The pH of the culture medium was adjusted to 7.2 which were found to improve the growth rate of the cells compared with pH 6.8. Cells to be used for mutagenicity assays were cleansed of TK-/- mutants by treatment with thymidine, hypoxanthine, methotrexate, and glycine.

Liver homogenate (S9) prepared from Aroclor 1254 pretreated male Sprague-Dawley rats and cofactors were added to prepare the metabolic activation mix. The mutagenicity assay was performed with the following modifications: following the 4-h treatment with the test compound the cells were resuspended in Fischer's medium containing 10% horse serum (F10p) to which Hepes (N-2-hydroxyethyl-N'-2-ethanesulphonic acid) was added to 10 mmol/l and the concentration of NaHCO3 was reduced to 0.8 g/1. The final pH in the medium was 7.2. The cell concentration was adjusted to 15 x 104cells/ml at the beginning of each day of the expression period which was 48 h.

At the end of the expression period 3.4x106cells from each treatment tube were centrifuged and resuspended in 1 ml of remaining medium by vigorous pipetting. The cells were diluted with 99ml F20P and three 29ml aliquots transferred to 50ml plastic tubes. One 500 microlitre aliquot was diluted to 25ml and three 500microlitre aliquots of this suspension were diluted to 30ml with F20p to give 340 cells per tube.

 

The selective agent trifluorothymidine was added to the three undiluted cell suspensions to a final concentration of 1.0 microgram/ml. The cells were cloned by adding 3.3ml 2% molten agar to give a final concentration of 0.2% in each tube, which was vortexed directly and poured onto 90mm petri dishes and allowed to solidify.

Colonies formed on the plates, containing the selective agent, were counted manually and those without using an Artek 880 automatic colony counter.

The modifications in the culture conditions, addition of Hepes and decreased cell density improved the plating efficiency of the cells, which was erratic and low in some experiments prior to the introduction of these changes. The resultant performances figures for the assay were: generation time, 11±2.8 (SD) h and plating efficiency, and 91±14% for n=55 experiments.

The spontaneous mutation frequency was 76±25 without and 86±33/106cells with metabolic activation (n=35 and 20) respectively.

Total survival was calculated from the suspension growth and cloning efficiency data and the mutation frequency expressed as the number of mutant cells/106viable cells. Compounds were generally tested up to a concentration that reduced total growth to 10-20% of the solvent control except in those cases when low solubility precluded the achievement of toxic concentrations. Assays which gave irregular dose responses or an absence of dose response were retested.

 

The results were subjected to statistical analysis. The number of colonies formed on the six replicate control agar plates and the three replicate plates from each treated culture was tested for normal distribution and found to be normally distributed in 92% of the cases (n=383). Further, the replicates were subjected to analysis of variance, which showed that the variance of the control and treated replicates was equal in 95%of the comparisons (n=317). Therefore, a pairwise two-tailed Students’s t-test was performed on each set of treated replicates versus the corresponding solvent control replicates.

   The application of linear, quadratic and cubic regression analysis to the results   provided data (not shown) which were in poor agreement with the subjective evaluation of the dose trends seen.The statistical analyses were performed using the SAS computer programming package.

 

The compound Sodium Chloride was positive in the test conditions at concentrations in the range 0.02-1 mol/l.      

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Two studies are available on calcium chloride. One OECD 471 study (Ishidate et al, 1984) and one OECD 473 study (Ishidate et al., 1984). The results are negative in these two studies. Furthermore, calcium chloride is a naturally present in cells. Therefore calcium chloride is not considered to be genotoxic.

For Sodium chloride several in vitro studies are available. On seven available studies, four showed positive results. The positive result observed on these studies is due to an indirect effect of sodium chloride due to the change of osmolality and is not a direct effect of the substance on the DNA. Therefore despite the vitro positive results, sodium chloride is not considered as genotoxic. 

Justification for classification or non-classification

Based on the available data and since calcium chloride is naturally present in cells, the two components of the reaction mass is not genotoxic. Therefore the reaction mass itself is not considered to be genotoxic.