Sodium chlorate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

IN VITRO GENETIC TOXICITY

Five Ames Test + one E.Coli DNA-Lethality

- NTP (eq. to OECD 471, 1999, K, rel. 2): not mutagenic with and without metabolic activation in S. typhimurium TA97 - TA98 - TA100 - TA102 - TA104 - TA1535

- May (OECD 471, 1989, GLP, SS, rel. 2): not mutagenic/ devoid of mutagenic activity under the conditions of the test with and without metabolic activation in S. typhimurium TA98, TA1538, TA100, TA1535 and TA1537

- Hossack (No guideline followed/publication, 1978, rel.3): not mutagenic/ failed to produce any clear evidence of mutagenic potential with and without metabolic activation in S. thyphimurium strains TA1537, TA98 & TA100

- Gocke (No guideline followed/publication, 1981, rel.4): mutagenic with and without metabolic activation in S. thyphimurium strains TA1535a, TA1535b, TA1538, TA1537, TA98 & TA100

- May (No guideline followed, 1989, rel.2): Clear positive responses were observed in WP67 and CM871 strains treated with the positive control in absence of S9. Sodium chlorate induced responses suggestive of DNA damage in E.coli strain WP67 and CM871

- Eckhardt (Publication/ Abstract only with limited information, 1982, rel.4): mutagenic activity in the Ames test on TA1535 + S9.

 

Two Mammalian Cell Gene Mutation Assays

- Clarke (OECD 476, 2007, HGPRT/CHO, GLP, K, rel. 1): not mutagenic up to cytotoxic concentrations with and without metabolic activation

- Hodson-Walker (comparable to OECD 476, 1989, HGPRT/CHO, GLP, K, rel. 1): not mutagenic up to cytotoxic concentrations with and without metabolic activation

 

One DNA damage and repair study

- Seeberg (OECD 482, GLP, K, rel. 1): not induce unscheduled DNA synthesis in HeLa S3 cells, either in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

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:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The data was retrieved from the NTP website. No study report avialable. No details on test substance composition. Protocol and results are available online. NTP study data is generally well trusted.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The study was performed accoding to standard NTP Protocol which is based on Zeiger (1992).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

other: Salmonella typhimurium TA97 - TA98 - TA100 - TA102 - TA104 - TA1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat or hamster

Test concentrations with justification for top dose:

100, 333, 1000, 3333 and 10000 microgram/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "any other information on materials...."

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 2 days
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): histidine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: In the methos it is stated: All chemicals were run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity
assay. The toxicity assay was performed using TA100 or the system developed by Waleh et al. [1982]. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
No data about a cytotoxicity test has been reported by the NTP.

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable

Evaluation criteria:

A chemical was judged mutagenic or weakly mutagenic if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a weakly mutagenic response, or if only single doses produced increases in his+ revertants in repeat trials.Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

Statistics:

-

Species / strain:

other: Salmonella typhimurium TA97 - TA98 - TA100 - TA102 - TA104 - TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: water soluble
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the criteria of this study sodium chlorate (100 to 10,000 µg/plate) was not mutagenic in S. typhimurium strains TA97, TA98, TA100, TA102, TA104, or TA1535, with or without induced rat or hamster liver S9 enzymes.

Executive summary:

Testing was performed as reported by Zeiger et al. (1992). Sodium chlorate was sent to the laboratory as a coded aliquot. It was incubated with the Salmonella typhimurium tester strains TA97, TA98, TA100, TA102, TA104, and TA1535 either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes at 37° C. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C. Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses of sodium chlorate. In the absence of toxicity, 10,000 µg/plate was selected as the high dose.Test concentrations were 100, 333, 1000, 3333 and 10000 microgram/plate. All trials were repeated at the same or a higher S9 fraction. In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive. In this assay sodium chlorate did not induce an increase in the number of revertant colonies. Sodium chlorate (100 to 10,000 µg/plate) was not mutagenic in S. typhimurium strains TA97, TA98, TA100, TA102, TA104, or TA1535, with or without induced rat or hamster liver S9 enzymes.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 February 1989 - 18 April 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed under GLP. The method used is not described but it is comparable to OECD476.

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
DMEM - Treatment medium
500 ml Dul becco's MEM ( 1x )
5 ml Penicillin: streptomycin solution (5000 IU per ml : 5000 µg/ml)

DMEM 10% FCS - complete medium
as for treatment medium plus 60 ml Foetal calf serum (FCS)

6-Thioguanine (Stock solution)
10 mg 6-Thioguanine
10 ml Sodium bicarbonate solution (0.5% w:v)
incubated at 50°c for at least 12 hours and filter sterilized.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

preliminary and main study: 8, 40, 200, 1000 and 5000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: not reported

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulphonate (EMS, Sigma) and dimethylbenzanthracene (DMBA, Sigma)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 6 days
- Fixation time (start of exposure up to fixation or harvest of cells): 6 days, resultant colonies were fixed in methanol and stained with 10% Giemsa.

SELECTION AGENT (mutation assays): 6-Thioguanine (6-TG)
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: Triplicate and an independent repeat was performed.

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: other: A preliminary toxicity test was first conducted: cells were exposed to five Sodium Chlorate concentrations ranging from 8 to 5000 µg/ml in the presence and absence of a rat-liver derived metabolic activating system (S-9 mix). All cultures were established in duplicate. After three hours of exposure cells were washed and seeded in the non-selective medium to assess toxicity: plating efficiency was determined by colony counts.

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable


OTHER:

Evaluation criteria:

Mutant frequency per 10E5 survivors = (No. of cells plated for PE/Mean No. of PE colonies)x Mean No. of 6-TGr colonies

Statistics:

-

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

not seen up to 5000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: the test substance in water soluble
- Precipitation: not observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: There was no evidence of toxicity of Sodium Chlorate at any concentration tested, up to a maximum o f 5000 µg/ml. The concentrations selected for use in the main mutation assays, therefore, were 8, 40, 200, 1000 and 5000 µg/ml both in the absence and presence o f S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

GENOTOXIC EFFECTS: After an expression time of 7 days, cells were plated out to assess survival and 6-Thioguanine resistance (6-TG r). 
- With metabolic activation: The positive controls induced a marked increase in the number of revertant colonies. Under the conditions of this this mammalian cell gene mutation
assay, sodium chlorate did not cause a positive response on any of the tested strains.
- Without metabolic activation: The positive controls induced a marked increase in the number of revertant colonies. Under the conditions of this mammalian cell gene
mutation assay, sodium chlorate did not cause a positive response on any of the tested strains.
FREQUENCY OF EFFECTS:
The positive control EMS had a mean mutaion frequency of 138.1 (compared to a solvent control value of 2.6) in the first assay and in the second assay 74.2 per 10e5 (compared
to a solvent control value of 0.5)
DMBA had a mean mutaion frequency of 55.9 (compared to a solvent control value of 1.1) in the first assay and in the second assay 46.3 per 10E5 (compared to a solvent control
value of 1.8)

In the first assay the sodium chlorate exposed cells without S9 -mix single cultures exposed at 8 and 40 µg/ml showed an increase in mutation frequency, 22.2 and 17.5 per 10e5
survivors, respectively. The control value was 2.6. In the second assay without S9 -mix only one culture exposed at 40 µg/ml gave a rise in mutation frequency, 8.7 per 10e5
survivors, compared to control values of 0.0, 1.5 and 0.0. In the first assay the sodium chlorate exposed cells with S9 -mix no increases in mutation frequency or mutaion
colonies were seen.
In the second assay the sodium chlorate exposed cells with S9 -mix at 200 µg/ml in one culure an increase in mutant colony numbers was seen (10.2 per 10E5 survivors compared to
control values of 4.4, 0.0, and 0.9). All mutations found in these studies fell well within the data range for the solvent control within this lab. 
   
PRECIPITATION CONCENTRATION: -
MITOTIC INDEX: Not applicable
CYTOTOXIC CONCENTRATION: No cytotoxicity observed
- With metabolic activation:  No cytotoxicity observed
- Without metabolic activation:  No cytotoxicity observed
TEST-SPECIFIC CONFOUNDING FACTORS: -
STATISTICAL RESULTS: -

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that, under the conditions of the test, Sodium Chlorate showed no evidence of mutagenic activity in either the absence or presence of S-9 mix.

Executive summary:

Sodium Chlorate was examined for mutagenic potential by measuring its ability to induce mutation in Chinese hamster (V79) cells at the hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) locus. The study was performed under GLP. The method used is not described but it is comparable to OECD476.

A preliminary toxicity test was first conducted: cells were exposed to five Sodium Chlorate concentrations ranging from 8 to 5000 µg/ml in the presence and absence of a rat-liver derived metabolic activating system (S-9 mix). All cultures were established in duplicate. After three hours of exposure, the cells were washed and seeded in non-selective medium to assess toxicity: plating efficiency was determined by colony counts.

Cultures of Chinese hamster cells were then exposed to five concentrations of Sodium Chlorate in each of two mutation assays. The selected concentrations were 8, 40, 200, 1000 and 5000 µg/ml in the absence and presence of S-9 mix. Treatments were established for three hours. Solvent control cultures treated with distilled water were included in all experiments. Ethylmethanesul phonate (EMS), a known direct-acting mutagen, and 7,-12 dimethylbenzanthracene (DMBA), a mutagen which requiresm metabolic activation to achieve optimal activity, were used as

positive controls. All cultures were establ ished in triplicate. After treatment, the cell sheet was washed and non-selective medium added. A sample of cells was taken from each culture and plated out to determine survival immediately post-treatment. The cultures were then incubated at 37°C. Cultures were studied each day and passaged when necessary to maintain subconfluence. After an expression time of 7 days, cells were plated out to assess survival and 6-Thioguanine resistance (6-TGr).

After 6 days of incubation at 37°C in a 5% CO2 atmosphere, the resultant colonies were counted. Survival was estimated from master non-selective plates, and mutants were scored in selective plates; these latter contained 6-TG which was added to the plates 2 -3 hours after seeding.

Cultures exposed to Sodium Chlorate showed no real increases in 6 -TGr colony numbers and no significantly increased mutant frequencies (per 10 survivors), compared to the solvent controls.

Under the same test conditions, EMS (1000 µg/ml) produced increases in the incidence of mutant colonies. DMBA (10 µg/ml) markedly increased the incidence of mutant colonies only in the presence of S-9 mix.

It is concluded that, under the conditions of the test, Sodium Chlorate showed no evidence of mutagenic activity in either the absence or presence of S-9 mix. The sensitivity of the assay system was proven by the observed responses to known mutagens.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2007 - 06 July 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed under GLP and accoding to OECD 476.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: F12 medium without hypoxanthine, supplemented with 5% dialyzed fetal bovine serum (F12FBS5-Hx)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, each freeze lot of cells has been tested and found to be free of mycoplasma contamination
- Periodically checked for karyotype stability: yes, cells used in the mutation assay were within four subpassages from cleansing in order to assure karyotypic stability.
- Periodically "cleansed" against high spontaneous background: yes, cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT).

The CHO/HGPRT assay was designed to select for mutant cells which have become resistant to such purine analogues as 6-thioguanine (TG) and 8-azaguanine as a result of mutation at the X-chromosome-linked HGPRT locus. This system has been demonstrated to be sensitive to the mutagenic action of a variety of chemicals.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 will be thawed and mixed with a cofactor pool to contain 100 µL S9/mL reaction mixture of approximately 4 mM NADP, 5 mM glucose-6-phosphate, 10 mM MgCl2, 30 mM KCI, 10 mM CaCI2, and 50 mM sodium phosphate buffer, pH 8.0.

Test concentrations with justification for top dose:

10, 33, 100, 333, 1000, and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: test medium
- Justification for choice of solvent/vehicle: indicated by the sponsor

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

test medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethyl methanesulfonate (EMS) for the non-activated test system and Benzo(a)pyrene (B(a)P) as the positive control for the S9-activated test system.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preparation of Target Cells: Exponentially growing CHO-K1-BH4 cells will be seeded in F12 medium without hypoxanthine, supplemented with 5% dialyzed fetal bovine serum (F12FBS5-Hx), at a density of 5 x 10E5 cells/25 cm2 surface area and will be incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 18-24 hours.
- Preincubation period: none
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 7-10 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): 6-thioguanine (TG, 2-aniino-6-mercaptopurine)

NUMBER OF REPLICATIONS: duplicate exposure

NUMBER OF CELLS EVALUATED: all colonies are counted

DETERMINATION OF CYTOTOXICITY
Evaluation of Cytotoxicity
For evaluation of cytotoxicity, the replicates from each treatment condition were detached using trypsin and subcultured independently in F12FBS-HX, in triplicate, at a density of 100 cells/60 mm dish. After 7-10 days incubation, the colonies were rinsed with HBSS, fixed with methanol, stained with 10% aqueous Giemsa, counted and cloning efficiency determined. Cytotoxicity was expressed relative to the solvent control-treated cultures.


OTHER: positive control details:
Ethyl methanesulfonate (EMS), CAS 62-50-0, lot 10101582, expiration August 2010, was obtained from Alfa Aesar and was used at a stock concentration of 20 µL/mL, for a final concentration of 0.2 µL/mL, as the positive control for the non-activated test system. Benzo(a)pyrene (B(a)P), CAS 50-32-8, lot 064K0688, expiration June 2008, was obtained from Sigma Chemical Company and was used at a stock concentration of 400 µg/mL, for a final concentration of 4 µg/mL, as the positive control for the S9-activated test system.

Mutagenesis Assay
The mutagenesis assay was used to evaluate the mutagenic potential of the test article. CHO cells were exposed for 5 hours at 37±1°C to the vehicle alone, appropriate positive controls and six concentrations oftest article in duplicate in both the absence and presence of S9 activation.

Treatment of the Target Cells
Exponentially growing CHO-K1 cells were seeded in F12FBS-HX at a density of 5x105 cells/25 cm2 surface area and were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 18-24 hours. The time of initiation of chemical treatment was designated as day 0. Treatment was carried out by refeeding the treatment flasks with 4.5 mL F12FBS-HX for the non-activated study, or 3.5 mL FI2FBS-HX and 1 mL S9 reaction mixture for the S9-activated study, to which was added 0.5 mL dosing solution of test or control article in vehicle or vehicle alone per 25 cm2 surface area. Duplicate flasks of cells were exposed to at least five concentrations of the test article for 5 hours at 37±1°C. No adjustment of pH was necessary. The osmolality of each treatment condition was determined. After the treatment period, all media were aspirated, the cells washed with Ca++ and Mg++-free Hanks’ balanced salt solution (CMF-HBSS) and cultured in F12FBS-HX for an additional 18-24 hours at 37±1°C. At this time, the cells were subcultured to assess cytotoxicity and to initiate the phenotypic expression period.

Expression of the Mutant Phenotype
For expression of the mutant phenotype, the replicates from each treatment condition were trypsinized and subcultured independently in F12FBS-HX, in duplicate, at a density no greater than 106 cells/100 mm dish. Subculturing by trypsinizing at 2-4 day intervals was employed for the 7-9 day expression period. At the end of the expression period, selection for the mutant phenotype was performed.

Selection of the Mutant Phenotype
For selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2x 10 cells/1100 mm dish in F12FBSHX containing 10 µM 6-thioguanine (TG, 2-amino-6-mercaptopurine). For cloning efficiency determinations at the time of selection, 100 cells/60 mm dish were plated in triplicate in medium without TG. After 7-10 days of incubation, the colonies were fixed, stained and counted for both cloning efficiency and mutant selection.

Evaluation criteria:

All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data:
• The test article was considered to induce a positive response if there was a concentration related increase in mutant frequencies with at least two consecutive concentrations showing mutant frequencies of> 40 mutants per 10E6 clonable cells.
• If a single point above 40 mutants per 10E6 clonable cells was observed at the highest concentration, the test article was considered suspect.
• If no culture exhibited a mutant frequency of> 40 mutants per 10E6 clonable cells, the test article was considered negative

Criteria for a Valid Test:
The cloning efficiency of the solvent control must be greater than 50%. The spontaneous mutant frequency in the solvent control must fall within the range of 0-25 mutants per 10E6 clonable cells. The positive control must induce a mutant frequency at least three times that of the solvent control and must exceed 40 mutants per 10E6 clonable cells. There must be at least four analyzable test article concentrations with mutant frequency data.

Statistics:

The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2x 10E6 cells: 10 plates at 2x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 10E6 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from concentrations giving < 10% relative survival are presented in the data but were not considered as valid data points. Because spontaneous mutant frequencies are very low for the CHO/HGPRT assay, calculation
of mutagenic response in terms of fold increase in mutant frequency above the background rate does not provide a reliable indication of the significance of the observed response. The wide acceptable range in spontaneous mutant frequency also suggests the need to set a minimum mutant frequency for a response to be considered positive. Li et al. (1988) refer to a level of 50 mutants per 10E6 clonable cells. In this laboratory, a more conservative approach is used which sets the minimum level at >40 mutants per 10E6 clonable cells.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: was determined does not need modification.
- Effects of osmolality: was determined.
- Evaporation from medium: not an issue
- Water solubility: not an issue
- Precipitation: not observed
- Other confounding effects: not applicable

RANGE-FINDING/SCREENING STUDIES: not performed

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, sovent and positive control were in line with hitrorical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Concurrent Cytotoxicity Test using Sodium chlorate

Non-activated –S9 mix
Treatment (μg/L)	subset	Plate counts			Cloning efficiency
		1	2	3	Total	Relative
solvent	A	70	69	63	71	100
	B	81	77	67
EMS	A	29	26	26	33	46
	B	32	38	45
10	A	60	69	86	68	95
	B	75	54	63
33	A	62	59	71	68	96
	B	57	73	88
100	A	69	67	77	64	90
	B	65	52	56
333	A	67	55	65	72	102
	B	68	97	82
1000	A	79	63	67	72	101
	B	75	67	81
5000	A	50	70	68	68	95
	B	77	70	70

Activated +S9 mix
Treatment (μg/L)	subset	Plate counts			Cloning efficiency
		1	2	3	Total	Relative
solvent	A	62	71	65	53	100
	B	25	64	32
B(a)P	A	7	15	18	9	18
	B	5	5	6
10	A	81	67	54	57	107
	B	45	41	54
33	A	52	44	41	62	117
	B	67	102	68
100	A	44	66	59	64	120
	B	78	68	68
333	A	48	47	41	63	118
	B	76	89	74
1000	A	68	82	74	69	130
	B	71	64	57
5000	A	33	60	51	73	138
	B	116	96	83

A and B are duplicate cultures

Cloning efficiency = total colonies counted/ (number of dishes x 100 cells/dish)

Relative cloning efficiency = (cloning efficiency of treatment group/ cloning efficiency of solvent group) X 100

Non-activated (-S9) Study using Sodium chlorate

Non-activated -S9 mix
Treatment (μg/L)	Subset	Plate counts			Average colonies	Cloning efficiency
		1	2	3
solvent	A	89	72	75	69.3	0.69
	B	55	56	69
EMS	A	45	73	50	55.3	0.55
	B	48	60	56
10	A	66	99	72	74.5	0.75
	B	69	88	53
33	A	78	107	94	75.2	0.75
	B	50	59	63
100	A	89	83	76	75.3	0.75
	B	65	73	66
333	A	77	60	85	68.2	0.68
	B	63	56	68
1000	A	73	66	56	65.5	0.66
	B	67	60	71
5000	A	75	69	83	64.7	0.65
	B	54	67	40

Selection (Mutation) plates

Treatment (μg/L)	subset	Plate counts
		1	2	3	4	5	Average colonies	Mutants/10E6 clonable cells
solvent	A	0	0	0	0	0	0	0
	B	0	0	0	0	0
EMS	A	48	44	43	42	47	43.6	394.0
	B	55	43	38	35	41
10	A	1	1	0	0	1	0.4	2.7
	B	1	0	0	0	0
33	A	0	0	0	0	0	0.4	2.7
	B	1	1	1	0	1
100	A	0	0	0	0	0	0.5	3.3
	B	0	0	2	1	2
333	A	0	1	0	1	0	0.2	1.5
	B	0	0	0	0	0
1000	A	0	0	0	0	0	0.1	0.8
	B	0	0	0	1	0
5000	A	0	0	0	0	0	0.5	3.9
	B	0	0	2	3	0

Activated (-S9) Study using Sodium chlorate

Non-activated -S9 mix
Treatment (μg/L)	Subset	Plate counts			Average colonies	Cloning efficiency
		1	2	3
solvent	A	65	74	73	64.8	0.65
	B	60	64	53
B(a)P	A	52	54	61	52.8	0.53
	B	46	56	48
10	A	89	88	91	76.7	0.77
	B	63	70	59
33	A	68	56	55	62.3	0.62
	B	68	75	52
100	A	90	62	77	69.5	0.70
	B	62	72	54
333	A	48	46	56	59.7	0.60
	B	59	78	71
1000	A	47	85	78	67.0	0.67
	B	70	60	62
5000	A	70	60	79	66.0	0.66
	B	62	57	68

Selection (Mutation) plates

Treatment (μg/L)	subset	Plate counts
		1	2	3	4	5	Average colonies	Mutants/10E6 clonable cells
solvent	A	0	0	0	0	1	0.4	3.1
	B	0	1	1	0	1
B(a)P	A	33	40	31	26	34	23.5	222.4
	B	14	9	20	8	20
10	A	0	0	0	0	0	0.5	3.3
	B	2	0	1	0	2
33	A	0	0	0	0	0	0.8	6.4
	B	1	2	2	1	2
100	A	0	0	0	0	0	0	0
	B	0	0	0	0	0
333	A	0	1	0	0	0	0.2	1.7
	B	0	0	0	1	0
1000	A	1	0	1	1	0	0.3	2.2
	B	0	0	0	0	0
5000	A	7	4	8	8	7	3.4	25.8
	B	0	0	0	0	0

Cloning efficiency = average colonies/ 00 cells/dish

Mutants/10E6 clonable cells = (average mutant colonies/cloning efficiency X 2x10E5cells) x10E5

A and B are duplicate cultures

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, sodium chlorate was concluded to be negative because no treatment conditions exhibited mutant frequencies of >40 mutants per 106 cells.

Executive summary:

The test article, sodium chlorate, was tested under GLP and accoring to OECD 476 in the CHO/HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9. The mutagenesis assay was used to evaluate the mutagenic potential of the test article. Culture medium was chosen by the Sponsor as the solvent for the test article. The test article was soluble in culture medium at a concentration of 50 mg/mL, the maximum concentration prepared for the mutation assay. The concentrations chosen by the Sponsor for the mutagenesis assay ranged from 10 to 5000 µg/mL for both the non-activated and S9 -activated cultures. In the mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 10⁶ clonable cells, were observed. No visible precipitate was observed in treatment medium at any concentration. Toxicity, i.e., cloning efficiency ≤50% of the solvent control, was not observed at any concentration. Under the conditions of this study, test article sodium chlorate was concluded to be negative in the CHO/HGPRT Mutation Assay.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 1989 - 14 June 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed under GLP and according to standard protocol.

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Qualifier:

according to guideline

Guideline:

EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5550 (DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

GLP compliance:

yes

Type of assay:

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

Target gene:

No specific

Species / strain / cell type:

mammalian cell line, other: HeLa S3 cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
The medium used for the growth of the cells has the following composition:
E.M.E.M. (10x)
(Minimal Eagle Medium with Earle's salts) 45.0 ml
Sterile Water 383.1 ml
Sodium Bicarbonate (7.5%) 12.0 ml
Non-essential amino acids (100x) 4.5 ml
Glutamine (200nM) 4.5 ml
Antibiotic solution 0.9 ml
Foetal Calf Serum 50.0ml
= 500 ml growth medium

The medium used to limit replicative DNA synthesis (arginine free medium) has the following composition:
Arginine-free E.M.E.M. 489.0 ml
Antibiotic solution 1.0 ml
L-Glutamine (200 mM) 5.0ml
Non Essential Amino Acids (100x) 5.0 ml
Dialysed Foetal Calf Serum 12.8 ml
= 518.8 ml arginine free medium

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Cytotoxicty assay: 625, 1250, 2500, 5000, 10000 µg/ml
Main study: 100, 316, 1000, 3160 and 10000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: not stated

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9-mix: 4-NQO (4-Nitroquinoline-N-oxide: 5.00 µg/ml. +S9-mix: B(a)P: (Benzo(a)pyrene) 2.50 µg/ml.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicate cultures per dose

NUMBER OF CELLS EVALUATED: DNA is extracted from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: other: Culture vessels are seeded with HeLa S3 cells and grown to near-confluence. A wide range of concentrations of the test substance are then added to the cultures in the presence of hydroxyurea, both with and without S9 metabolism. The cultures are incubated for three hours, after which the treatment media are removed. The cells are washed with PBS (Phosphate Buffered Saline) and stained with trypan blue. The cells are examined by microscope for evidence of gross toxicity, loss of cells by detachment or lysis, morphological alterations and trypan blue exclusion index; cell counts are also performed. These parameters are used to detect different mechanisms of toxicity; for example, dye exclusion is an index of increased membrane permeability. Cell detachment is often a precursor of death.

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable

Evaluation criteria:

DNA Synthesis if a 50% increase in tritiated thymidine incorporation (per microgram of DNA) compared with the solvent control values is observed:
(i) at two consecutive dose-levels.
(ii) at a single dose-level if that is the highest dose-level which can be tested (as the result of solubility, toxicity, formulation, etc.).
The increases must be reproduced in an independent experiment. Evidence of a dose-response relationship will be considered as supportive evidence for genotoxic activity but will not be a requirement for a positive result.

Statistics:

none

Species / strain:

mammalian cell line, other: HeLa S3 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

not cytotoxic up to 10000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

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: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: No signs of toxixty were observed up to 10000 µg/ml

COMPARISON WITH HISTORICAL CONTROL DATA: The values for the solvent control were well within the mean values for the historical solvent control for this lab.

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

GENOTOXIC EFFECTS: 
- With metabolic activation: In the first experiment  radiolabel incorporation decreased mildly over the entire dose-range, indicating a toxic effect of the test substance
treatment. In the second experiment radiolabel uptake was similar to or slightly below the control values at all dose-levels. The positive controls induced marked increases.
- Without metabolic activation: In the first experiment incorporation of radiolabel decreased over the entire dose-range reaching approximately 40% of the control value
after treatment at the maximum dose-level. This depression in radiolabel uptake is indicative of test substance induced toxicity. In the second experiment the same tendency was
observed but the depression was less pronounced. The positive controls induced marked increases.
FREQUENCY OF EFFECTS: -
PRECIPITATION CONCENTRATION: -
MITOTIC INDEX: Not applicable
CYTOTOXIC CONCENTRATION: Sodium chlorate was not markeldy cytotoxic at any dose level, highest 10000 µg/ml.  
- With metabolic activation: Sodium chlorate was not markedly cytotoxic at any dose level, highest 10000 µg/ml
- Without metabolic activation: Sodium chlorate was not markedly cytotoxic at any dose level, highest 10000 µg/ml
dose
(microg/ml) S9 % cell loss % viable cells
0           -    0           100
625         -    0            98
1250        -    0            99 
2500        -    0           100
5000        -    10          100 
10000       -    12          100 

dose
(microg/ml) S9 % cell loss % viable cells
0           +    0            99
313         +    3           100 
625         +    7           100
1250        +    9           100 
2500        +    14           99
5000        +    19          100 
10000       +    17          100 

TEST-SPECIFIC CONFOUNDING FACTORS: -
STATISTICAL RESULTS:
statistically significant effects,  Experiment (in absence and presence of S9-mix): Incorporation was generally higher in the second experiment.
Dose level (in absence and presence of S9-mix): Incorporation decreased in a dose dependent matter, indicating a toxic effect of the test material.

The test substance did not induce any icrease in tritiated thymidine incorporation to a value 50% greater than the control values.

Conclusions:

Treatment with the test substance did not induce increases in UDS to values 50% greater than the controls at any dose-level in either experiment. Treatment with the test material in both the absence and presence of S9 metabolism caused some depression of radiolabel uptake, indicating a toxic effect on the cells. Marked increases in UDS were observed following treatment with the positive control compounds, indicating the correct functioning of the assay system. It is concluded that Sodium Chlorate does not induce unscheduled DNA synthesis in HeLa S3 cells in vitro, either in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test material Sodium Chlorate was examined for genotoxic properties by assay for the induction of unscheduled DNA synthesis (UDS) in cultured human cells (HeLa S3) both in the absence and presence of S9 metabolic activation. The study was performed under GLP and according to internationally accepted guidelined.

A preliminary cytotoxicity test was performed. The test substance was assayed at a maximum dose-level of 10000 µg/ml (the highest concentration indicated for testing in the Study Protocol ) and a wide range of lower dose-levels spaced by two-fold dilutions. Treatment with the test substance was not toxic and the same concentration was selected as the highest dose-level s to be used in both UDS assays in the absence and presence of S9 metabolism.

Two independent assays for the induction of UDS were performed using dose-levels of 10000, 3160, 1000, 316 and 100 µg/ml. The same dose-levels were used both i n the absence and presence of S9 metabolic activation. Cultures of HeLa S3 cells were pre-treated with low-serum

arginine-free medium and prior to treatment replicative DNA synthesis was blocked with hydroxyurea. After treatment with the test material in the presence of tritiated thymidine, DNA was extracted from the cultures, and radiolabel incorporation was measured by scintillation counting. The values for incorporation were corrected for the amount of DNA recovered, and unscheduled DNA synthesis was expressed as tritiated thymidine incorporation per microgram DNA.

Treatment with the test substance did not induce increases in UDS to values 50% greater than the controls at any dose-level in either experiment. Treatment with the test material in both the absence and presence of S9 metabolism caused some depression of radiolabel uptake, indicating a toxic effect on the cells. Marked increases in UDS were observed following treatment with the positive control compounds, indicating the correct functioning of the assay system.

It is concluded that Sodium Chlorate does not induce unscheduled DNA synthesis in HeLa S3 cells in vitro, either in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

IN VIVO GENETIC TOXICITY

Four Micronucleus studies

- Mackay (OECD 474, 1989, GLP, K, rel. 1): no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of sodium chlorate

- NTP (eq. to OECD 474, 1999, SS, rel. 3): no increases in the frequencies of micronucleated NCEs were seen in peripheral blood samples from male and female B6C3F1 mice exposed to concentrations of 125 to 2,000 mg/L sodium chlorate in drinking water for 3 weeks.

- Meier (No guideline followed/publication, 1985, rel.3): Negative

- Gocke (No guideline followed/publication, 1981, rel.4): Negative

 

A Chromosome aberration test

Meier (No guideline followed/publication, 1985, rel.3): Negative

 

A chromosomal damage and sperm-head abnormalities in mice

Meier (No guideline followed/publication, 1985, rel.3): Negative

 

A Drosophila T34-12 Basic test

(Gocke, 1981, rel.4) : Positive

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 1989 - 10 March 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to internationally accepted guidelines and under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK) , Margate, Kent, England.
- Age at study initiation: 4-5 weeks
- Weight at study initiation: preliminary study: 27-30 g for males and 19-24 g for females. main study: 23-28 g for males and 19-25 g for females.
- Assigned to test groups randomly: Yes, under following basis: All mice were identified by ear-notch number codes, numbers being assigned using a set of computer-generated random numbers. Cage labels, identifying the occupants by experiment, animal numbers, sex and treatment group, were attached to all cages.
- Fasting period before study: no data
- Housing: single-sex groups of two or five in high density polypropylene cages with stainless steel tops.
- Diet (e.g. ad libitum): Laboratory animal diet LAD 1 (Manea, Cambridgeshire, England) was fed ad libitum throughout the study.
- Water (e.g. ad libitum): Drinking water was supplied to each cage via a polythene bottle and sipper-tube.
- Acclimation period: at least 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 15 February 1989 - 10 March 1989

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: dustilled water
- Justification for choice of solvent/vehicle: sodium chlorate is soluble in water
- Concentration of test material in vehicle: 20 g/L, 100 g/L, 5000 g/L.
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): not applicable
- Purity:not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was found to be soluble in distilled water to a concentration in excess of 700 mg/ml. Consequently, dosing solutions were freshly prepared in distilled water on the day of dosing; the solution of maximum concentration was initially prepared and the lower concentrations required were prepared from this by serial dilution in distilled water.

Duration of treatment / exposure:

Examination 24, 48 and 72h after administration

Frequency of treatment:

Single dose

Post exposure period:

None

Remarks:

Doses / Concentrations:
200, 1000 and 5000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Positive control(s):

chlorambucil (Sigma Chemical Company)
- Justification for choice of positive control(s): know positive chemical
- Route of administration: oral
- Doses / concentrations: A suspension of chlorambucil (Sigma Chemical Company) in aqueous 10% ethanol was prepared immediately prior to dosing, for use as a positive control. The positive control agent, chlorambucil, was administered orally, at a dosage of 30 mg/kg.

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary toxixty test was performed.
The toxicity of sodium chlorate to dividing bone marrow erythrocytes was examined : slide evaluation was restricted to determination of numbers of polychromatic and mature erythrocytes and calculation of the ratio of polychromatic to mature cells for each animal, and for each group. The highest level tested was the maximum stated in the Study Protocol. Dosing was by the oral route, on one occasion and at a volume-dosage of 10 ml/kg.
animals were killed 72 hours after treatment. Four groups with 2/animals per sex were dosed with 625, 1250, 2500 or 5000 mg/kg bw sodium chlorate.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Preparations of the test compound or dosing vehicle (distilled water) were administered once, by the oral route, to groups of male and female mice. The volume-dosage was 10 ml/kg. The positive control agent, chlorambucil, was administered orally, at a dosage of 30 mg/kg in aqueous 10% ethanol.Five male and five female mice per group were killed 24 hours after treatment; further lots of five males and five females from groups 1 to 4 were kiled 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION:
Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the
cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa) staining technique. When air-dried, permanent mounts were made using DPX mountant, after clearing for five minutes in xylene.

METHOD OF ANALYSIS:
Preliminary toxicity test
The slides were examined under the light microscope, and regions judged to be of adequate technical quality to permit scoring were selected under low magnification. At high magnification (x 1000, oil immersion) a total of at least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature: polychromatic cells stain blue/pink and the older cells stain red/pink. At least 1000 cells of each type were scored from each animal where possible, but where there was an appreciable deviation from unity in the ratio of polychromatic to mature until a minimum of 2000 of counted.

Main micronucleus test
One slide from each animal not subsequently involved erythrocytes, scoring continued the predominant cell type were was randomly coded by a person n the scoring o f the study. Care was taken to ensure that no unique slide identifications remained visible in order to eliminate bias. Slides were examined as detailed for the preliminary toxicity test, but in addition each erythrocyte scored was examined for the presence or absence of micronuclei. When examination had been completed, the slides were decoded. The resultant data were used to calculate the number of micronucleated cells per 1000 erythrocytes. The ratio of polychromatic to mature cells was also determined; a decrease in this may indicate inhibition of cell division following treatment, and the incidence of micronuclei in the mature cell population 24 hours after treatment reflects the pretreatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.
OTHER: Animals were inspected daily throughout the acclimatisation period and the dosing period for signs o f ill-health or reaction to treatment. Any deviation from normal was recorded. All animals were weighed on the day of treatment and again immediately before termination, and bodyweights were recorded. In addition, the animals in the preliminary toxicity test were weighed immediately prior to dosing and daily thereafter until termination.

Evaluation criteria:

See above and below.

Statistics:

Using the frequency of micronucleated cells per 1000 polychromatic erythrocytes scored, the data were subjected to statistical analysis by the Mann-Whitney procedure. (Mann and Whitney, 1942). A computer-based version of this test was employed and significance was determined by reference to tabulated values of R1. Data from males and females within each group were compared. As there was no significant difference within each group, the sexes were pooled for further analysis. For each sampling time (24, 48 or 72 hours), each treated group was compared with concurrent vehicle controls.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

No indication of bone marrow toxicity was noted in any group treated with sodium chlorate. However clinical signs at 2500 and 5000 mg/kg bw in the prelimininary test and at 5000 mg/kg bw in the main test demonstrated systemic exposure to the test item.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Evidence of toxicity of sodium chlorate was noted in both males dosed with sodium chlorate at 5000 mg/kg; signs included hunched posture, rapid respiration and piloerection on days 3 and 4 of the study. Hunched posture and piloerection on days 3 and 4 were also noted in one male dosed with sodium chlorate at 2500 mg/kg.
Bodyweights were recorded at dosing and daily there after until termination; incidences o f weight loss were noted, but these were small and not dose-related. The ratios o f polychromatic: mature erythrocytes as evidenced by depression in bone marrow proliferation) to the bone marrow of the treated mice. There was no evidence of toxicity of sodium chlorate. After consideration of these data, the highest sodium chlorate dosage selected for the main micronucleus test was 5000 mg/kg.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): No significant differences in the frequencies of micronucleated polychromatic cells were seen between sexes, in any group.
Among mice killed 24 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells scored) was 0.5 for the vehicle control group, with a range of 0.0-2.0. Corresponding values for animals given sodium chlorate at 200, 1000 or 5000 mg/kg were closely similar: 0.5, 0.8 or 0.7, with ranges of 0.0-1.9, 0.0-3.0 and 0.0-2.8 respectively.
Among mice killed 48 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells scored) was 0.4 for the vehicle control group, with a range of 0.0-1.0. Corresponding values for animals given sodium chlorate at 200, 1000 or 5000 mg/kg were 0.5, 0.5 or 0.4, with ranges of 0.0-1.8, 0.0-1.0 and 0.0-1.0 respectively.
Among mice killed 72 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells scored) was 0.2 for the vehicle control group, with a range of 0.0-1.0. Corresponding values for animals given sodium chlorate at 200, 1000 or 5000 mg/kg were 0.6, 0.6 or 1.0, with ranges of 0.0-1.9, 0.0-2.0 and 0.0-2.0 respectively.
Thus, mean values for sodium chlorate treated groups were closely similar to mean control group values at all termination times.

Chlorambucil treatment produced a range of micronucleated cells per 1000 polychromatic erythrocytes from 31 .0-62.9 with a mean of 43.4. Statistical analysis showed that animal s treated with chlorambucil had significantly more micronucleated polychromatic cells than vehicle controls (p < 0.01). This increase in chromosomal damage after exposure to a known mutagen demonstrates the sensitivity of the test system.

- Ratio of PCE/NCE (for Micronucleus assay):
The recorded incidence of micronuclei per 1000 mature erythrocytes varied between 0.0 and 2.7 throughout all groups. These findings demonstrate the normal status of the animals used in the study: in particular, the low incidence in animals killed 24 hours after treatment shows the absence of any pre-treatment abnormality in the bone marrow.

The ratio of polychromatic to mature erythrocytes was 1.0 in the vehicle control group at 24 hours. Ratios for groups given sodium chlorate at 200, 1000 or 5000 mg/kg and terminated 24 hours post-dose were also 1.0. Forty eight hours after treatment, the ratio of the vehicle control group was 1.0, and in animals given sodium chlorate at 200, 1000 or 5000 mg/kg it was 1.0, 0.9 and 0.9 respectively.
Seventy two hours after treatment, the ratio of polychromatic to mature erythrocytes in the vehicle control group was 0.9, and in animals given sodium chlorate at 200, 1000 or 5000 mg/kg it was 0.9, 0.9 and 1.0 respectively. Ratios for all treatment groups were therefore closely similar to those of their respective vehicle control groups.
In animals treated with chlorambucil, the ratio between polychromatic and mature erythrocytes was reduced to 0.7.

- Appropriateness of dose levels and route: Toxicty to the bone marrow was not observed however e.g. no direct proof of exposure was provided but the presence of clinical signs at 2500 and 5000 mg/kg bw in the prelimininary test and at 5000 mg/kg bw in the main test was considered to demonstrate systemic exposure to the test item.

- Statistical evaluation: Statistical analysis confirmed that there was no significant difference between the vehicle control group and any sodium chlorate treated group, at any termination time (p > 0.05).

Conclusions:

It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of sodium chlorate. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.

Executive summary:

The effect of sodium chlorate on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. The study was performed under GLP and according to OECD474. Chromosome damage was measured indirectly by counting micronuclei. A preliminary toxicity test was first conducted, using dosages of 625, 1250, 2500 and 5000 mg/kg. Subsequently, male and female mice were given a single dose of sodium chlorate at 200, 1000 or 5000 mg/kg. In all cases sodium chlorate was dosed orally, dissolved in distilled water. Concurrent vehicle and positive control groups of mice were similarly dosed with distilled water or chlorambucil (30 mg/kg) respectively. Five males and five

females from each group were killed 24 hours after treatment; further lots of five males and five females, given sodium chlorate at 200, 1000 or 5000 mg/kg or the vehicle control, were killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.

A total of at least 2000 erythrocytes per animal was then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. The ratio of polychromatic:mature cells was also calculated for each animal, as an indicator of gross toxicity.

No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with sodium chlorate. Evidence of toxicity was noted in five male animals dosed with sodium chlorate at 5000 mg/kg; signs included hunched posture and slight piloerection on day 4 of the study. No adverse reactions to treatment were recorded for any other group treated with either sodium chlorate or the vehicle control.

Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of sodium chlorate were similar to those in concurrent controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Statistically significant increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg (p<0.01).

It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of sodium chlorate. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of in vitro genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 NTP	Ames Test (eq. to OECD 471) K, rel. 2, 1999	Bacterial gene mutation	TA97,TA98 TA100, TA102, TA104, TA1535	-S9 +S9	100 to 10,000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
2 May	Ames Test (OECD 471, 1989, GLP, SS, rel. 2)	Bacterial gene mutation	TA98, TA1538, TA100,TA1535 and TA1537	-S9 +S9	Up to 5000 micrograms/plate	-S9 : not mutagenic +S9 : not mutagenic
3 Hossack	Ames Test (No guideline followed/ publication, 1978, rel.3)	Bacterial gene mutation	TA1537, TA98 & TA100	-S9 +S9	10, 100, 1000, 10000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
4 Gocke	Ames Test (No guideline followed/ publication, 1981, rel.4	Bacterial gene mutation	TA1535a, TA1535b, TA1538, TA1537, TA98 & TA100	-S9 +S9	Up to 3600 microgram/plate	-S9 : mutagenic +S9 : mutagenic The frequency of revertants in strains TA1535 A and B was approximately doubled on VB medium at a dose of 12 µmol/plate
5 May	E.Coli-DNA-Lethality (No guideline followed, 1989, rel.2)	Gene mutation	E.coli strain WP2 WP67 and CM871	-S9 +S9 : not validated	10, 316, 1000, 3160 and 10000 µg/ml	-S9 : responses suggestive of DNA damage in E.coli strain WP67 and CM871 +S9 : not validated
6 Eckhardt	Publication/ Abstract only with limited information, 1982, rel.4	Ames test Micronucleus test on mouse bone marrow Recessive-lethal test on Drosophila	Not specified	-S9 +S9	Not specified	Ames test : Positive on TA1535 with metabolic activation Drosophila test system: Positive Micronucleus test: Negative
7 Clarke	HGPRT/CHO (OECD 476, 2007, GLP, K, rel. 1)	Mammalian gene mutation	Chinese Hamster ovary cells	-S9 +S9	10, 33, 100, 333, 1000, and 5000 µg/mL	-S9 : non mutagenic +S9 : non mutagenic
8 Hodson-Walker	HGPRT/CHO (comparable to OECD 476, 1989, GLP, K, rel.1)	Mammalian gene mutation	Chinese Hamster lungs fibroblasts cells (V79)	-S9 +S9	8, 40, 200, 1000 and 5000 µg/ml	-S9 : non mutagenic +S9 : non mutagenic
9 Seeberg	DNA damage and/or repair study (OECD 482, GLP, K, rel. 1)	UDS	HeLa S3 cells	-S9 +S9	100, 316, 1000, 3160 and 10000 µg/ml	-S9 : non mutagenic +S9 : non mutagenic

 

Table 7.6/1: Summary of in vivo genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 Mackay	Micronucleus assay (OECD 474,1989, GLP, K, rel. 1)	Chromosomal aberration	Bone marrow erythrocytes cells	NA	200, 1000 or 5000 mg/kg	Not clastogenic
2 NTP	Micronucleus assay (eq. to OECD 474, 1999, SS, rel. 3)	Chromosomal aberration	Bone marrow cells	NA	125 to 2,000 mg/L	Not clastogenic
3 Gocke	Micronucleus assay (No guideline followed/ publication, 1981, rel.4)	Chromosomal aberration	Bone marrow cells	NA	530, 1060 and 2120 mg/kg ip and 2128, 3192 and 4265 mg/kg oral	Not clastogenic
4 Meier	Micronucleus assay (No guideline followed/ publication, 1985, rel.3)	Chromosomal aberration	Bone marrow cells	NA	200, 500, 1000 mg/L (6.67, 16.67 and 33.33  mg/kg bw)	Not clastogenic
5 Meier	Chromosome abberation (No guideline followed/ publication, 1985, rel.3)	Chromosomal aberration	Human lymphocytes	-S9 +S9	200, 500, 1000 mg/L (6.67, 16.67 and 33.33  mg/kg bw)	-S9 : not clastogenic +S9 : not clastogenic
6 Meier	Sex-linked recessive lethal test (No guideline followed/ publication, 1981, rel.4)	Chromosomal aberration	Drosophila Melanogaster	NA	Not specified	Positive

Sodium chlorate was not mutagenic in the majority of in vitro studies and in all in vivo systems tested. Sodium chlorate did not induce gene mutations in either bacteria or mammalian cells but the results in E.coli are suggestive of a primary DNA damage with no requirement for metabolic activation. This effect was not confirmed in mammalian HeLa S3 cells in vitro where there was no evidence of unscheduled DNA synthesis. Mechanisms of DNA reparation in bacteria being different from those in mammals, the results observed in mammals are considered more relevant for risk assessment.

In the absence of in vivo genotoxicity in somatic, it was therefore concluded that limited evidence of genotoxicity in vitro in bacteria was not relevant to the in vivo situation.

Therefore, based on both in vitro and in vivo studies, there are sufficient reliable data indicating that sodium chlorate has no genotoxic potential.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

 

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.