Sodium dichromate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sodium dichromate dihydrate and other soluble chromium (VI) substances are mutagenic in the bacterial reverse mutation test, the in vitro mammalian cell gene mutation assay as well as in clastogenicity assays.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: NTP protocol

Deviations:

yes

Remarks:

deviates from standard protocols with respect to the number of tested strains

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

deviates from standard protocols with respect to the number of tested strains

Principles of method if other than guideline:

The NTP protocol is broadly similar to standard methods (e.g. OECD 471) with the exception of the numbers of strains investigated.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Reversion to histidine independence

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Type: 10% S9 mix (metabolic activation enzymes and cofactors
- source of S9: from Aroclor 1254-induced male Sprague Dawley rat liver

Test concentrations with justification for top dose:

5-300 µg/plate

Vehicle / solvent:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine

Remarks:

Positive control for metabolic activation was 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: all trials were repeated (number not stated)

METHOD OF TREATMENT/ EXPOSURE:
- test substance was dissolved in water and preincubated with bacterial tester strains 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 surface of minimal glucose agar plates.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C
- Exposure duration/duration of treatment: 2 days at 37°C

Evaluation criteria:

A positive response was defined as a reproducible, dose-realted increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as an increase in revertants that was not dose-related, was not reproducible, or was not sufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment. There was no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Sodium dichromate dihydrate was mutagenic in Salmonella typhimurium TA100 and TA98 and in Escherichia coli strain WP2 uvrA pKM101 with and without 10% induced rat liver S9 enzymes. Responses were stronger in the strains that mutate via base substitution (TA100, E. coli WP2); in all three tester strains, mutagenicity was more pronounced in the absence of S9, based on the lowest concentration that elicted a significant mutagenic response.
In the tester strain TA98 the high doses were limited by cytotoxicity (50-300 µg/plate). Cytotoxicity was not observed in tester strain TA100 and E.coli WP2.

Details on results please refer to attached background material.

Conclusions:

The results of this study show that sodium dichromate is mutagenic in the Ames test.

Executive summary:

Sodium dichromate dihydrate (5 to 300 µg/plate) was mutagenic in Salmonella typhimurium strains TA100 and TA98 and in Escherichia coli strain WP2 uvrA pKM101 with and without 10% induced rat liver S9 enzymes. Responses were stronger in the strains that mutate via base substitution (TA100, E. coli WP2); in all three tester strains, mutagenicity was more pronounced in the absence of S9, based on the lowest concentration that elicited a significant mutagenic response.

This guideline study is considered to be valid, only minor deviations were noted:

Test substance identification data were missing (e.g. CAS, purity, etc). Test substance solubility tests prior to study start were not reported. No details about bacterial growth phase (number of cells per culture) and preincubation method (amount of bacterial cells mixed with amount of test substance and S9 fraction) were provided. Only three instead of five stains of bacteria have been used. and it remains unknown which media have been used for bacterial culture. Individual plate counts were not reported and it was not described which cytotoxicity test system has been used. The number of independent experiments was reported and the revertants were presented as mean ± SD from three plates only, which raises the question whether the experiment has been conducted only once. Historical control data were not reported.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

study was conducted before implementation of the OECD TG for the In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene

Deviations:

yes

Remarks:

individual data missing, exposure conditions, metabolic activation unclear, number of replicates unclear

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

potassium dichromate and potassium chromate were tested

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK+/- -3.7.2 heterozygote of L5178Y mouse lymphoma cells

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver homogenate of Aroclor-induced rats
- method of preparation of S9 mix : Liver homogenate was prepared from the livers of Sprague-Dawley rats weighing 200-250 g. Livers were homogenized in KCI, pooled, and centrifuged for 10 min at 9000 Xg,
- concentration or volume of S9 mix and S9 in the final culture medium: 10% dilution in medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 concentrations were routinely verified by using the promutagen AAF as a test chemical

Test concentrations with justification for top dose:

1, 2, 4, 6, 8 µg/mL (used for both substances KCrO4 and K2Cr2O7)

Vehicle / solvent:

water, 0.1 ml of each dilution was added to a 10-ml cell-medium suspension

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 6 * 10E6
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hrs

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar
- selective agent: 2 µg/mL trifluorothymidine
- Number of cells seeded: 5 * 10E6/mL
- Criteria for small (slow growing) and large (fast growing) colonies: not investigated

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Total survival was determined by the method of Clive and Spector (1975) which combines growth in suspension culture and soft cloning efficiency data.

Evaluation criteria:

not stated

Statistics:

not performed

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

K2CrO4

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

K2Cr2O7

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Values for the solvent and positive controls fall within the range established by previous experiments. K2CrO4 elicited a favorable dose response in total survival and MF, with significant mutation seen at 6, 4, and 2 µg/mL. In comparison, K2Cr2O7 elicited increased responsiveness in toxicity and mutation, resulting in an excessive cytotoxicity as of 2 µg/mM, resulting in only one scorable concentration.

Conclusions:

Potassium chromate followed a concentration-response relationship and was judged positive in the in vitro mouse lymphoma assay, whereas the results for potassium dichromate were judged equivocal as only one analysable concentration was available due to high cytotoxicity. Results with metabolic activation were not reported. Authors did not report whether experiments were run in duplicates or triplicates or whether the experiment was repeated. Colony sizing was not reported, thus no conclusion can be made whether the findings were due to gene mutation or chromosomal mutation.
The experiments were conducted prior to the implementation of the OECD technical guideline of the In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene. The study was well documented, meets generally accepted scientific principles and was therefore deemed acceptable for assessment.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

There is equivocal evidence for clastogenic effects of sodium dichromate in mice exposed via drinking water over a period of 90 days.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Two independent studies were conducted to evaluate the effect of sodium dichromate dihydrate, administered in drinking water for 3 months, on the frequency of micronuclei in peripheral blood erythrocytes of mice. Micronucleus frequencies were evaluated in male am3-C57BL/6 mice administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 250 mg/L in drinking water for 3 months.
The am3-C57BL/6 strain is a C57BL/6 mouse transgenic for the phiX174am3 gene. This is a nonessential integrated transgene that provides a target for forward and backward mutations and can be recovered from cells and scored for mutation frequency and type (Malling and Burkhart, 1989). Due to technical difficulties, these mutation studies could not be completed, and no results are included in this Toxicity Study Report.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

C57BL

Details on species / strain selection:

The am3-C57BL/6 strain is a C57BL/6 mouse transgenic for the phiX174am3 gene. This is a nonessential integrated transgene that provides a target for forward and backward mutations and can be recovered from cells and scored for mutation frequency and type (Malling and Burkhart, 1989).

Sex:

male

Route of administration:

oral: drinking water

Details on exposure:

The dose formulations were prepared five times during the 3-month studies in male am3-C57BL/6 mice (study 2). Formulations used in study 2 were stored in NALGENE® containers and refrigerated at approximately 5° C.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuous

Post exposure period:

no performed

Dose / conc.:

62.5 mg/L drinking water

Dose / conc.:

125 mg/L drinking water

Dose / conc.:

250 mg/L drinking water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

peripheral blood

Details of tissue and slide preparation:

At the ends of the 3-month exposure periods, peripheral blood samples were obtained from mice, and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

Evaluation criteria:

An individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.

Statistics:

The frequency of micronucleated cells among NCEs and PCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group (ILS, 1990).

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Conclusions:

Micronucleus frequencies were determined in peripheral blood erythrocytes of male am3-C57BL/6 mice administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 250 mg/L for 3 months. A significant exposure concentration-related increase (P<0.001) in micronucleated erythrocytes was noted in male am3-C57BL/6 mice. In this study, two of three dose groups were significantly (P<0.008) elevated over the control group. No significant effect of chemical exposure on the percentage of polychromatic erythrocytes was observed.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Two independent studies were conducted to evaluate the effect of sodium dichromate dihydrate, administered in drinking water for 3 months, on the frequency of micronuclei in peripheral blood erythrocytes of mice. Male and female B6C3F1 mice were administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 1,000 mg/L for 3 months.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Route of administration:

oral: drinking water

Details on exposure:

The dose formulations were prepared four times during the 3-month studies in B6C3F1 mice (study 1) Formulations were stored in NALGENE® containers at room temperature and protected from light.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuous

Post exposure period:

no performed

Dose / conc.:

62.5 mg/L drinking water

Dose / conc.:

125 mg/L drinking water

Dose / conc.:

250 mg/L drinking water

Dose / conc.:

500 mg/L drinking water

Dose / conc.:

1 000 mg/L drinking water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

peripheral blood

Details of tissue and slide preparation:

At the ends of the 3-month exposure periods, peripheral blood samples were obtained from mice, and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

Evaluation criteria:

An individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.

Statistics:

The frequency of micronucleated cells among NCEs and PCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group (ILS, 1990).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Conclusions:

Micronucleus frequencies were determined in peripheral blood erythrocytes of male and female B6C3F1 mice administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 1,000 mg/L for 3 months. No significant increases were seen in micronucleated normochromatic erythrocytes in male or female mice over the exposure concentration range tested; there was a decrease in the percentage of polychromatic erythrocytes among total erythrocytes (an indication of bone marrow toxicity), but the changes were small and not well correlated with exposure concentrations

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Two independent studies were conducted to evaluate the effect of sodium dichromate dihydrate, administered in drinking water for 3 months, on the frequency of micronuclei in peripheral blood erythrocytes of mice. Micronucleus frequencies were evaluated in male B6C3F1 mice administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 250 mg/L in drinking water for 3 months.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

oral: drinking water

Details on exposure:

The dose formulations were prepared five times during the 3-month studies in male B6C3F1 mice (study 2). Formulations used in study 2 were stored in NALGENE® containers and refrigerated at approximately 5° C.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuous

Post exposure period:

no performed

Dose / conc.:

62.5 mg/L drinking water

Dose / conc.:

125 mg/L drinking water

Dose / conc.:

250 mg/L drinking water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

peripheral blood

Details of tissue and slide preparation:

At the ends of the 3-month exposure periods, peripheral blood samples were obtained from mice, and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

Evaluation criteria:

An individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.

Statistics:

The frequency of micronucleated cells among NCEs and PCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group (ILS, 1990).

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Conclusions:

Micronucleus frequencies were determined in peripheral blood erythrocytes of male and female B6C3F1 mice administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 250 mg/L for 3 months. An increase in micronucleated erythrocytes that was judged to be negative was noted in male B6C3F1 mice, the trend test (P=0.031), which showed an increase in micronucleated normochromatic erythrocytes that did not reach statistical significance (required P value of 0.025); no exposed groups were significantly increased over the control group in this study. No significant effect of chemical exposure on the percentage of polychromatic erythrocytes was observed.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Two independent studies were conducted to evaluate the effect of sodium dichromate dihydrate, administered in drinking water for 3 months, on the frequency of micronuclei in peripheral blood erythrocytes of mice. Micronucleus frequencies were evaluated in male BALB/c mice administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 250 mg/L in drinking water for 3 months.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Balb/c

Sex:

male

Route of administration:

oral: drinking water

Details on exposure:

The dose formulations were prepared five times during the 3-month studies in male BALB/c mice (study 2). Formulations used in study 2 were stored in NALGENE® containers and refrigerated at approximately 5° C.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuous

Post exposure period:

no performed

Dose / conc.:

62.5 mg/L drinking water

Dose / conc.:

125 mg/L drinking water

Dose / conc.:

250 mg/L drinking water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

peripheral blood

Details of tissue and slide preparation:

At the ends of the 3-month exposure periods, peripheral blood samples were obtained from mice, and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

Evaluation criteria:

An individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.

Statistics:

The frequency of micronucleated cells among NCEs and PCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group (ILS, 1990).

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Conclusions:

Micronucleus frequencies were determined in peripheral blood erythrocytes of male BALB/c mice administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 250 mg/L for 3 months. No increase in micronucleated normochromatic erythrocytes was observed in male BALB/c mice. No significant effect of chemical exposure on the percentage of polychromatic erythrocytes was observed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Bacterial mutation

In the study by NTP (2007) sodium dichromate dihydrate (5 to 300 µg/plate) was mutagenic in Salmonella typhimurium strains TA100 and TA98 and in Escherichia coli strain WP2 uvrA pKM101 with and without 10% induced rat liver S9 enzymes. Responses were stronger in the strains that mutate via base substitution (TA100, E. coli WP2); in all three tester strains, mutagenicity was more pronounced in the absence of S9, based on the lowest concentration that elicited a significant mutagenic response.

This guideline study is considered to be valid, only minor deviations as follows: test substance identification data were missing (e.g. CAS, purity, etc). Test substance solubility tests prior to study start were not reported. No details about bacterial growth phase (number of cells per culture) and preincubation method (amount of bacterial cells mixed with amount of test substance and S9 fraction) were provided. Only three instead of five stains of bacteria have been used. and it remains unknown which media have been used for bacterial culture. Individual plate counts were not reported and it was not described which cytotoxicity test system has been used. The number of independent experiments was reported and the revertants were presented as mean ± SD from three plates only, which raises the question whether the experiment has been conducted only once. Historical control data were not reported.

 

The results primarily non-standard bacterial reverse mutation assays performed with water-soluble hexavalent chromium compounds have been reviewed by the UK HSE (1989), UK IOH (1997) and in the EU RAR (2005). Findings in these studies are largely positive.

Chromosomal aberration

A number of references are reported in the section for in vitro genetic toxicity, addressing the endpoint clastogenicity, such as the In Vitro Sister Chromatid Exchange Assay in Mammalian Cells and the In Vitro Mammalian Chromosome Aberration Test. However, none of the references fulfils the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only. Further in vitro testing for clastogenicity is not considered to be required, since reliable and relevant in vivo studies are available.

Results of further predominantly non-standard cytogenicity assays conducted with water-soluble hexavalent chromium compounds have been reviewed by the UK HSE (1989), UK IOH (1997) and in the EU RAR (2005). Findings in these studies are largely positive.

Mammalian cell mutation

Oberly et al (1982) investigated the induction of mutations in mouse lymphoma L5178Y cells in vitro In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene with potassium dichromate and potassium chromate. The mutation frequency for potassium chromate followed a concentration-response relationship and was judged positive in the in vitro mouse lymphoma assay, whereas the results for potassium dichromate were judged equivocal as only one analysable concentration was available due to high cytotoxicity. Results with metabolic activation were not reported. Authors did not report whether experiments were run in duplicates or triplicates or whether the experiment was repeated. Colony sizing was not reported, thus no conclusion can be made whether the findings were due to gene mutation or chromosomal mutation. The experiments were conducted prior to the implementation of the OECD technical guideline of the In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene and is therefore not in compliance. However, the study was well documented, meets generally accepted scientific principles and was therefore deemed acceptable for hazard assessment purposes.

The study by Tindall et al. (1980) is available as abstract only. Authors report that potassium chromate induced mutations in CHO cells in a concentration-related manner over a range of 0.25-2 µg/mL at approx. 20% cloning efficiency. Due to the lack of details on materials, methods and results, the reference is of supporting nature only.

Results of further predominantly non-standard cytogenicity assays conducted with water-soluble hexavalent chromium compounds have been reviewed by the UK HSE (1989), UK IOH (1997) and in the EU RAR (2005). Findings in these studies are largely positive.

Additional investigations

The reuslts of a large number of non-standard study types using different test systems and endpoints are also reviewed by the UK HSE (1989), UK IOH (1997) and in the EU RAR (2005). These results of these studies are largely positive, indicating that water-soluble hexavalent chromium compounds have the potential to cause DNA damage, mutagenicity and chromosomal damage in vitro. These additional studies therefore further support the conclusion that the water-soluble hexavalent chromium compounds are genotoxic in vitro.

Genotoxicity in vivo

In the NTP report (2007) two independent studies were conducted to evaluate the effect of sodium dichromate dihydrate. In study 1, male and female B6C3F1 mice were administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 1,000 mg/L for 3 months. In study 2, micronucleus frequencies were evaluated in male B6C3F1, BALB/c, and am3-C57BL/6 mice administered sodium dichromate dihydrate over an exposure concentration range of 62.5 to 250 mg/L in drinking water for 3 months.

In study 1, no significant increases were seen in micronucleated normochromatic erythrocytes in male or female mice over the exposure concentration range tested; there was a decrease in the percentage of polychromatic erythrocytes among total erythrocytes (an indication of bone marrow toxicity), but the changes were small and not well correlated with exposure concentrations.

In study 2, an increase in micronucleated erythrocytes that was judged to be negative was noted in male B6C3F1 mice, the trend test (P=0.031), which showed an increase in micronucleated normochromatic erythrocytes that did not reach statistical significance (required P value of 0.025); no exposed groups were significantly increased over the control group in this study. No increase in micronucleated normochromatic erythrocytes was observed in male BALB/c mice (Table B4). A significant exposure concentration-related increase (P<0.001) in micronucleated erythrocytes was noted in male am3-C57BL/6 mice. In this study, two of three dose groups were significantly (P<0.008) elevated over the control group. No significant effect of chemical exposure on the percentage of polychromatic erythrocytes was observed in any of the three micronucleus tests conducted in study 2.

 

No proprietary studies of genotoxicity performed using the water-soluble chromium (VI) compounds in this group are available. The genotoxicity of chromium (VI) trioxide and other chromium (VI) salts have been extensively reviewed by the UK Health and Safety Executive (HSE, 1989); the UK Institute of Occupational Health (IOH, 1997) and most recently in the EU RAR (2005). The results of studies using parenteral administration are generally positive. The results of studies using oral administration are less clearly positive, however the low gastrointestinal absorption and rapid reduction of Cr (VI) to Cr (III) in the gastrointestinal tract and plasma act to reduce systemic exposure to Cr (VI). The EU RAR concluded that 'there is a very large body of evidence indicating that the Cr (VI) ion in solution is directly mutagenic in in vitro systems. The in vitro genotoxicity of Cr (VI) was diminished considerably by the presence of reducing agents, in the form of tissue S9 or S12 fractions, gastric juice or reducing agents such as glutathione, ascorbate or sulphite. These all serve to reduce Cr (VI) to Cr (III) outside the cell therefore greatly reducing entry of chromium into the cell.

The genotoxicity of Cr (VI) compounds in vivo has been less extensively studied. Parenteral administration of sodium or potassium dichromate or potassium chromate to rats or mice resulted in significant increases in chromosome aberrations and micronucleated cells in the bone marrow and DNA single-strand breaks, interstrand cross-links and DNA-protein cross-links in the liver, kidneys and lung. A mouse spot test involving intraperitoneal injection of potassium chromate gave positive results. Oral studies have been negative but these employed lower dose levels and absorption is known to be poor by the oral route. Overall, water soluble Cr (VI) compounds are in vivo somatic cell mutagens in animal studies. A significant increase in post-implantation deaths in a dominant lethal assay was reported in mice following intraperitoneal injection of potassium dichromate.

 

Mode of action for genotoxicity/carcinogenicity

The SCOEL (2017) discussed in its recent recommendation for chromium VI compounds (SCOEL/REC/386), the mode of action as follows: “As opposed to many other metal compounds, in case of Cr VI the formation of DNA adducts appears to play an important role in generating genomic instability and thus tumor formation (Hartwig, 2013; Wise & Wise, 2012). Under physiological conditions, Cr VI enters the cell as the anionic tetrahedral species chromate, CrO42-, via anion transport systems such as the sulfate carrier, and is intracellularly reduced to Cr III, described by the so-called “uptake-reduction” model. Within the cell, reduction does not require enzymatic steps but is mediated by direct electron transfer from ascorbate and non-protein thiols such as glutathione and cysteine; during this process, potentially toxic intermediates such as oxygen and sulfur radicals are generated, dependent on the intracellular reductant. In case of poorly water soluble Cr VI compounds such as barium chromate and lead chromate, uptake is mediated via endocytosis. DNA lesions generated after exposure towards Cr VI consist of two categories, namely oxidatively induced DNA damage and DNA lesions resulting from Cr III-DNA interactions. With respect to the formation of ROS during the intracellular reduction process, the induction of oxidatively damaged DNA by Cr VI appears to be restricted to high exposure concentrations, and its relevance on physiological conditions has been questioned. In contrast, especially ternary Cr-DNA adducts may be of special importance for chromate-induced carcinogenicity, where Cr bridges DNA and small molecules such as cysteine, histidine, glutathione or ascorbate, presumably arising from preformed Cr-ligand complexes during the reduction process. Under physiological conditions, ascorbate appears to be the major reductant, and especially ternary adducts formed from Cr-ascorbate are potent premutagenic DNA lesions. One other additional aspect of chromate-induced carcinogenicity is the induction of genomic instability, as evident by simultaneous occurrence of microsatellite instability and chromosome instability in Cr VI-induced tumors. This may be a consequence of disturbed DNA mismatch repair (MMR). Thus, Cr VI-induced DNA lesions lead to aberrant MMR, and upon chronic exposure to toxic doses of Cr VI the selective outgrowth of MMR-deficient clones exerting a high degree of genomic instability has been postulated. In summary, Cr VI acts directly genotoxic by inducing specific DNA lesions, which are not easily repaired; genomic instability is increased via mismatch-repair deficient cell clones which survive on the expense of hypermutability. Even though a fraction a Cr VI may be reduced extracellularly to Cr III, this fraction is currently not quantifiable. Due to the direct genotoxic mode of action, a linear dose-response relationship is assumed.”

 

Reviews of chromium genotoxicity

An additional comprehensive review of the genotoxicity of chromium compounds by De Flora et al., (Mut. Res. 238 (1990) 99 -172) discusses the results of approximately 130 studies performed with 32 different compounds. For the water-soluble hexavalent chromium compounds, the authors conclude that the results of genotoxicity studies in cellular systems are consistently positive. It is noted that an 'impressive' number' of bacterial mutagenicity studies are positive due to the induction of frameshift and substitution mutation. Compounds were also found to cause unscheduled DNA synthesis, DNA damage, forward mutation, sister chromatid exchanges, micronuclei and chromosomal aberrations in cultured mammalian cells. Several endpoints of genotoxicity including sister chromatid exchanges, micronuclei and chromosomal aberrations are reported to have been observed in studies in vivo. The authors conclude, however, that the evidence for a dominant lethal effect in mice was 'conflicting'.

Justification for classification or non-classification

The genotoxicity of Cr VI compounds has been widely investigated in assays for different genetic endpoints and has, with a few possible exceptions, been uniformly positive in in vitro assays for mutagenicity and clastogenicity, with evidence of in vivo expression of these effects in some compounds. Based on this, the legal classification of sodium dichromate (Index 024 -004 -00 -7) as mutagenic category 1B (H340) is adopted.