Chromium (III) oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No information on the in vitro genotoxicity of chromium (III) oxide is available. Therefore, a conservative read-across to the soluble chromium(III) salts category is applied as detailed in the read-across document attached to IUCLID section 13.

Soluble chromium(III) substances were assessed in a number of in vitro genotoxicity assays showing no genotoxic potential, clastogenic or aneugenic potency. Consequently, soluble chromium (III) salts are considered non genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18/09/1990-25/02/1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983-05-26

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator

Target gene:

TA1535: his G 46
TA100: his G 46 & pKM 101
TA98: his D 3052 & pKM 101
TA1537: his C 3076

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (containing 30 % S9 (v/v)): phosphate buffer (100 mM); NADP, disodium salt (315.0 mg); glucose-6-phosphate; disodium salt (179.1 mg); MgCl2 x 6H2O (162.6 mg); KCl (246.0 mg)

Test concentrations with justification for top dose:

8, 40, 200, 1000 and 5000 ug/plate (with and without S9; concentrations in both independent experiments)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the used vehicle was chosen out of the following vehicles, in the order given: water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: Nitrofurantonin (pos. control w/out S9; conc.: 0.2 µg/plate; strain: TA100), 4-nitro-1,2-phenylene diamine (pos. control w/ S9; conc.: 0.5 and 10 µg/plate; strains:TA1537 & TA98) & 2-aminoanthracene (pos. control w/ S9; conc.: 3 µg/plate; all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)/ two independent experiments were performed

NUMBER OF REPLICATIONS: quadruplicate

DURATION
The count was made after the plates had been incubated for 48 hours at 37°C. If no immediate count was possible, plates were temporarily stored in a refrigerator.

DETERMINATION OF CYTOTOXICITY
- Method: the toxicity of the substance was assessed in three ways, as follows:
1) gross appraisal of background growth on the plates for mutant determination.
2) toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.
3) titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive resul t. For TA1535, TA100 and TA98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

Not applicable

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Precipitation was seen at 1000 and 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 1000 μg per plate, the substance started to precipitate.

CYTOTOXICITY:
As may be seen, there was no indication of a bacteriotoxic effect of chromiumhydroxide at doses of up to and including 5000 μg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted.

EXPERIMENTS:
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the repeat tests.
Please also refer to the field "Any other information on results incl. tables" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Attached background material" below.

Exposure to the test material did not induce any increase in the number of revertant colonies of any bacterial strain. Positive control compounds induced large increased in the number of revertant colonies, confirming the sensitivity of the assay.

Treatment	-S9
	Initial assay				Confirmatory assay
	TA98	TA100	TA1535	TA1537	TA98	TA100	TA1535	TA1537
Vehicle	20	114	12	9	34	130	15	11
8	16	125	13	9	36	123	18	9
40	26	127	15	9	34	141	20	13
200	21	133	16	7	29	138	21	12
1000	22	109	11	7	34	132	17	13
5000	20	127	12	9	34	146	21	14
Nitrofurantoin		382				416
4-NPDA	86			56	105			58
Sodium azide			631				793
	+S9
	Initial assay				Confirmatory assay
	TA98	TA100	TA1535	TA1537	TA98	TA100	TA1535	TA1537
Vehicle	31	134	14	9	48	162	27	16
8	29	127	14	9	51	173	25	14
40	30	105	18	8	43	164	27	15
200	25	124	16	6	37	163	26	12
1000	25	125	16	9	35	174	23	14
5000	30	121	13	8	41	176	22	20
2-AA	427	817	99	134	570	1297	242	68

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Executive summary:

The mutagenicity of chromium hydroxide was investigated in an Ames test (plate incorporation assay) using S. typhimurium strains TA98, TA100, TA1535 and TA1537. Four replicate plates of each strain were exposed to the test material (suspended in deionised water) at concentrations of 0, 8, 40, 200, 1000 or 5000 ug/plate in the presence and absence of an exogenous metabolic activation system (Aroclor 1254 -induced male rat liver S9 fraction). There was no evidence of cytotoxicity at the limit concentration; precipitation of the test material was seen at 1000 and 5000 ug/plate. Exposure to the test material did not induce increased numbers of revertant colonies of any strain. Appropriate positive control compounds induced large increases in the numbers of revertant colonies, confirming the sensitivity of the assay. Results were confirmed in an independently-repeated assay. No evidence of mutagenicity was seen under the conditions of this study. The results of this study, performed using chromium (III) hydroxide, can be extrapolated to the similarly water-insoluble chromium (III) oxide salt.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

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

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)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

purity/physical description of the test item missing; solvent used was not defined; tubes were not aerated during pre-incubation by using a shaker; individual data missing; historical control data missing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

not specified

Target gene:

TA98: his D 3052
TA100 & TA1535: his G 46
TA97: hisD6610 hisO1242
TA102 & TA104: hisG428

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium, other: TA104

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix (containing S9 obtained from male Sprague-Dawley rats or male Syrian hamster livers; both types of S9 were used in the study; both types of S9 mix contained either 10 % or 30 % of S9)

Test concentrations with justification for top dose:

Experiment 1:
- 33 (TA98 only),100, 333, 667 (TA97 only), 1000, 2000 (TA97 only) 3333 and 10000 µg/plate (with and without metabolic activation; strains TA97, TA98, TA100, TA102, TA104 &TA1535)

Experiment 2:
- 100, 333, 666 (TA102 only), 1000, 1666, 3333, 6666 and 10000 µg/plate (with and without metabolic activation; strains TA100, TA102 and TA1535)
- 100, 333, 1000, 3333 and 10000 µg/plate (with and without metabolic activation; strains TA97, TA98 and TA104)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: yes (it was stated that a solvent was used, but the solvent was not further specified)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

mitomycin C

other: 4-nitro-o-phenylenediamine (without metabolic activation) & 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (as described in Haworth et al. (1983)*

EXPERIMENTAL PROTOCOL:
- test chemical (0.05 mL), overnight culture of Salmonella (0.05 mL), and S-9 mix or buffer (0.50 mL), were incubated at 37°C, without shaking, for 20 minutes.
- top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium.
- histidine-independent (his+) colonies arising on the plates were counted following two days incubation at 37°C.
- plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.
- at the discretion of the investigators, plates with low numbers of colonies, containing precipitated test chemical, or having excessively reduced contrast because of chemical color, were counted by hand.

Two or three trials were conducted for each concentration with and without metabolic activation.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity assay was performed using TA100 or the system developed by Waleh et al. (1982)*. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

* Reference:
- Haworth, S., Lawlor T., Mortelsmans, K., Speck, W., Zeiger E., (1983): Salmonella mutagenicity results for 250 chemicals. Environ Mutagen 7 [Suppl 1]: 3 - 142.
- Waleh NS, Rapport SJ, Mortelmans K (1982): Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains. Mutat Res 97:247-256.

Rationale for test conditions:

The high dose was set at 10000 μg/plate by experimental design, because no toxicity was observed.

Evaluation criteria:

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

Statistics:

mean ± standard error

Key result

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA102, TA104 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

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:

No induction of gene mutations was observed in two independent studies conducted with chromium picolinate (up to 10000 μg/plate) in several strains of S. typhimurium with and without hamster or rat liver S9.

Precipitation was observed as follows:
TA97:
- 10000 µg/plate (with 10 and 30 % hamster S9)
TA100 & TA1535:
- 3333 µg/plate (without S9 as well as with 10 and 30 % hamster and rat S9)

Please also refer for results to the field "Attached background material" below.

Conclusions:

No induction of gene mutations was observed in two independent studies conducted with chromium picolinate (up to 10000 μg/plate) in several strains of S. typhimurium with and without hamster or rat liver S9.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

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

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)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

three test strains only; solvent used was not defined; tubes were not aerated during pre-incubation by using a shaker; individual data missing; historical control data missing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (~25 °C), protected from light, in sealed plastic buckets.

Target gene:

TA100: his G 46
TA98: his D 3052
E. coli WP2 uvrA/pKM101: trpE

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:

S9 mix (containing 10 % rat S9)

Test concentrations with justification for top dose:

100, 500, 1000, 5000 and 10000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: yes (it was stated that a solvent was used, but the solvent was not further specified)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine (without metabolic activation; strain TA98) & 2-aminoanthracene (with metabolic activation; all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (as described in Haworth et al. (1983)*

EXPERIMENTAL PROTOCOL:
- test chemical (0.05 mL), overnight culture of Salmonella (0.05 mL), and S-9 mix or buffer (0.50 mL), were incubated at 37°C, without shaking, for 20 minutes.
- top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium.
- histidine-independent (his+) colonies arising on the plates were counted following two days incubation at 37°C.
- plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.
- at the discretion of the investigators, plates with low numbers of colonies, containing precipitated test chemical, or having excessively reduced contrast because of chemical color, were counted by hand.

Two or three trials were conducted for each concentration with and without metabolic activation.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity assay was performed using TA100 or the system developed by Waleh et al. (1982)*. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

* Reference:
- Haworth, S., Lawlor T., Mortelsmans, K., Speck, W., Zeiger E., (1983): Salmonella mutagenicity results for 250 chemicals. Environ Mutagen 7 [Suppl 1]: 3 - 142.
- Waleh NS, Rapport SJ, Mortelmans K (1982): Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains. Mutat Res 97:247-256.

Rationale for test conditions:

The high dose was set at 10000 μg/plate by experimental design, because no toxicity was observed.

Evaluation criteria:

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

Statistics:

mean ± standard error

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

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:

Over a concentration range of 100 to 10,000 μg/plate, no evidence of mutagenicity was observed in S. typhimurium strains TA100 or TA98 or E. coli strain WP2 uvrA/pKM101 when chromium picolinate monohydrate was tested with or without exogenous metabolic activation (S9).
Precipitation was observed in one trial without metabolic activation and one trial with metabolic activation at the highest concentration (10000 µg/plate) tested with S. typhimurium strain TA98.

Please also refer for results to the field "Attached background material" below.

Conclusions:

Over a concentration range of 100 to 10,000 μg/plate, no evidence of mutagenicity was observed in S. typhimurium strains TA100 or TA98 or E. coli strain WP2 uvrA/pKM101 with chromium picolinate monohydrate with or without exogenous metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

check for mycoplasma contamination of cells missing; changes in pH and osmolality were not measured /reported; individual culture data missing; historical control data missing

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay

Specific details on test material used for the study:

not specified

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

K1 cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture COllection, Manassas, VA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254-induced rat liver S9 was prepared by the laboratory; final S9 concentration was 10 % in the S9-cofactor mix and 2 % in treatment medium))

Test concentrations with justification for top dose:

Initial assay: 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL (with and without metabolic activation)
Independent repeat assay: 31.3, 62.5, 125, 250 and 500 µg/mL (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
The concentrations of the test item were exposed in a final concentration of 1 % dimethyl sulfoxide (DMSO).

- Justification for choice of solvent/vehicle: preparations of stock solutions of 500 mg/mL of the test item in organic solvents and 50 mg/mL in water showed that dimethyl sulfoxide appeared to be the best solvent for dissolving chromium picolinate and for attaining a solution that would result in maximum exposure of cells to the test material. DMSO exhibited a significantly deep red color from the solubilized test item when compared to distinct absence of color and rapid precipitation of the test item in the other solvents evaluated (distilled water, ethyl alcohol and acetone). Only a slight, fine precipitate could be discerned in the DMSO tubes compared to essentially complete precipitation of test item in the tubes containing the other solvents. The undissolved test item also stayed in suspension longer in DMSO and produced a visibly smaller amount of precipitate on the bottom of the tube than seen with the other solvents. Based on these visual solubility tests and the previous experience with compatibility of DMSO in the CHO test, DMSO was utilized as the solvent for testing of test item cytotoxicity and mutagenicity.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

MUTAGENESIS ASSAY:
Duplicate cultures of exponentially growing CHO–K1 cells were seeded in Hams F12 medium (without hypoxanthine) containing 5% dialyzed fetal bovine serum, 100 units of penicillin/mL, 100 µg streptomycin/mL and 2mM l-glutamine/mL at a density of 5×10E5 cells/25 cm² culture flask and incubated at 37 ± 1 °C in a humidified 5 ± 1 % CO2 atmosphere for 18 – 24 h. The cultures were exposed for 5 hours to concentrations of the test item and control materials with and without S9 activation. After the 5 hour treatment with the test and control materials, the media was removed, the cells washed with calcium and magnesium free Hanks balanced salt solution (HBSS) and fresh Hams F12 added to the cultures. After 18 – 24 h, cells were removed as above with rinsing and trypsinization and approx. 1×10E6 cells/100 mm dish were seeded into fresh culture medium to initiate the post-treatment period for expression of the mutant phenotype and 100 cells were seeded in triplicate in 60 mm culture dishes for cytotoxicity assessment. The cytotoxicity dishes were incubated at 37 ± 1 °C in a humidified 5 ± 1 % CO2 atmosphere for 7 – 10 days. Following this incubation period, medium was removed, cells were washed with HBSS, fixed with methanol and stained with 10 % Giemsa to allow counting of colonies. For determination of mutant frequencies, cultures, approx. 1×10E6 cells/100 mm dish, were sub-cultured every 2 – 4 days into fresh medium and after a 7 – 9 day expression period, 2×10E5 cells were seeded in each of five 100 mm plates for mutant selection in medium containing 10 µM of 6-thioguanine. Cultures were incubated at 37 ± 1 °C in a humidified 5 ± 1 % CO2 atmosphere for 7 – 10 days. Cells were also plated in triplicate, at a density of 100 cells/60 mm culture dish, for cloning efficiency in culture medium without 6-thioguanine and incubated at 37 ± 1 °C in a humidified 5 ± 1 % CO2 atmosphere. After incubation, the mutant selection and cloning efficiency plates were scored for the number of colonies and the cloning efficiency and mutant frequencies were calculated (Manchanoff et al. (1981); O'Neill et al. (1977); Hsie et al. (1981); Li et al. (1987, 1988))*.

ASSESSMENT OF CELLULAR CYTOTOXICITY:
Ranger-finder:
An initial stock solution of 50 mg/mL of the test item in DMSO was used to prepare subsequent dilutions that would deliver a constant volume of 50 µL of the test solution/5mL of the culture medium. CHO cells were treated with the solvent alone and nine concentrations of the test item ranging from 0.05 to 500 µg/mL in the absence and presence of S9 reaction mixture.

Mutagenesis assay:
In both the non-activated and S9-activated systems, 100 cells/plate, from cultures treated with concentrations of 15.6, 31.3, 62.5, 125, 250, and 500 µg/mL of the test item, were cloned in medium without 6-TG to determine concurrent cytotoxicity.

ACCEPTABILITY CRITERIA:
The following criteria for a valid test were required:
- cloning efficiency of the solvent control (media minus 6-thioguanine media) was greater than 50%
- spontaneous mutant frequency of the cells treated with solvent (media plus 6-thioguanine) was within the range of 0 – 25 mutants/10E6 clonable cells
- positive control induced a mutant frequency at least three times that of the solvent control and exceeded 40 mutants/10E6 clonable cells
- minimum of four analyzable concentrations with mutant frequency data was obtained.

*References:
- R. Machanoff, J.P. O’Neill, A.W. Hsie, Quantitative analysis of cytotoxicity and mutagenicity of benzo(a)pyrene in mammalian cells (CHO/HPRT), Chem. Biol. Interact. 34 (1981) 1–10.
- J.P. O’Neill, P.A. Brimer, R. Machanoff, G.P. Hirschand, A.W. Hsie, A quantitative assay of mutation induction at the hypoxanthine–guanine phosphoribosyl transferase gene in
Chinese hamster ovary cells (CHO/HPRT system): development and definition of the system, Mutat. Res. 45 (1977) 91 – 101.
- A.W. Hsie, D.A. Casciano, D.B. Couch, B.F. Krahn, J.P. O’Neill, B.L. Whitfield, The use of Chinese hamster ovary cells to quantify specific gene mutation and to determine mutagenicity of chemicals: a report of the Gene–Tox program, Mutat. Res. 86 (1981) 193 – 214.
- A.P. Li, J.H. Carver, W.N. Choy, A.W. Hsie, R.S. Gupta, K.S. Loveday, J.P. O’Neill, J.C. Riddle, L.F. Stankowski, L.L. Yang, A guide for the performance of Chinese hamster
ovary cell/hypoxanthine–guanine phosphoribosyl transferase gene mutation assay, Mutat. Res. 189 (1987) 135–141.
- A.P. Li, R.S. Gupta, R.H. Heflich, J.S. Wassom, A review and analysis of the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase assay to determine the mutagenicity of chemical agents. A report of phase III of the U.S. Environmental Protection Agency Gene–Tox program, Mutat. Res. 196 (1988) 17 – 36.
- A.W. Hsie, Structure–mutagenicity analysis with CHO/HPRT system, Food Cosmet. Toxicol. 19 (1981) 617–621.

Rationale for test conditions:

Based on the results of the toxicity test and solubility test, the concentrations chosen for the mutagenesis assay ranged from 15.6 to 500 µg/mL for both the non-activated and S9- activated cultures with a 5 h exposure. Higher doses were not tested due to the limited solubility of the test item.

Evaluation criteria:

The result was considered to be a positive mutagenic effect, if a concentration-related increase in mutant frequency was observed and two consecutive dose levels exhibited mutant frequencies of >40 mutants/10E6 clonable cells. A result was considered equivocal, if a single dose exhibited a mutant frequency of >40 mutants/10E6 clonable cells. If no dose level produced a mutant frequency of 40 mutants/10E6 clonable cells over the background level, the result was concluded to be negative, based upon experience at the laboratory with historical variability of the spontaneous frequency of mutants in this test system.

Statistics:

The method of Snee and Irr (1981)* was used to describe, if the highest concentration exhibited a mutant frequency of 40 or more mutants/10E6 clonable cells over the background. Following power transformation of the data, a Student’s t-test (for significance of observed increases) and analysis of variance (ANOVA) was conducted to determine, if there was a dose–responsive increase in mutant frequency. A trend analysis was considered, but not used, because none of the test doses produced positive effects as judged by mutant frequencies >40 mutants/10E6 clonable cells over the background.

*Reference:
- R.D. Snee, J.D. Irr, Design of a statistical method for the analysis of mutagenesis at the hypoxanthine–guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells, Mutat. Res. 85 (1981) 77–93.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

K1 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field "Additonal information on results" below.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Initial assay: there was a visible precipitate in the treatment medium only at 500 µg/mL.
Independent repeat assay: there was a visible precipitate in the treatment medium only at 500 µg/mL.

RANGE-FINDING STUDY:
Toxicity (i.e., cloning efficiency ≤ 50 % of the solvent control) was observed in the presence of a visible precipitate in the treatment medium only at 500 µg/mL. Cloning efficiency relative to the solvent controls (RCE) at 500 µg/mL was 50 % without activation with a 5 h exposure, 66 % with S9 activation with a 5 h exposure. Based on the results of the toxicity test, the concentrations chosen for the mutagenesis assay ranged from 15.6 to 500 µg/mL for both the non-activated and S9-activated cultures with a 5 h exposure.

MUTAGENESIS ASSAY.
- Initial assay: in a total of 1×10E6 cells seeded into medium containing 6-thioguanine (2×10E5 cells in each of 5100 mm dishes), none of the treated cultures exhibited mutant frequencies of greater than 40 mutants/10E6 clonable cells. Although there were slight increases in mutant frequency over the solvent control value in some cultures, these small increases were not consistently dose-related and were within the range of variability of spontaneous mutant frequencies in CHO cells; these increases were not considered indicative of a positive response in the assay.
- Independent repeat assay: none of the treated cultures seeded into 6-thioguanine selection medium exhibited mutant frequencies of greater than 40 mutants/10E6 clonable cells, the basal increase considered to represent a positive mutagenic effect. Although some concentrations produced slight increases in mutant frequency over the solvent control value, these small increases were not consistently dose-related and were within the normal range of variability seen in this test system.

INFORMATION ON CYTOTOXICITY (Mutagenesis assay):
- Initial assay: cloning efficiency relative to the solvent controls was 78 and 68% at the highest concentration tested in the nonactivated and S9-activated systems, respectively.
- Independent repeat assay: relative cloning efficiency was 89 and 74% at the highest concentration tested in the non-activated and S9-activated systems, respectively.

Please also refer to the field "Attached background material" below.

NOTE: a second second study evaluated the effect of the test item in a CHO/Hprt gene mutation test with a 48 hour exposure period in the absence of metabolic activation to duplicate treatment conditions employed by Stearns et al. (2002)*.

The test conditions, sub-culturing and expression periods were the same as for the 5 hour exposure to the test item and control agents, as described in this endpoint study summary.

Results:

There was visible precipitate in the treatment medium only at 500 µg/mL. Relative cloning efficiency was determined with 100 cells/plate from cultures treated with concentrations of 31.3, 62.5, 125, 250, and 500 µg test item/mL and in medium without 6-TG. The lowest Relative cloning efficiency was 25% at 500 µg/mL. No mutant frequencies of greater than 40 mutants/10E6 clonable cells were observed at any dose level and all increases above the concurrent solvent control values were within the normal range of variation seen in this test system.

*Reference:

- D.M. Stearns, S.M. Silveira, K.K.Wolf, A.M. Luke, Chromium (III) tris(picolinate) is mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase gene in Chinese hamster ovary cells, Mutat. Res. 513 (2002) 135–142.

Conclusions:

Chromium picolinate was tested in the in vitro gene mutation assay in the CHO/hprt cellline. No increase mutation frequency was seen when tested up up to precipitating concentrations with and without metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

physical description of test item missing; info. on pH, osmolality & precipitation missing; confirmatory assay, individual data & historical control data missing; justification for choice of solvent missing; vol. of vehicle & test substance added missing

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay

Specific details on test material used for the study:

not specified

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y TK+/- 3.7.C mouse lymphoma cells were obtained from Ms. Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC.

MEDIA USED
- Type and identity of media: cells were grown in Fischer's medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.
- Periodically checked for Mycoplasma contamination: yes

New cultures were initiated at approx. three-month intervals from cells stored in liquid N2.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (containing liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats)

Test concentrations with justification for top dose:

500, 600, 650, 700 and 750 µg/mL (equivalent to 97.5, 117.0, 126.8, 136.5 and 146.3 µg Cr/mL) (without metabolic activation)
1700, 1750, 1800, 1900 and 2000 µg/mL (equivalent to 331.5, 341.3, 351.0, 370.5 and 390.0 µg Cr/mL) (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
Stock solutions of the compound were prepared immediately prior to use. The final concentration of solvent was 10 % water.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

MUTAGENICITY ASSAY:
The mutagenicity assay was performed as described (Clive and Spector, 1975)*. A total of 1.2 x 10E7 cells in duplicate cultures were exposed to the test chemical and controls for 4 hours at 37 ± 1 °C, washed twice with growth medium and maintained at 37 ± 1 °C for 48 hours in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 x 10E5 cells/mL at 24 hours intervals. They were then cloned (1 x 10E6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft-agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68 and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration: 3 µg/mL) to the cloning medium for mutant selection. Plates were incubated at 37 ± 1 °C in 5% CO2 in air for 10 – 12 days and then counted with a ProtoCOL automated colony counter.

The size of mutant mouse lymphoma colonies was also determined using an Artek or a ProtoCOL colony counter/sizer. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in increments of approx. 0.1 mm in colony diameter. The size range used was approx. 0.2 – 1.1 mm.

DETERMINATION OF CYTOTOXICITY
The toxicity of the chemical was determined both with and without metabolic activation. Cells at a concentration of 6 x 10E5/mL (6 x10E6 cells total) were exposed for 4 hours to a range of concentrations of the chemical. The cells were then washed, resuspended in growth medium and incubated at 37 ± 1 °C for 24 hours. Cells in the cultures were then adjusted to 3 x 10E5 cells/mL and incubated at 37 ± 1 °C for an additional 24 hours. The rate of cell growth was determined for each of the treated cultures and compared with the rate of growth of the solvent controls. The doses of the chemical selected for testing were within the range yielding approx. 0 – 90 % cytotoxicity.

*Reference:
- Clive, D., Spector, J.F.S., 1975. Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y TK+/- mouse lymphoma cells. Mutat. Res. 31, 17 – 29.

Rationale for test conditions:

Concentrations were selected based on results from a cytotoxicity test (please refer to the field "Details on test system and conditions" above). The doses of the chemical selected for testing were within the range yielding approx. 0 – 90 % cytotoxicity.

Evaluation criteria:

The results were evaluated according to the guidelines in Clive et al. (1995)*. A data set would be considered to show an indication of mutagenic activity if there was evidence of a dose response with at least one concentration giving an increase in mutant frequency of at least 100 mutants per 10E6 surviving cells above the concurrent solvent control value. Only colonies larger than approximately 0.1 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10E6 surviving cells. Only doses yielding total growth values of ≥10% were used in the analysis of induced mutant frequency.

*Reference:
- Clive, D., Bolcsfoldi, G., Clements, J., Cole, J., Homn, M., Majeska, J., Moore, M., Muller, L., Myhr, B., Oberly, T., Oudelhkim, M., Rudd, C., Shimada, H., Sofuni, T., Thybaud, V., Wilcox, P., 1995. Consensus agreement regarding protocol issues discussed during the mouse lymphoma workshop: Portland, Oregon, May 7, 1994.
Environ. Mol. Mutagen. 25, 165–168.

Statistics:

not specified

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity between 26.5 % and 82.5 % was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity between 29.5 % and 79.5 % was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY ASSAY
Chromium chloride was negative in the absence of metabolic activation and induced a questionable response with metabolic activation. The amount of Cr/mL at the highest dose tested was 390 µg.
Please also refer to the field "Attached background material" below.

Conclusions:

Chromium chloride was negative in the in vitro mouse lymphoma assay (tk mutation) in L5178Y cells, when tested up to cytotoxic concentrations (RTG <20%) in the absence and presence of metabolic activation. A slight but not statistically significant increase in the mutant frequency was seen in the highest dose tested with metabolic activation. Since this increased mutation frequency was seen only at the highes dose with severe cytotoxicty, it is regarded as not biologically relevant. Consequently chromium(III) chroride is considered negative in the in vitro mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo test systems: negative (OECD 474; GLP compliant)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10/09/1991-23/10/1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983-05-26

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 29 - 42 g
- Assigned to test groups randomly: yes
- Housing: males: kept singly in Makrolon type I cages; females: groups of a maximum of three animals in Makrolon type I cages; bedding material: soft wood granules, type S 8/15 (J. Rettenmaier &Söhne, Füllstoff-Fabriken, 7092 Ellwangen-Holzmühle)
- Diet (ad libitum): Altromin 1324 Standard diet (supplier: Altromin GmbH, Lage)
- Water (ad libitum)
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 - 23.0
- Humidity (%): 47 - 54
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil (suspension); 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Chromoxid-Grün GN was suspended in corn oil.

Duration of treatment / exposure:

Groups administered the test material were sacrificed at 16, 24 or 48 hours.
Negative and positive control groups were sacrificed at 24 hours.

Frequency of treatment:

Single intraperitoneal dose.

Post exposure period:

Groups administered the test material were sacrificed at 16, 24 or 48 hours.
Negative and positive control groups were sacrificed at 24 hours.

Dose / conc.:

10 000 other: mg/kg bw (actual dose received)

No. of animals per sex per dose:

5 males / 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control animals were administered a single ip dose of cyclophosphamide (20 mg/kg bw).

Tissues and cell types examined:

1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
The number of normochromatic erythrocytes per 1000 polychromatic ones was noted.
The number of normochromatic erythrocytes showing micronuclei was also established.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The selection of the Chromoxid-Grün GN dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 5000 mg/kg and 10000 mg/kg Chromoxid-Grün GN. The following symptoms were recorded for up to 48 hours, starting at 5000 mg/kg: apathy, stretching of body, roughened fur, staggering gait and spasm. None of the animals died.
Based on these results, 10000 mg/kg Chromoxid-Grün GN was chosen as MTD for this test.

SAMPLING TIMES / DETAILS OF SLIDE PREPARATION / METHOD OF ANALYSIS:
Groups administered the test material were sacrificed at 16, 24 or 48 hours.
At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor) and the proximal end of the femur was opened at its extreme end. A cannula containing foetal calf serum was pushed into the open end of the marrow cavity. The femure was then compeletly immersed in the calf serum. The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension. Finally, the flushing might be repeated from the other end, after it had been opened.
The tube containing the serum and bone marrow was centrifuged (approx. 1000 rpm) and the supernant was removed. The sediment was mixed to produce a homogeneous suspension.
One drop of the viscous suspension was placed on a well-cleaned slide and spread with a suitable object. The labeled slides were dried overnight.

The smears were stained automatically with an Ames HemaTek Slide Stainer from the Miles Company. The slides were then "destained'' with methanol, rinsed with deionized water, and left to dry.

Following this treatment, the smears were transferred to a holder. A cuvette was filled with xylene, into which the holder was immersed for approximately ten minutes. The slides were removed singly to be covered.

A small amount of covering agent was taken from a bottle and applied to the coated side of the slide. A cover glass was then placed in position without trapping bubbles. The slides were not evaluated until the covering agent bad dried.

Slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus.

Evaluation criteria:

A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or signif icant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant. In this case, a second test had to be performed at the most sensitive interval.

Statistics:

Wilcoxon's non-parametric rank sum test; one sided chi-squared test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

(systemic toxicity and cytotoxicity)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Since there were no relevant variations in results between males and females, they were evaluated jointly.
The proportion of NCEs was increased in all groups receiving the test substance; significantly at the 16 hour sacrifice. The proportion of micronucleated NCEs was comparable in all groups. The proportion of micronucleated PCEs in all groups receiving the test material was comparable in the vehicle control and test substance groups; a significantly higher proportion of micronucleated PCEs was seen in the positive control group.

Please refer for the results and the historical control data to the field "Attached background material" below.
Please also refer for results to the field "Any other information on results incl. tables" below.

OBSERVATIONS.
Chromoxid-Grün GN, treated animals showed the following compound-related symptoms until sacrifice: apathy, stretching of body, roughened fur, staggering gait, spasm and difficulty in breathing. Their feeding behavior was normal. There were no substance-induced mortalities. No symptoms were recorded for the control groups. No animals died in these groups.

Summary of results:

Since there were no relevant variations in results between males and females, they were evaluated jointly.

Treatment	Sacrifice	Cells (#)	NCEs (#)	Micronuclei (#)
				/1000 NCEs	/1000 PCEs
Vehicle control	24h	10000	763	1.4	1.8
Test substance	16h	10000	2082*	1.0	1.1
Test substance	24h	10000	1537	1.3	1.9
Test substance	48h	10000	1497	1.3	0.6
Cyclophosphamide	24h	10000	675	1.8	15.7*

*P<0.01

The proportion of NCEs was increased in all groups receiving the test substance (significantly at the 16 hour sacrifice), indicating adequate exposure of the bone marrow. The incidence of micronucleated PCEs was comparable in the vehicle controls and groups receiving the test substance. A significantly increased incidence of micronucleated PCEs was seen in the positive control group.

Conclusions:

In conclusion, there was no indication of a clastogenic effect of an intraperitoneal dose of 10000 mg/kg Chromoxid-Grün GN in the micronucleus test on the mouse:

Executive summary:

The genotoxicity of chromium (III) oxide was investigated in a mouse micronucleus assay. Mice (5/sex/group) were administered single intraperitoneal doses of the test material at a dose level of 10000 mg/kg bw and femoral bone marrow harvested following sacrifice at 16, 24 or 48 hours. Negative (vehicle) and positive control (cyclophosphamide) groups were sacrificed at 24 hours. 1000 polychromatic erythrocytes per animal were assessed for the numbers of micronuclei and the proportion of normochromatic cells. The proportion of NCEs was increased in all groups receiving the test substance (significantly at the 16 hour sacrifice), indicating adequate exposure of the bone marrow. The incidence of micronucleated PCEs was comparable in the vehicle controls and groups receiving the test substance. A significantly increased incidence of micronucleated PCEs was seen in the positive control group, confirming the sensitivity of the assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There is not sufficient information available on the in vitro and in vivo genotoxicity of dichromium trioxide to fulfil the formal data requirements in accordance with the annexes of regulation (EC) 1907/2008. One in vivo micronucleus study in mice with dichromium trioxide is available, showing no evidence for clastogenic or aneugenic activity after i.p. administration. The remaining evidence for the lack of genotoxicity of dichromium trioxide is taken from read-across studies with chromium(III) substance with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (March 2017) attached to IUCLID section 13.

 

 

in vitro studies

In two NTP studies (NTP, 2010) chromium picolinate was evaluated for bacterial mutagenicity in strains of Salmonella typhimurium (TA98, TA100, TA1535, TA97, TA102, TA104 and TA100, TA98, E. coli WP2 uvrA/pKM101) using the pre-incubation method. Five concentrations were tested in triplicate in the absence and presence of S9 in 2 independent experiments. Chromium picolinate (up to 10,000 μg/plate) gave a negative response in all strains in the presence and absence of S9 activation mix.

 

Whittaker et al. (2005) tested chromium(III) chloride at 100, 333, 1000, 3333 and 10000 µg/plate with and without metabolic activation in Salmonella typhimurium TA98, TA100 and TA1535 in triplicate cultures using the pre-incubation method. Chromium(III) chloride gave a negative response in all strains in the presence and absence of S9 activation mix.

 

Chromium(III) chloride has been tested for induction of tk mutations in mouse lymphoma L5178Y cells (Whittaker 2005). Treatments were for 4 hr hrs in the absence and presence of S9. Chromium chloride was negative in the in vitro mouse lymphoma assay (tk mutation) in L5178Y cells, when tested up to cytotoxic concentrations (RTG <20%) in the absence and presence of metabolic activation. A slight but not statistically significant increase in the mutant frequency was seen in the highest dose tested with metabolic activation. Since this increased mutation frequency was seen only at the highest dose with severe cytotoxicty, it is regarded as not biologically relevant. Consequently chromium(III) chloride is considered negative in the in vitro mouse lymphoma assay.

 

Slesinski, R.S. et al. (2005) assessed the mutagenic potential of chromium(III) chloride. Duplicate cultures of exponentially growing CHO–K1 cells were exposed for 5 hours to concentrations of chromium(III) chloride and control materials with and without S9 activation. In both the non-activated and S9-activated systems, 100 cells/plate, from cultures treated with concentrations of 15.6, 31.3, 62.5, 125, 250, and 500 µg/mL of the test item, were cloned in medium without 6-TG to determine concurrent cytotoxicity. Visible precipitate was visible only at 500 µg/mL. The lowest relative cloning efficiency was 25% at 500 µg/mL. No mutant frequencies of greater than 40 mutants/10E6 clonable cells were observed at any dose level and all increases above the concurrent solvent control values were within the normal range of variation seen in this test system. No increase mutation frequency was seen when tested up to precipitating concentrations with and without metabolic activation.

 

in vivo studies

The genotoxicity of dichromium trioxide was investigated in an in vivo mouse micronucleus assay. Mice (5/sex/group) were administered single intraperitoneal doses of the test material at a dose level of 10000 mg/kg bw and femoral bone marrow harvested following sacrifice at 16, 24 or 48 hours. Negative (vehicle) and positive control (cyclophosphamide) groups were sacrificed at 24 hours. 1000 polychromatic erythrocytes per animal were assessed for the numbers of micronuclei and the proportion of normochromatic cells. The proportion of NCEs was increased in all groups receiving the test substance (significantly at the 16 hour sacrifice), indicating adequate exposure of the bone marrow. The incidence of micronucleated PCEs was comparable in the vehicle controls and groups receiving the test substance. A significantly increased incidence of micronucleated PCEs was seen in the positive control group, confirming the sensitivity of the assay.

 

In a 13 week (5 days per week) oral repeated dose toxicity study with 5 different doses of chromium picolinate, the MN frequency was measured in the peripheral blood of mice (NTP, 2010) . The frequency of micronucleated cells in 1000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group was determined. No increase in the frequency of micronucleated normochromatic erythrocytes was observed in male B6C3F1 mice administered chromium picolinate monohydrate in feed for 3 months, indicating no potential for inducation of chromosomal alterations. In female mice, however, the small increase in micronucleated normochromatic erythrocytes noted in the highest exposure concentration group (50,000 ppm) was not significant at P=0.0396. There were no clinical findings in male or female animals, related to exposure to chromium picolinate monohydrate; reddish-colored faeces of 50000 ppm animals were believed to be due to excretion of the test article and were not considered a sign of toxicity. All mice survived to the end of the study. Consequently, chromium picolinate is considered non clastogenic and non aneugenic in this in vivo assay.

 

Five male rats were treated with chromium picolinate three times during 72 hrs orally and sacrificed 24 hrs after final treatment. 2000 polychromatic erythrocytes were scored for the frequency of micronucleated cells in each of five rats per dose group. No induction of micronucleated polychromatic erythrocytes was observed in bone marrow of male F344/N rats treated with chromium picolinate (156 to 2,500 mg/kg) by oral gavage three times at 24-hour intervals, and no significant alterations in the percentage of polychromatic erythrocytes among total erythrocytes was observed in dosed rats, indicating that these doses of chromium picolinate did not induce bone marrow toxicity. Consequently, chromium picolinate is considered non clastogenic and non aneugenic in this in vivo assay.

 

In an in vivo chromosome aberration assay in bone marrow, male and female SD rats were treated once with chromium picolinate orally via gavage at concentrations of 33, 250 and 2000 mg/kg bw. Animals were sacrificed 18 and 42 hrs after dosing. In all test item-treated groups there were no differences in frequency of chromosomal aberrations when compared to vehicle controls. The rats treated with the test item presented no significant differences in mean mitotic index compared with the vehicle control group. The mean total number of chromosomal aberrations in test item groups treated rats displayed no significant differences. The positive control, induced a significant amount of chromosomal damage in both males (30%) and females (37%) that were statistically different from all test item doses and from the vehicle control. Consequently, chromium picolinate is considered non clastogenic in this in vivo assay.

 

 

Conclusion

The available data on genetic toxicity allow a conclusive statement on the genetic toxicity for the soluble chromium(III) salts category. There is no convincing evidence that the chromium(III) salts category induce gene, chromosome or genome mutations either in bacterial or in mammalian cells let alone in in vivo systems. Overall, there is no consistent evidence of induction of genetic toxicity with relevance to humans for the soluble chromium(III) salts category.

Justification for classification or non-classification

In consideration of the negative test results in highly reliable genotoxicity assays with the soluble chromium(III) salts category, no genotoxicity needs to be expected from exposure to the target substance dichromium trioxide. In consequence, no classification is required.