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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read across approach

In the absence of substance-specific data, the genetic toxicity of diarsenic trioxide is assessed based on reviews and/or data for inorganic arsenic compounds.

Diarsenic trioxide is readily soluble in water (17.8 g/L at 20°C). Upon dissolution in water, it reacts acidically to trivalent arsenite ions which are not subject to any relevant degree of oxidation for up to 72 hours (Klawonn, 2010). Read-across from toxicological data on inorganic arsenites to diarsenic trioxide is justified without restrictions. However, it is also known that in the human body, inorganic arsenic compounds are converted apart from As(III) also to As(V). Upon becoming systemically available, As(V) is rapidly partly converted to As(III). As(III) species are considered to be more toxic and bioactive than As(V) species. The difference in toxicological potency between As(III) and As(V) cannot be quantified exactly and may vary between routes of exposure and/or type of toxicological effects. Generally, risk assessments are conducted for "inorganic arsenic compounds" as a group, and do not differentiate between various species.Following a conservative approach, the toxicity of diarsenic trioxide is therefore considered to be determined by the release of soluble inorganic species (trivalent arsenites and pentavalent arsenates) which do not differ substantially in potency and may be interconverted both in the environment and in the body. Consequently, it is justified to apply read across to soluble inorganic arsenic compounds to evaluate the systemic effects, including genetic toxicity, of diarsenic trioxide.

General remarks

A large number of investigations on the genetic toxicity of inorganic arsenic compounds are available, and these have been reviewed on several occasions by renowned scientific organizations. Given the overwhelming volume of information, individual Robust Study Summaries (RSS) were not developed. Instead, the following paragraphs summarise the outcome of existing in vitro and in vivo studies.

In vitro genetic toxicity

Mutagenesis in bacterial and mammalian cells:

Studies evaluating direct mutagenesis such as the Ames assay have generally been negative, particularly those conducted under guidelines currently in place (Tsuji et al., 2019).

Readily bioavailable arsenicals such as trivalent arsenites for example were not mutagenic when tested in Salmonella typhimurium (Löfroth and Ames, 1978) or Escherichia coli (Rossman et al., 1980). Arsenite also does not induce point mutation in mammalian cell systems. The overwhelming majority of assays conducted in mouse lymphoma cells, Chinese hamster V79 cells, Chinese hamster ovary cells and Syrian hamster embryo cells yielded negative results (see for example Lee et al., 1985) (ATSDR, 2007).

Furthermore, Nesnow et al. (2002) clearly demonstrated that arsenicals do not react directly with DNA and based on chemical principles, such a reaction is highly unlikely (interaction with DNA requires reactive electrophiles and in some instances reactive free radicals, whereas inorganic arsenic and its metabolites have an anionic structure and therefore cannot react with the ionic nucleotides of the DNA (Cohen et al., 2013)).

Cytogenicity in mammalian cells:

A vast amount of positive results obtained in vitro in cytogenicity assays with human fibroblasts, lymphocytes and leukocytes, mouse lymphoma cells, Chinese hamster ovary cells and Syrian hamster embryo cells demonstrate that trivalent inorganic arsenic compounds can induce indirect damage to DNA, including chromosomal aberrations, micronucleus formation and sister chromatid exchange (see for example Oberly et al., 1982 and Lee et al., 1985). Likewise, in vitro studies in human, mouse and hamster cells have also been positive for DNA damage and repair, as well as enhancement or inhibition of DNA synthesis.

Positive results for arsenic in these assays are manifested at cytotoxic concentration. Cytotoxicity is not always demonstrated in the specific assays, or is considered low (<10%), but the assays are usually being performed for relatively short periods of time (4-24 h), which are too short for cell death to be completed. Cell death following toxicity is evidenced by the presence of necrosis, when treatment or observation is extended to longer periods of time (Cohen et al., 2013).

In vivo genetic toxicity

Numerous published data are available on in vivo studies in animals via the oral route, including rat and mouse bone marrow assays. There is also human (oral exposure) data investigating chromosomal aberrations or micronuclei formation and sister chromatid exchanges in peripheral lymphocytes (see for example Das et al., 2016 and Deknudt et al., 1986). The overall positive results of these studies confirm the clastogenicity of trivalent inorganic arsenic species (ATSDR, 2007).

Human and animal data are similarly available indicating that inorganic arsenic is clastogenic also via inhalation exposure. Most noteworthy, workers exposed to unspecified concentrations of arsenic trioxide at the Ronnskar copper smelter in Sweden were found to have a significant increase in the frequency of chromosomal aberrations in peripheral lymphocytes (Beckman et al., 1977; Nordenson et al., 1978).

Overall conclusion

Based on a wide body of available evidence, inorganic arsenic compounds do not appear to be directly mutagenic but may cause indirect DNA damage, including chromosomal aberrations, micronucleus formation and sister chromatid exchanges in vitro and in vivo.

The two most plausible processes that could lead to indirect genotoxicity are inhibition of DNA repair and interaction with the protein involved in the formation of the mitotic spindle (Cohen et al., 2013). Induction of oxidative stress and changes in DNA methylation patterns have also been discussed as underlying mechanisms (Beyersmann and Hartwig, 2008).

Overall, it is assumed that a threshold exists for these indirect effects (Hassauer and Kalberlah, 2010). This is supported by a meta-analysis of available genotoxicity data by Rudel et al. (1996), who concluded that the dose-response relationships for the majority of the observed genotoxic effects are seemingly sublinear.

Finally, the indirect toxicity was observed to occur generally at high concentrations, above those that would be attained systemically in animals and humans. Therefore, as further discussed in the following section, this does not seem to be the basis for the carcinogenicity of arsenicals, either in animals or in humans, particularly at lower doses (Tsuji et al., 2019).

​References not cited in ATSDR (2007)

Beyersmann D and Hartwig A (2008). Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms. Arch. Toxicol. 82:493-512.

Cohen SM et al. (2013). Evaluation of the carcinogenicity of inorganic arsenic. Crit. Rev. Toxicol. 43(9):711-752.

Hassauer M and Kalberlah F (2010). Erarbeitung einer Expositions-Risiko-Beziehung (ERB) für Arsen (review article or handbook), Forschungs- und Beratungsinstitut Gefahrstoffe GmbH (FoBiG), Freiburg, Germany, on behalf of Berufsgenossenschaft der chemischen Industrie, April 2010 (1st draft).

Nesnow S et al. (2002).DNA damage induced by methylated trivalent arsenicals is mediated by reactive oxygen species. Chem. Res. Toxicol. 15:1627-1634.

Rudel R et al. (1996). Implications of arsenic genotoxicity for dose response of carcinogenic effects. Reg. Toxicol. Pharmacol. 23:87-105.

Tsuji, JS et al.(2019). Dose-response for assessing the cancer risk of inorganic arsenic in drinking water: the scientific basis for use as a threshold. Crit. Rev. Toxicol. https://doi.org/10.1080/10408444.2019.1573804.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Studies described in the publication are acceptable for assessment.
Principles of method if other than guideline:
An investigation of the mutagenicity of sodium arsenite in E. coli was performed. Spot tests, treat and plate protocols, and fluctuation test were conducted. Also, the potential of λ prophage induction was tested.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
E. coli, other: WP2 (trpE), WP2s (trpE, uvrA), WP6 (trpE, polA1), WP10 (trpE, recA1), WP44s-NF (trpE, uvrA, tif-1/sfi-), WP44s-NF amp^r and WP2s (λ)
Metabolic activation:
not specified
Test concentrations with justification for top dose:
Spot test: 10 µL of 0.01M sodium arsenite (WP44s-NF (tif-1); 50 µL of 0.1 M sodium arsenite WP44s-NF, WP2, WP6, or WP10
Spot test: 50 and 100 µL of 0.1 M sodium arsenite
Treat and Plate Protocol: 25 mM sodium arsenite (WP2 & WP6)
Fluctuation test: 0.4, 1.0 and 2.0 mM arsenite (WP2)
Prophage induction: 1 and 10 mM arsenite (WP2s (λ))
Details on test system and experimental conditions:
MEDIA:
Cultures were grown in MST broth, consisting of Minimal Broth Davis (Difco) containing 0.2% glucose and 20 µg/mL tryptophan. Semi-enriched (SEM) plates contained Minimal Broth Davis (Difco), 0.2% glucose, and 5% nutrient broth (BBL), solidified with 1.5% agar. Nutrient plates contained nutrient broth solidified with 1.5% agar. Soft agar containing 6.5 mg Difco agar per liter of distilled water was ued to plate phage and bacteria.

BACTERIAL MUTAGENESIS
Plate assays for Trp+ revertants were performed by plating cells on SEM agar, according to the method of Witkin(1974)*. Plates were scored after three days of growth. Unless otherwise stated, the incubation temperature was 37°C.
Spot test for Trp+ revertants were performed by placing a sterile 12.7 mm diameter filter paper disc in the center of an SEM agar plate spread with approximately 2 X 10^7 bacteria. The test substance is added to the disc, and plates are scored for Trp+ revertants after three days of incubation at 37°C unless otherwise stated.
When toxic doses of arsenite are used, the bacteria can be exposed for only short periods of time and are then plated in the absence of arsenite (treat and plate protocol). these experiments were performed by treating a culture in exponential growth in MST broth with arsenite, removing samples at various times, centrifuging the cells, resuspending in the same volume of minimal A salts, and spreading 100 µL onto SEM agar plates.
Fluctuation tests for Trp+ revertants were carried out according to the method of Green et al. (1977)*.

INDUCTION OF λ PROPHAGE
The lysogen WP2s (λ) and indicator strain WP44s-NF-amp°r are grown in MST to midexponential phase. After treatment of the lysogen, infectious centers are assayed using a soft agar overlay on nutrient broth plates with or without 10 µg/mL ampicillin. Since the indicator strain WP44s-NF-amp^r carries the tif-1 mutation, lysogeny is not maintained well at 37°C and clear plaques result, allowing ease of scoring.

* References:
Witkin EM (1974): Thermal enhancement of ultraviolet mutability in a Tif-1 uvrA derivative of Escherichia coli B/r: Evidence that ultraviolet mutability in a Tif-1 uvrA derivative of function. Proc Natl Acad Sci 71: 1930-1934.
Green MHL, Rogers AM, Muriel WJ, Ward AC, McCalls DR (1977): Use of a simplified fluctuation test to detect and characterize mutagenesis by nitrofurans. Mutation Res 44: 139-143.
Species / strain:
E. coli, other: WP2, WP6, WP10 and WP44s-NF in spot test
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
E. coli, other: WP2s (λ) & WP44s-NF in spot test
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
E. coli, other: WP2 and WP6 in treat and plate protocol
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
This concentration of arsenite causes a complete inhibition of growth, and a decrease in viable count to 35% of the control value in one hour.
Species / strain:
E. coli, other: WP2 in fluctuation test
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
E. coli, other: WP2s (λ) in test on prophage induction
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
Interpretation of results (migrated information):
negative

Arsenite is not mutagenic to E. coli.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: No guideline was followed
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Arsenic trioxide
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
4 concentrations of arsenic trioxide with the top dose of 10 µg/ml were tested.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Cells
The mouse lymphoma L5178Y tk+/- cell clone 3.7.2C, supplied by Dr D. Clive (Burroughs Wellcome Co., Research Triangle Park, NC), was used in this study.

Experimental method
The cytotoxicity of the test substance was determined by relative survival (RS) after 3 h treatment at concentrations up to 5000 µg/mL with and without S9 mix, regardless of solubility. The highest concentration chosen was one with a 10-20% RS. There was no perceived need to test concentrations >5000 µg/ml. Each experiment consisted of one solvent control, one positive control and three test substance concentrations in duplicate cultures. As a rule, 2-fold serial dilutions were prepared from the highest concentration to obtain at least three graded concentration levels.

Mutation experiment
Cultures were exposed to the test substance for 3 h, then cultured for 2 days before plating in 96-well plates at 2000 cells/well with trifluorothymidine for mutant selection and at 1.6 cells/well for cell viability. Subsequently, the number of wells containing colonies was counted on day 12 after plating, and large and small colonies were scored and mutation frequencies were analysed.
Evaluation criteria:
The acceptable ranges of mean absolute plating efficiency (PE) for solvent control were 60-140% for survival and 70-130% for viability.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Conclusions:
Under the study conditions, arsenic trioxide was positive in a mouse lymphoma assay.
Executive summary:

A mouse lymphoma assay (MLA) was conducted by Japanese laboratories and seven overseas laboratories to clarify the performance of the MLA for the detection of in vitro clastogens and spindle poisons. The tests performed with arsenic trioxide (with the top dose of 10 µg/ml) were included in this study. Under the study conditions, arsenic trioxide was positive in a mouse lymphoma assay (Sofuni, 1996).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: No guideline was followed
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Sodium arsenite and sodium arsenate
Species / strain / cell type:
other: Syrian hamster embryo cell cultures (strain LSH/ss LAK)
Metabolic activation:
not specified
Test concentrations with justification for top dose:
4 concentrations of sodium arsenite and sodium arsenate were tested: 0.8, 3.0, 6.2, 10 µM and 10, 32, 64, 96 µM, respectively.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Details on test system and experimental conditions:
Cell culture
Syrian hamster embryo cell cultures were established from 13-day gestation fetuses collected aseptically by Caesarean section from inbred Syrian hamsters, strain LSH/ss LAK (Lakeview Hamster Colony, NJ). Cultures were routinely tested and found to be free of mycoplasma.

Cytogenetic assays
Tertiary-passage cells were inoculated into 75-cm2 flasks at 5 - 1 0 x 10^5 cells/flask. After overnight incubation, the culture medium was removed, 10 ml of either culture medium or medium containing 0.8, 3.0, 6.2 or 10 µM of sodium arsenite, or 10, 32, 64 or 96 µM of sodium arsenate were added to the flasks, and the cultures were then incubated. Chromosome preparations were made following colcemid (0.4 µ/ml) addition for the last 3 h of culture. The cells were trypsinized, collected by centrifugation, treated with 0.075 M KCl for 10 min, and fixed in methanol:acetic acid (3:1). The cells were dropped onto slides and stained with Giemsa (15% in 0.05 M phosphate buffer, pH 6.8, for 15 min). At least 100 metaphases at each dose were scored for numerical and structural changes. The experiments were repeated twice.
Sister chromatid exchanges were also examined 24 — 27 h after the initiation of 5-bromo-2'-deoxyuridine (BrdU) (Sigma Co., St. Louis, MO) treatment; BrdU (10 µg/ml) was added at the same time as the chemical treatment. Slides were stained by FPG treatment. The experiment was repeated three times.
Key result
Species / strain:
other: Syrian hamster embryo cell cultures (strain LSH/ss LAK)
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the study conditions, cell transformation and cytogenetic effects, including endoreduplication, chromosome aberrations, and sister chromatid exchanges were induced by the test substances, with similar dose-responses.
Executive summary:

A study was conducted to determine the potential cytogenetic effects of sodium arsenite and sodium arsenate in Syrian hamster embryo cell cultures. Four concentrations were tested: 0.8, 3.0, 6.2, 10 µM and 10, 32, 64, 96 µM, respectively. Cells in metaphase were harvested from cultures treated with the indicated dose of either chemical for 24 h (endoreduplication and chromosome aberrations) or 48 h (chromosome number and polyploid cells) and then fixed and scored for the abnormalities. The experiment was repeated three times. Under the study conditions, cell transformation and cytogenetic effects, including endoreduplication, chromosome aberrations, and sister chromatid exchanges were induced by the test substances, with similar dose-responses (Lee, 1985).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Abstract only, documentation not sufficient for assessment.
Principles of method if other than guideline:
Arsenite, As(III), or arsenate, As(V), have been tested for mutagenicity in the Salmonella plate incorporation test using different histidine-requiring strains which are reverted to prototrophy by different mechanisms.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium
Details on test system and experimental conditions:
Arsenite, As(III), or arsenate, As(V), have been tested for mutagenicity in the Salmonella plate incorporation test using different histidine-requiring strains which are reverted to prototrophy by different mechanisms.
Species / strain:
other: Salmonella typhimurium
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
No mutagenicity has been detected for arsenite, As(III), or arsenate, As(V); i.e. <0.01 revertants/nmole.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: No guideline was followed
GLP compliance:
not specified
Type of assay:
other: Syrian hamster embryo cells
Specific details on test material used for the study:
Sodium arsenite and sodium arsenate
Species / strain / cell type:
other: Syrian hamster embryo cell cultures (strain LSH/ss LAK)
Metabolic activation:
not specified
Test concentrations with justification for top dose:
4 concentrations of sodium arsenite and sodium arsenate were tested: 1, 3.1, 6.2, 10 µM and 10, 32, 64, 100 µM, respectively.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Cell culture
Syrian hamster embryo cell cultures were established from 13-day gestation fetuses collected aseptically by Caesarean section from inbred Syrian hamsters, strain LSH/ss LAK (Lakeview Hamster Colony, NJ). Cultures were routinely tested and found to be free of mycoplasma.

Gene mutation assays
Cells (2.5-5 x 10^5) were seeded into 75-cm2 flasks and after overnight incubation, treated with sodium arsenite, sodium arsenate, or benxo(a)pyrene in complete medium for 48 h. After treatment the cultures were washed twice with 10 ml of medium and replenished with fresh medium. After 24 h, the cells were sub-cultured at a split ratio of 1 —10. Following 3 days cultivation (4 days after treatment), 5000 cells were plated on 100-mm dishes and incubated for 7 days for colony formation. For mutation experiments, 10^5 cells were plated on each of 10-20 dishes (100 mm in diameter) with medium containing 3 ug/ml 6-thioguarune or 1.2 mM ouabain and incubated 7 days for colony formation.
Cells were also plated in new flasks at 5 x 10^5 cells/75-cm2 flask and grown for an additional 3 days expression time and then assayed for colony formation and thioguanine-, or ouabain-resistant mutants. Subsequently, the mutation frequency was calculated. The experiments were repeated three times.
Key result
Species / strain:
other: Syrian hamster embryo cell cultures (strain LSH/ss LAK)
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
Under the study conditions, no arsenic induced gene mutations were detected at two genetic loci.
Executive summary:

A study was conducted to determine gene mutation potential of sodium arsenite and sodium arsenate in Syrian hamster embryo cell cultures. Four concentrations ere tested: 1, 3.1, 6.2, 10 µM and 10, 32, 64, 100 µM, respectively. The cells were treated for 48 h with the indicated dose of chemicals, grown for 4 and 7 d, and then plated in selective media. Mutant colonies were selected in this media for 7 - 1 0 d and then the cells were fixed and stained and surviving TGr or OuaR colonies enumerated. Subsequently, the mutation frequency was measured and calculated. The experiment was repeated three times. Sodium arsenite and sodium arsenatewere observed to induce morphological transformation of Syrian hamster embryo cells in a dose-dependent manner. The trivalent sodium arsenite was greater than 10-fold more potent than the pentavalent sodium arsenate. The compounds also exhibited toxicity; however, transformation was observed at non-toxic as well as toxic doses. At low doses, enhanced colony-forming efficiency of the cells was observed. Under the study conditions, no arsenic-induced gene mutations were detected at two genetic loci (Lee, 1985).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: No guideline was followed
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Sodium arsenite and disodium hydrogen arsenate
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
5 concentrations of sodium arsenite and disodium hydrogen arsenate were tested: 0.5, 1.0, 1.5, 2.0, 2.5 µg/ml and 14, 18, 22, 26, 50 µg/ml, respectively.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile deionized glass-distilled water
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: N-2-fluorenylacetamide
Details on test system and experimental conditions:
Cell Culture

The TK+/- -3.7.2 heterozygote of L5178Y mouse lymphoma cells was used in this study. All cells were thawed from frozen stock and maintained in Fischer's medium for leukemic cells of mice containing 10% heat-inactivated horse serum, Pluronic F68, sodium pyruvate, penicillin G and streptomycin sulfate.
Background spontaneous TK+/- mutant frequencies were reduced weekly by 24-h treatment of the cells with medium containing thymidine, hypoxanthene, methotrexate and glycine.

Forward Mutation Assay

The test system was based on the procedure described by Clive et al. (1975, 1979) with modifications to the cloning procedure.
Sodium arsenite and disodium hydrogen arsenate were diluted in sterile glass-distilled water, and 0.1 ml of each dilution was added to a 10-ml suspension containing 6 X 10^6 cells from a culture recently cleansed of TK+/- cells. When testing with activation, the 10-ml suspension included 4 ml of an appropriate dilution of S9 with cofactor mix.
Cultures containing either test chemical, positive or negative controls were incubated for 4 h at 37°C. After exposure, the cells were washed twice, fresh medium was added, and the cultures were carried through a 2-day expression period and were counted after the first day. On the second day a modified cloning procedure was followed. The plates were then incubated at 37°C for approximately 12 d before they were counted.
Statistics:
The mutation frequency (MF) was calculated as the number of mutants per 10^5 colony-forming cells.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Additional information on results:
A weak positive responses, yielding 2- to 3-fold increases in mutation frequency above the solvent control at greater than 10% survival, were seen for both sodium arsenite and disodium hydrogen arsenate.

NaAsO2 evoked a weak response at survivals greater than 10% but did not require a metabolic activation system.
Na2HAs04 yielded no significant mutation in the absence of S9, yet a weak response was observed with S9 metabolic activation at 22 and 18 ug/ml.
Conclusions:
Under the study conditions, weak positive responses, yielding 2- to 3-fold increases in mutation frequency above the solvent control at greater than 10% survival, were seen for both sodium arsenite and disodium hydrogen arsenate.
Executive summary:

A study was conducted to determine potential of sodium arsenite and disodium hydrogen arsenate to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. Five concentrations of sodium arsenite and disodium hydrogen arsenate were tested: 0.5, 1.0, 1.5, 2.0, 2.5 µg/ml and 14, 18, 22, 26, 50 µg/ml, respectively. Cultures containing either test treatments, positive or negative controls were incubated for 4 h at 37°C. After exposure, the cells were washed twice, fresh medium was added, and the cultures were carried through a 2 d expression period and were counted after the first day. On the second day, a modified cloning procedure was followed. The plates were then incubated at 37°C for approximately 12 d before they were counted. Under the study conditions, weak positive responses, yielding 2- to 3-fold increases in mutation frequency above the solvent control at greater than 10% survival, were seen for both sodium arsenite and disodium hydrogen arsenate (Oberly, 1982).

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

See section on genetic toxicity in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: No guideline was followed
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
Sodium arsenite (CAS 7784-46-5)
Species:
mouse
Strain:
Balb/c
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Pasteur Institute, Shillong, Meghalaya, India
- Age at study initiation: 10–12 weeks
- Weight at study initiation: 20–25 g body weight
- Assigned to test groups randomly: yes, n = 6, 3 males and 3 females in each group
- Diet (e.g. ad libitum): Amrut Laboratory Animal Feeds, New Delhi
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 5°C
- Photoperiod (hrs dark / hrs light): 12 L:12 D cycles
Route of administration:
oral: drinking water
Vehicle:
The mode of administration of sodium arsenite was through drinking water.
Duration of treatment / exposure:
90 d
Dose / conc.:
0.2 mg/kg bw/day (nominal)
Remarks:
sodium arsenite
Dose / conc.:
2 mg/kg bw/day (nominal)
Remarks:
sodium arsenite
Details of tissue and slide preparation:
MN test in femur bone marrow cells were carried according to the method of Schmid with minor modifications.
Key result
Sex:
male/female
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the study conditions, both tested doses of sodium arsenite induced statistically significant micronucleated polychromatic erythrocytes as compared to the control group.

Executive summary:

A study was conducted to determine the genotoxicity of sodium arsenite by using the bone marrow micronucleus assay in Balb/c mice. The test substance was given to animals for a period of 90 days through drinking water at doses of 0.2 and 2 mg/kg bw/d. Genotoxicity was evaluated by studying the incidence of micronucleated polychromatic erythrocytes from bone marrow. Under the study conditions, both tested doses of sodium arsenite induced statistically significant micronucleated polychromatic erythrocytes as compared to the control group (Das, 2016).

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: No guideline was followed
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
Sodium meta-arsenite (NaAsO2)
Species:
mouse
Strain:
Balb/c
Details on test animals or test system and environmental conditions:
For all experiments, 10-week-old mice of the BALB/c strain, ranging in weight between 27 and 29 g, were used.
Route of administration:
intraperitoneal
Details on exposure:
Bone marrow preparations were made according to the method described by Schmid (1976). In a first experiment, animals were killed 24, 30 or 48 h after intraperitoneal injection with 10 mg/kg NaAsO2. In a second experiment the observations were performed 30 h after increasing doses of NaAsO2 ranging from 0, 0.5, 2.5, 5.0 to 10.0 mg/kg or with cyclophosphamide (150 mg/kg).
Duration of treatment / exposure:
1st experiment: 24 h; 30 h; or 48 h
2nd experiment: 30 h
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
NaAsO2 (1st experiment)
Dose / conc.:
0.5 mg/kg bw (total dose)
Remarks:
NaAsO2 (2nd experiment)
Dose / conc.:
2.5 mg/kg bw (total dose)
Remarks:
NaAsO2 (2nd experiment)
Dose / conc.:
5 mg/kg bw (total dose)
Remarks:
NaAsO2 (2nd experiment)
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
NaAsO2 (2nd experiment)
No. of animals per sex per dose:
4 animals per group
Control animals:
yes, concurrent no treatment
Positive control(s):
cyclophosphamide (150 mg/kg)
Tissues and cell types examined:
1000 polychromatic erythrocytes per mouse were analysed for the induction of micronuclei.
Key result
Sex:
not specified
Genotoxicity:
positive
Toxicity:
no effects
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the study conditions, the dose-related linear increase of micronuclei observed in somatic cells, indicated that the test substance displays clastogenic properties in vivo.
Executive summary:

A study was conducted to determine the ability of sodium meta-arsenite (NaAsO2) to produce genetic damage in vivo in mice by the micronucleus test on bone marrow cells. Four 10-week-old animals were treated per group. In a first experiment, animals were killed 24, 30 or 48 h after intraperitoneal injection with 10 mg/kg NaAsO2. In a second experiment the observations were performed 30 h after increasing doses of NaAsO2 ranging from 0, 0.5, 2.5, 5.0 to 10.0 mg/kg. Under the study conditions, the dose-related linear increase of micronuclei observed in somatic cells, indicated that the test substance displays clastogenic properties in vivo (Deknudt, 1986).

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: No guideline was followed
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
Arsenic trioxide (CAS No. 1327-53-2)
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: six to eight weeks old
- Assigned to test groups randomly: yes, the animals were segregated into various experimental groups with six animals (three males and the three females) in each group
Route of administration:
oral: gavage
Details on exposure:
The arsenic trioxide the most widely used commercial compound of arsenic, was used as source of arsenic exposure in mice. The stock solution of arsenic trioxide was prepared in 1 M NaOH which was further diluted with water to obtain aqueous solutions of three different concentrations of arsenic (30 ug/L, 150 ug/L and 1.5 mg/L). The measurement of arsenic contents in the aqueous solutions of arsenic trioxide was done by the atomic absorption spectrophotometric method (APHA, 1998).
Duration of treatment / exposure:
15 d
Frequency of treatment:
daily
Dose / conc.:
0.3 other: µg/kg/d
Dose / conc.:
1.5 other: µg/kg/d
Dose / conc.:
15 other: µg/kg/d
No. of animals per sex per dose:
3
Control animals:
yes, concurrent no treatment
Details of tissue and slide preparation:
Slides were prepared directly by smears of the suspension of bone marrow cells of mice (Schmid, 1975; Das and Kar, 1980). Animals were sacrificed 24 h after the last treatment, and bone marrow cells were harvested from their femura in chilled hypotonic KCl solution. The cell suspension was centrifuged immediately and all but a little portion of the supernatant was discarded. Cells were resuspended in the remaining fluid. Smears of bone marrow cells were made by using the slide-drawn method, air dried and then fixed in methanol. Slides were first stained in May-Gruenwald’s solutions followed by Giemsa staining, rinsed in running tap water and then air-dried again.
Evaluation criteria:
Maturing enucleated erythrocytes were screened for the presence of micronuclei (MN) in them because of the unambiguity of MN in otherwise empty erythrocytes. Staining with May-Gruenwald-cum-Giemsa solution further allowed the unequivocal identification of chromatin containing micronuclei as well as the differentiation of RNA containing newly formed erythrocytes from the older erythrocytes lacking RNA.

The slides were initially prescreened for areas rich in polychromatic erythrocytes of correct morphology, staining and spacing. About 1000 PCEs and corresponding number of NCEs (encountered in the same optical field) were recorded for each animal of an experimental group for the presence of micronuclei in them.
For the purpose of quantification, a micronucleated cell was considered as a unit irrespective of the number of micronuclei present in it. PCEs/NCEs ratio was also taken into consideration for the each group to register any cytotoxic effect on the turnover of PCEs.
Key result
Sex:
male/female
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
not specified
Conclusions:
Under the study conditions, significant increases in the frequency of micronucleated erythrocytes were observed in mice in a dose-dependent manner.
Executive summary:

The genotoxic potential of diarsenic trioxide was evaluated in a micronucleus (MN) assay in mice. The animals were exposed to different doses of the substance through oral gavaging for 15 consecutive days. For treatment, animals were segregated into three groups (3 males and 3 females per group), which were then exposed to low, moderate and high doses of 0.3, 1.5 and 15 µg arsenic/kg/d, respectively. One group of animals was maintained as control. Under the study conditions, significant increases in the frequency of micronucleated erythrocytes were observed in mice in a dose-dependent manner (Khan, 2013).

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: No guideline was followed
GLP compliance:
not specified
Type of assay:
other: inhalation
Specific details on test material used for the study:
Arsenic trioxide
Species:
mouse
Strain:
other: CFLP strain
Sex:
not specified
Route of administration:
inhalation: aerosol
Details on exposure:
CFLP strain mice were exposed to arsenic trioxide as an aerosol in an inhalation chamber. The aerosol was generated in the chamber by spraying aqueous solution of arsenic trioxide. Animals were exposed 4 h daily on the 9th, 10th and 12th days of gestation. Arsenic atmospheric concentration in the inhalation chamber was measured at least once daily during each exposure.
Duration of treatment / exposure:
4 h per day on the 9th, 10th, 11th and 12th days of gestation
Dose / conc.:
28.5 mg/m³ air (analytical)
Dose / conc.:
2.9 mg/L air (analytical)
Dose / conc.:
0.26 mg/m³ air (analytical)
No. of animals per sex per dose:
Chromosome preparations were made from the liver of ten fetuses per each exposure group.
Control animals:
yes, concurrent no treatment
Details of tissue and slide preparation:
On the 18th day of gestation, mice were killed by an overdose of ether and fetuses were removed. The number of live and dead fetuses were recorded, all fetuses were weighed and then examined under a stereomicroscope.
Chromosome preparations were made from the liver of ten fetuses per each exposure group according to Datta et. al. 1970. Twenty mitoses in each fetus (200 in each group) were scored for chromosomal damage and 10% of these were karyotyped.
Statistics:
With the exception of fetal weight, all data were analysed for statistical significance with Fisher’s exact probability test. Fetal weight was analysed with the Dunnett multiple comparison t-test for comparing several treatment groups with one control group.
Key result
Sex:
not specified
Genotoxicity:
positive
Remarks:
conc. of 28.5 mg/m3
Toxicity:
yes
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the study conditions, exposure to the test substance at 28.5 mg/m3 caused fetotoxic effects and chromosomal damage, while the two lower concentrations produced no significant changes, with the exception of a slight decrease (9.9 and 3.1%, respectively) in fetal weight.
Executive summary:

A study was conducted to determine the genotoxicity of diarsenic trioxide in a mouse inhalation study. Fetal chromosomal damage and toxicity were assessed in mice exposed to atmospheric concentrations of 28.5, 2.9 and 0.26 mg/m3 of arsenic for 4 h / day on the 9th, 10th, 11th and 12th day of gestation. On Day 18 of gestation, the fetuses were removed and the following parameters were examined: number of dead fetuses, retardation in growth, osteogenesis and chromosomal aberrations in liver cells. Under the study conditions, exposure to the test substance at 28.5 mg/m3 caused fetotoxic effects and chromosomal damage, while the two lower concentrations produced no significant changes, with the exception of a slight decrease (9.9 and 3.1%, respectively) in fetal weight (Nagymajtényi, 1985).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Justification for classification or non-classification

Diarsenic trioxide is subject to harmonised classification as Carc. 1A – H350 (May cause cancer) according to Regulation (EC) No. 1272/2008 (see Carcinogenicity section). Based on a wide body of available evidence, inorganic arsenic compounds do not appear to be mutagenic but may cause indirect DNA damage, including chromosomal aberrations, micronucleus formation and sister chromatid exchanges in vitro and in vivo. The indirect toxicity is thought to be caused by threshold effects such as inhibition of DNA repair, interaction with the protein involved in the formation of the mitotic spindle, induction of oxidative stress and/or changes in DNA methylation patterns. Also, they were observed to occur generally at high concentrations, above those that would be attained systemically in animals and humans and do not seem to be the basis for the carcinogenicity of arsenicals, either in animals or in humans, particularly at lower doses. For these reasons, a separate classification for genotoxicity of diarsenic trioxide is not considered warranted.