Chromium trioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacterial cells

Sodium dichromate dihydrate was shown to be mutagenic in S. typhimurium strain TA98 and TA100 and E. coli WP2 uvr A pKM 101 with and without metabolic activation, whereas mutagenicity was more pronounced in the absence of S9 (NTP, 2007).

Cytogenicity in mammalian cells

Chromium trioxide was able to induce chromosome aberrations in CHO cells (Levis and Majone, 1979).

Gene mutation in mammalian cells

Potassium dichromate was shown to be mutagenic in mammalian cells, inducing forward mutations at the TK locus in mouse lymphoma L5178Y cells (Oberley, 1982)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

NTP guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

deviates with respect to the number of tested strains

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

5-300 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine; 2-aminoanthracene

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:

not specified

Species / strain:

S. typhimurium TA 100

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:

not specified

Species / strain:

E. coli WP2 uvr A pKM 101

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:

not specified

Sodium dichromate dihydrate (5 to 300 µg/plate) was clearly 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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

The inhibition of cell growth, the reduction of cell survival and the induction of chromosome aberrations and of sister chromatid exchange (SCE) have been determined in cultured hamster cell lines (BHK and CHO) treated with 11 water-soluble compounds of hexavalent and trivalent chromium.

GLP compliance:

no

Remarks:

Literature study

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Hexavalent chromium (Cr6+) was tested as CrO3 (chromic acid) (Merck, Darmstadt, Germany); with analytical-grade and soluble in water at concentrations up to 10^-3M. Concentrated solutions were made in twice-distilled water, sterilized by filtration through 0.22 µm porous Millipore filters, and kept frozen at - 30°C.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cultures of the established pseudodiploid CHO Chinese hamster ovary fibroblast line were grown at 37°C as monolayers, in Eagle's minimal essential medium supplemented with 10% calf serum. Cultures in glass Petri dishes were maintained in a humidified 5% CO2 atmosphere. 025% trypsin (Difco 1:250, Detroit, Mich., U.S.A.) was routinely used for subculturing. The cultures were periodically tested for the presence of Mycoplasma by Dr L. Conventi (Institute of Microbiology, University of Padua) with standard selective culture media.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

0.1; 0.25 µg/mL
The LD50 for CrVI compounds is approx. 0.15 µg/mL; top dose was selected based on pre-tests on cell survival

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Suspensions of CHO cells were prepared by trypsinization of log-phase cultures and diluted with growth medium in 100mm Petri plates. Each plate received about 8 x 105 cells in 10 ml of medium, which was changed 24 h after seeding. At this time 3 x 10-5 M bromodeoxyuridine (BUdR, Sigma, St Louis, Mo., U.S.A.) was added and the cells were allowed to incorporate the analogue for two division cycles (30 h). Cr compound or mitomycin C (Sigma) were added at the same time as BUdR. The medium was not changed until the cells were collected 30 h later. During the last 4 h of treatment, 0.4 µg/ml of colchicine (Merck, Darmstadt, Germany) were added. At the end of treatment, metaphase cells were dislodged by gently pipetting the overlaying medium and collected by centrifuging the suspension at 900 rev/min for 5 min. The cell pellet was suspended in 5 ml hypotonic buffer (10% sodium citrate) at 37°C for 10 min and fixed in ethanol/acetic acid (3/1). Fixed cells were heated at 89°C for 10 min in 1M NaH2PO4 (pH 8) and stained with Giemsa (Korenberg & Freedlender, 1974). All cultures were treated with BUdR so that sister chromatid exchanges (SCE) and chromosome aberrations were scored on the same cell preparations: 1st and 2nd division metaphases could be distinguished because bifilarly BUdR-substituted chromatids stain paler with Giemsa.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

Table III shows the percentages of 1st division metaphases in CHO cells treated for 30 h with CrO3. All Cr6+ compounds induce a mitotic delay proportional to the chromium dose: with the concentration of 0.5-1.0 µg/ml Cr6+ a very marked delay of the cell cycle during the 1st division is observed. The mean number of chromosome aberrations is significantly increased after treatment with CrO3, in proportion to concentration (Tab. III). A detailed analysis of chromosome aberrations shows that single chromatid gaps, breaks and interchanges prevail, the frequencies of which increase in proportion to the concentration of Cr. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings are also induced, but their frequency does not increase linearly with the Cr concentration.

Table III. Mitotic delays and chromosome aberrations induced by CrO3 in CHO cultures

Treatment	[Cr] µg/mL	Metaphases counted*	1st division (%)	Aberrations per 100 metaphases
				Chromosome aberrations		Chromatid aberrations				Iso chromatid breaks#	Total
				Dicentrics	Rings	Gaps	Breaks	Rings	Inter-changes
-	-	99	15.1	2	1	6	4	-	1	3	17
CrO3	0.1	80	82.5	8	1	15	25	4	2	5	60
	0.25	100	94.3	3	3	27	19	-	1	9	62

* Total at 1st and 2nd divison. # Including replicated single-chromatid breaks.

Conclusions:

The present data on the clastogenic effects of Cr show that Cr(VI) compounds, such as CrO3 are able to induce chromosome aberrations.

Executive summary:

The inhibition of cell growth, the reduction of cell survival and the induction of chromosome aberrations and of sister chromatid exchange (SCE) have been determined in cultured hamster cell lines (BHK and CHO) treated with 11 water-soluble compounds of hexavalent and trivalent chromium.

All Cr(VI) compounds reduce survival of CHO cells, whereas the cytotoxicity curves obtained with the different Cr(VI) compounds are almost overlapping. All Cr(VI) compounds were able to induce chromosome aberrations in CHO cells.

The present data confirm that Cr(VI) compounds are characterized by a marked cytotoxicity and clastogenic action on mammalian cell cultures.

Reference 3

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Investigation of forward mutations at the TK locus in mouse lymphoma L5178Y cells

GLP compliance:

no

Remarks:

literature study

Type of assay:

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

Specific details on test material used for the study:

K2 Cr2 O7 was purchased from Fisher Scientific Co. (Pittsburgh Pa.). All salts were certified ACS grade. Examination of the lot analysis of the metals indicated that amounts of contaminants, if present, were insufficient to induce mutagenesis.

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The TK-/-.3.7.2 heterozygote of L5178Y mouse lymphoma cells was used. All cells were thawed from frozen stock and maintained in Fischer's medium for leukemic cells of mice (Gibco Laboratories, Grand Island, N.Y.) containing 10% heat-inactivated horse serum (Flow Laboratories, McLean, Va.), Pluronic F68 (Wyandotte, Wyandotte, Mich.), sodium pyruvate (Gibco), penicillin G (Eli Lilly, Indianapolis, Ind.), and streptomycin sulfate (Lilly). Background spontaneous TK-/- mutant frequencies were reduced weekly by 24-h treatment of the cells with medium containing thymidine (Gibco), hypoxanthene (Gibco), methotrexate (Sigma Chemical Co., St. Louis, Mo.), and glycine (Sigma).

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate was prepared from the livers of Sprague-Dawley rats weighing 200-250 g (Aroclor-induced). Animals were killed by decapitation, bled, and livers removed aseptically. The livers were homogenized in KCI, pooled, and centrifuged for 10 min at 9000 X g, and the supernatant (S9 fraction) was decanted. The protein concentration of the S9 was found to be 34 mg/ml. The S9 fraction was stored in 2-dram vials at -80°C. As needed, S9 fraction was thawed and added in appropriate dilution with cofactor mix containing NADP (Calbiochem, San Diego, Calif.) at 8 mg/ml and isocitric acid (Sigma) at 15 mg/ml in Fischer's medium.
Optimal induced rat liver S9 concentrations were routinely verified by using the promutagen AAF as a test chemical. With the batch of S9 available for these assays, a 10% dilution of S9 in medium was utilized.

However, data from tests with S9 metabolic activation were omitted unless it was necessary to show a positive response

Test concentrations with justification for top dose:

1, 2, 4, 6, 8 µg/mL
cytotoxicty observed

Vehicle / solvent:

sterile deionized glass-distilled water

Details on test system and experimental conditions:

Metal compounds 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. EMS and AAF were used as positive controls in the absence and presence of S9 metabolic activation, respectively, while the negative controls received the solvent, sterile deionized glass-distilled water. 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-d expression period. The cultures were counted after d 1 and readjusted to 3 X 10^5 cells per milliliter if necessary.
On d 2 a modified cloning procedure was followed. A sample from each culture was centrifuged and the cells resuspended at 500,000 viable cells per milliliter in Fischer's medium. The concentrated cells were serially diluted and appropriate dilutions plated in triplicate in cloning medium with and without trifluorothymidine (TFT). Approximately 500,000 viable cells (as determined by exclusion of trypan blue) were plated on each of three selective medium plates containing 2 pg/ml TFT (Sigma), and 100 cells were cloned on each of three nonselective plates for each test and control tube. Cell inocula were added directly into 100-mm tissue culture plates (Costar, Vineland, N. J.), followed by the addition of about 30 ml cloning medium. The Plates were swirled gently to ensure even dispersal of the inocula, allowed to gel, and then incubated at 37°C for approximately 12 d before they were counted.

Rationale for test conditions:

The test system was based on the procedure described by Clive et al. (1975, 1979) with modifications to the cloning procedure

Evaluation criteria:

A New Brunswick Scientific automatic colony counter was used to determine the number of colonies per plate. Total survival was determined by the method of Clive and Spector (1975) which combines growth in suspension culture and soft cloning efficiency data. The mutation frequency (MF) was calculated as the number of mutants per 10^6 colony-forming cells.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Data from tests with S9 metabolic activation were omitted unless it was necessary to show a positive response.
K2Cr2O7 elicited increased responsiveness in toxicity and mutation with survival >10% at 2 and 1 µg/mL; the mutagenic response remained prominent with 20.9- and 10.3-fold increases over background.

Chemical	Dose (µg/mL)	Percent total survival	Mutation frequency	Increase over solvent (-fold)
Solvent	0	100	3.6
EMS	620	1	600.0	166.7
K2Cr2O7	8	1	62.1	17.3
	6	1	233.8	64.9
	4	4	98.9	27.5
	2	10	75.3	20.9
	1	14	37.2	10.3

Conclusions:

Potassium dichromate has been shown to be mutagenic in mammalian cells, inducing forward mutations at the TK locus in mouse lymphoma L5178Y cells

Executive summary:

Eleven metals were examined for their potential to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. Potassium dichromate has been shown to be mutagenic in mammalian cells, inducing forward mutations at the TK locus in mouse lymphoma L5178Y cells

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Cytogenicity in vivo mammalian somatic cells

Sodium dichromate administered to three strains of mice via drinking water was able to incude micronuclei in one (transgenic) mouse strain, the findings were not apparent in the other strains (NTP, 2007).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

NTP guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The induction of peripheral blood erythrocyte micronuclei was investigated in mice following the oral administration of sodium dichromate for 3 months. 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.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: B6C3F1, BALB/c, and am3-C57BL/6

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
B6C3F1: Taconic Laboratory Animals and Services (Germantown, NY)
BALB/c: Charles River Laboratory (Portage, MI)
am3-C57BL/6: Charles River Laboratory (Wilmington, MA)
- Age at study initiation: 6 (B6C3F1 and BALB/c mice) or 7 (am3-C57BL/6 mice) weeks
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: 1 animal per cage, Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed at least once weekly; Bedding: Irradiated hardwood bedding chips (P.J. Murphy Forest Products, Inc., Montville, NJ), changed at least once weekly; Cage Filters: Spun-bonded polyester (Snow Filtration Co., Cincinnati, OH); Racks: Stainless Steel (Lab Products, Inc., Seaford, DE), changed every 2 weeks
- Diet (e.g. ad libitum): Irradiated NTP-2000 wafer rodent feed (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water (e.g. ad libitum): Tap water (Columbus, OH, municipal supply) via glass bottles with Teflon®-lined septa and stainless steel sipper tubes (Wheaton, Millville, NJ), available ad libitum
- Acclimation period:
B6C3F1: 12 days
BALB/c: 16 days
am3-C57BL/6: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° +/- 3° F (20.6 - 23.9 °C)
- Humidity (%): 50% +/- 15%
- Air changes (per hr): 18/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day light, 12 hours dark

IN-LIFE DATES: From: (first exposure)
B6C3F1: August 20, 2002
BALB/c: August 22, 2002
am3-C57BL/6: August 23, 2002
To: (necropsy)
B6C3F1: November 19, 2002
BALB/c: November 21, 2002
am3-C57BL/6: November 22, 2002

Route of administration:

oral: drinking water

Vehicle:

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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared fore times during the 3-month studies in B6C3F1 mice (study 1) and five times during the 3-month studies in male B6C3F1, BALB/c, and am3-C57BL/6 mice (study 2). Formulations used in study 1 were stored in NALGENE® containers at room temperature and protected from light. Formulations used in study 2 were stored in NALGENE® containers and refrigerated at approximately 5° C.

Duration of treatment / exposure:

90 days (3 months)

Frequency of treatment:

Continuous

Post exposure period:

None

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

Remarks:

in study 1 only

Dose / conc.:

1 000 mg/L drinking water

Remarks:

in study 1 only

No. of animals per sex per dose:

study 1: 5 animals/sex/dose
study 2: 5 B6C3F1 mice/dose, 5 BALB/c mice/dose, and 10 am3-C57BL/6 mice /dose (males only)

Control animals:

yes, concurrent no treatment

Tissues and cell types examined:

Peripheral blood erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses were seleted based on effects reported in previous studies and the present study was performed primarily to aid the design and dose selection for the 2-year carcinogenicity studies.

DETAILS OF 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. Slides were scanned to determine the frequency of micronucleated cells in 2,000 normochromatic erythrocytes (NCEs) in each of five mice per treatment group for all except the am3-C57BL/6 strain; for the am3-C57BL/6 mice, five core study and four or five mutagenicity study mice per treatment group were evaluated. In addition to assessment of micronucleus frequencies, the percentage of polychromatic erythrocytes (PCEs) in a population of 1,000 erythrocytes was determined as a measure of bone marrow toxicity.

METHOD OF ANALYSIS:
The results were tabulated as the mean of the pooled results from all animals within an exposure group plus or minus the standard error of the mean.
A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Because additional test data could not be obtained, results of the 3-month studies were accepted without repeat tests. Ultimately, the final
call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

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).
In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, 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.

Sex:

male/female

Genotoxicity:

ambiguous

Remarks:

Positive in one strain

Toxicity:

yes

Remarks:

Reduced PCE count in one assay

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see any other information on results

The results of the micronucleus tests conducted in three strains of mice were variable:

Study 1

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 1000 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 did not clearly correlate with exposure concentration.

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. An increase in micronucleated erythrocytes that was judged to be equivocal was noted in male B6C3F1 mice, based on 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.

Study 1	B6C3F1
	Male	Female	Male	Female
	Micronucleated NCEs (/1000)		PCEs (%)
0	2.70	1.70	4.1	3.6
62.5	2.60	1.20	3.5	2.5
125	2.20	1.60	3.1	3.4
250	3.70	1.80	3.3	3.9
500	2.50	2.10	2.7	3.2
1000	2.00	1.90	3.3	2.7
Study 2	B6C3F1
0	2.20	-	3.3	-
62.5	3.20	-	3.6	-
125	3.00	-	3.2	-
250	3.80*	-	2.8	-
BALB/c
0	4.70	-	3.7	-
62.5	3.90	-	4.0	-
125	3.30	-	3.3	-
250	4.20	-	3.5	-
am3-C57BL/6
0	1.65	-	2.9	-
62.5	2.50	-	2.8	-
125	3.05	-	2.9	-
250	3.72*	-	2.6	-

*significant by the trend test

Conclusions:

A significant increase in the frequency of peripheral blood erythrocyte micronuclei was seen in one (transgenic) mouse strain under the conditions of this study; similar findings were not apparent in the other strains. The results of this study are therefore equivocal.

Executive summary:

The potential of sodium dichromate to induce micronuclei was investigated in the peripheral blood erythrocytes of three strains of mice following administration via drinking water for 3 months. A significant increase in the frequency of micronuclei was seen in the transgenic am3 -C57/BL6 strain, however similar findings were not apparent in B6C3F1 of BALB/c mice. The results of this study are therefore equivocal.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

Mechanism of genotoxicity

Cr (VI) readily enters cells, in contrast to Cr (III) which cannot easily pass through the cell membrane. Because it has been shown to be relatively nonreactive, Cr (VI) is believed to exert its genotoxic effects, at least in part, through the generation of oxygen radicals during metabolic transformation from the hexavalent form through the more reactive Cr (V) and Cr (IV) valences to Cr (III). In studies with laboratory animals, administration of radical scavengers simultaneously with or prior to administration of Cr (VI) salts reduced clastogenic potency, thus providing support for the oxygen radical mechanism of action. The results of in vitro mammalian cell studies of Cr (VI)-induced DNA damage in the presence of a variety of oxygen radical scavengers and reducing agents provides additional support for this mechanism. Cr (III), the product of intracellular reduction of Cr (VI), has been shown to interact directly with DNA and other macromolecules to induce chromosomal alterations and mutational changes in DNA.

Additional information

Genotoxicity in vitro

Evidence for positive genotoxicity is shown in in vitro studies using bacterial and mammalian cells in the available literature.

In addition, the results 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 an NTP peripheral blood erythrocyte micronucleus study, sodium dichromate administered in drinking water for 90 days to mice was found to cause a significant positive response in a transgenic strain, but not in standard strains of mice. 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 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. Extensive in vitro testing of highly water-soluble Cr(VI) compounds has produced positive results for point mutations and DNA damage in bacteria, point mutations, mitotic crossing-over, gene conversion, disomy and diploid in yeasts, and gene mutation, DNA damage, chromosome aberrations, sister chromatid exchanges and unscheduled DNA synthesis in mammalian cells. 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. Toxicokinetic data for water soluble Cr(VI) compounds indicate that chromium will reach the germ cells following inhalation exposure (a relevant route of exposure for humans). Therefore taking these two observations together, it can be concluded that water-soluble Cr(VI) compounds have the potential to produce germ cell mutagenicity.

A few studies have been conducted in which circulating lymphocytes have been isolated from chromium-exposed workers and examined for chromosome aberrations, micronuclei, SCE and changes in chromosome numbers. In general, the results from the better-conducted and reported studies including chromium plating workers in Japan and SS-MMA welders in Scandinavia have been negative. Evidence of genotoxicity has been reported in several other studies of chromate production workers in Eastern Europe and chromium plating workers in Italy. However the manner in which these were conducted and reported precludes full assessment of the significance of the findings.'

Given the available database on the genotoxicity of chromium (VI) compounds and the conclusion that this group of compounds is mutagenic, it is considered that further testing (specifically in guideline and GLP-compliant studies) is not required.

Justification for classification or non-classification

Chromium (VI) trioxide, sodium chromate, sodium dichromate and potassium dichromate are classified under Annex I to directive 67/548/EEC. Chromium (VI) trioxide is listed in Annex VI to Regulation (EC) No 1272/2008 under Index No 024-001-00-0 with the following harmonised classification:

Muta. 1B, H340 ‘May cause genetic defects’

This classification is consistent with the data available in the literature and reviewed in the EU RAR. No change to this classification is proposed.