Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria

Ames test, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 +/-S9, negative

Ames test, S. typhimurium TA 98 and TA 100, E. coli WP2 uvrA pKM 101, +/-S9, negative

Gene mutation in mammalian cells

HPRT, primary human fibroblast cells, -S9, ambiguous

Chromosomal aberration

CA, Chinese hamser ovary cells, -S9, positive

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:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only Salmonella typhimurium strains TA98, TA100 and Escherichia coli strain WP2 uvrA/pKM101 were tested

GLP compliance:

no

Remarks:

data from apublication

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver of Aroclor 1254-induced male Sprague-Dawley rats
- concentration or volume of S9 mix and S9 in the final culture medium: 10 %
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): positive control for metabolic activation used (2-aminoanthracene)

Test concentrations with justification for top dose:

0, 100, 500, 1000, 5000, 10000 µg/plate
The high dose was set at 10,000 μg/plate by experimental design, because no toxicity was observed.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine (TA98, -S9), 2-aminoanthracene (all strains, +S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : 2 (S. typhimurium strain TA 100, E. coli WP2 uvrA), 3 (S. typhimurium strain TA 98)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in buffer (preincubation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Exposure duration/duration of treatment: 2 d

Test articles were incubated with the bacterial tester strains either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rats) for 20 minutes at 37° C. Top agar supplemented with L-histidine and d-biotin was added, and the
contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.

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.

Species / strain:

S. typhimurium 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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

not measured/tested

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Species / strain:

S. typhimurium TA 1537

Remarks:

not measured/tested

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: precipitation observed in strain TA 98 in highest dose group with and without metabolic activation

STUDY RESULTS
- Concurrent vehicle negative and positive control data : please refer to table attached

Ames test:
- Mean number of revertant colonies per plate and standard deviation : please refer to table attached

Conclusions:

In the standard bacterial reverse mutation screening assays conducted by the NTP, chromium picolinate monohydrate showed no clear evidence of genotoxicity. Over a concentration range of 100 to 10,000 μg/plate, no evidence of mutagenicity was observed in S. typhimurium strains TA100 and TA98 and E. coli strain WP2 uvrA/pKM101 when chromium picolinate monohydrate was tested with or without exogenous metabolic activation (S9).

Executive summary:

Chromium picolinate monohydrate was tested in a bacterial reverse mutation test in in Salmonella typhimurium strains TA98, TA100 and Escherichia coli strain WP2 uvrA/pKM101 at a concentration range of 100 to 10,000 μg/plate with and without exogenous metabolic activation (S9). The substance was negative in all tested strains with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sigma-Aldrich
- Purity, including information on contaminants, isomers, etc.: 99 %

Target gene:

his

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Liver S9 homogenate was prepared from male Sprague–Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight.
- method of preparation of S9 mix : 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4) and the appropriate S9 homogenate at a concentration of 0.1 ml/ml of mix.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 ml S9 mix per plate

Test concentrations with justification for top dose:

100, 333, 1000, 3333, 10000 µg/plate

The doses to be tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (1.0 μg/plate) for all strains +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1–2 x 10^9 cells/ml.
- Test substance added to 2.5 ml of molten selective top agar at 45 ± 2 °C (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h


All plates were counted with a Minicount automated colony counter (Imaging Products International, Chantilly, VA), which was calibrated prior to use.

Evaluation criteria:

For a test article to be considered positive, it must induce at least a doubling (TA98 and TA100) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate must be accompanied by a dose response to increasing concentrations of the test chemical. If the study shows a dose–response but with a less than threefold increase on TA1535 or TA1537, the response must be confirmed in a repeat experiment.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Please refer to attached table.

Ames test:
- Signs of toxicity : not specified
- Individual plate counts : please refer to attached table
- Mean number of revertant colonies per plate and standard deviation : please refer to attached table

Conclusions:

Chromium chloride did not induce a mutagenic response in S. typhimurium strains TA100, TA98, TA1535 and TA1537.

Executive summary:

A bacterial reverse mutation assay similar to OECD Guideline 471 was performed using the plate incorporation method. In this study chromium (III) chloride was tested in the S. typhimurium plate incorporation assay with tester strains TA100, TA98, TA1535 and TA1537 at dose levels up to and including 10,000 µg/plate without metabolic activation and with liver S9 preparations from Sprague- Dawley rats and Syrian golden hamsters. As a result, chromium chloride did not induce a mutagenic response in any of the tested strains of S. typhimurium.

Reference 3

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:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The frequencies of chromosome aberrations and SCE have been determined in CHO cells after incubation for 2 division cycles (32 h) with different concentrations of Cr compounds added directly to the growth medium. Bromodeoxyuridine was incorporated in the CHO cell cultures. The metaphase cells were prepared, so that chromosome aberrations were scored on the cell preparations.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

name in publication: Neochromium

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

maintained in vitro as monolayers, grown in Eagle's minimal essential medium (MEM) supplemented with 10% calf serum

Metabolic activation:

without

Test concentrations with justification for top dose:

5, 25, 150 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : single
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 32 h (two division cycles)
- Harvest time after the end of treatment (sampling/recovery times): cells were analysed directly after end of exposure

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Bromodeoxyuridine was incorporated in the CHO cell cultures for 32 h
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 30 metaphases scored

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cell growth (% of control) - Cell growth was estimated on the basis of the DNA + RNA content of treated cultures.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

please refer to 'Any other information on results.

Table 1: Chromosome aberrations and sister chromatid exchanges induced by Cr(III) compounds in CHO cell cultures
Treatment*	Concentrations (µg/mL)	Cell growth (% control)^f	Meta-phases counted¹	Chromosome and chromatid aberrations per 100 metaphases	SCE/ metaphase	t for SCE/ metaphase ^§	P
Neochromium (Chromium hydroxide sulphate) (III)	5	100	30	20.0	7.46 ± 0.22	0.05	> 0.7
	25	100	30	23.3	7.57 ± 0.18	0.50	> 0.5
	150	100	30	26.7	7.62 ± 0.22	0.69	> 0.4
* Monolayers were treated for 32 h in MEM.
^fCell growth was estimated on the basis of the DANN + RNAcontent of treated cultures.
¹ Chromosome aberrations and SCE were scored on the same 2nd division metaphases.
^§ t values for comparison with control

Conclusions:

The frequency of chromosome aberrations is increased after treatment with Cr(III) compounds (i.e. chromium hydroxide sulphate), being about doubled after exposure to 25-150 µg/ml.

Executive summary:

The frequencies of chromosome aberrations and SCE have been determined in CHO cells after incubation for 2 division cycles with different concentrations of Cr compounds added directly to the growth medium. The frequency of chromosome aberrations is increased after treatment with Cr(III) compounds (i.e. chromium hydroxide sulphate), being about doubled after exposure to 25-150 µg/ml. On the other hand, the frequencies of SCE are not significantly increased.

Reference 4

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:

guideline study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: induction of 6-Thioguanine Resistance as indication for mutation
- Short description of test conditions: HFC were plated at 10E5 cells/60-mm dish into Ham's F10 medium containing 15% fetal calf serum, 10 dishes for each concentration were studied. The positive control MNNG or the test item was added 1 day later for 24 h. Treated cells were then reseeded 6 days later at a density of 3 x 10E4 cells/60-mm dish, 40 to 60 dishes for each concentration of each compound studied, so that approximately 1.5 x 10E6 cells were examined for each treatment group per experiment, into medium containing 6-thioguanine (3 x 10E-5 M).
- Parameters analysed / observed: All mutant colonies were fixed, stained, and counted 14 days after plating. Colonies containing 25 cells were scored and mutation frequencies (MF) were calculated as:
MF = (Total no. of 6-TG' colonies) / (Plating efficiency) x no. of cells seeded/dish

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyltransferase locus)

Species / strain / cell type:

primary culture, other: human fibroblast cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human infants were circumcised and the foreskin tissues were obtained through the courtesy of the Huntington Memorial Hospital in Pasadena, CA, and were used to prepare primary cultures of HFC

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's minimum essential media supplemented with 0.1 µM sodium pyruvate, 1 x nonessential
amino acids, 0.2% sodium bicarbonate, 2.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, and 15% fetal calf serum was used for cell culturing. All HFC used in these studies
were routinely checked for Mycoplasma contamination by growth on Mycoplasma agar and were found to be negative.

Metabolic activation:

without

Test concentrations with justification for top dose:

50, 100, 250, 750 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : 10
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3 x 10E4 cells/60-mm dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 24 h
- Harvest time after the end of treatment (sampling/recovery times): 6 d

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 6 d
- Selection time (if incubation with a selective agent): 14 d
- Fixation time (start of exposure up to fixation or harvest of cells): 21 d
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 6-thioguanine (3 x 10E-5 M in medium), 14 days of exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 3 x 10E4 cells/60-mm dish, Colonies containing 25 cells were scored

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative survival

Statistics:

chi square test using a 2 x 2 table

Species / strain:

primary culture, other: human fibroblast cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Of the trivalent compounds studied, soluble CrCl3-6H2O was not mutagenic, while insoluble CrCl3 induced up to 4-fold increases in the mutation frequency.

Table 1 Induction of anchorage independence in human fibroblasts by chromium(III) compounds
Treatment (µM)	Cytotoxicity: relative survival (%)	AI colonies/ dish	AI frequency/1 x 10^5survivors	Fold increase in AI frequency
Control*	100	6±2	26 ±9
Insoluble chromium
Anhydrous CrCl3^a
0.05	88	10 ±4	54 ±22	2
0.1	80	18 ±3	75 ± 13*	3
1.0	87	17 ±4	113 ±27*	4
2.5	84	15 ±3	107 ± 21*	4
5.0	78	18 ±4	138 ±31*	5
10.0	65	20 ±2	222 ± 22*	9
Soluble chromium
CrCl36H2O
2.5	75	11 ±3	73 ±20	3
5.0	87	15±3	83 ± 17*	3
10.0	75	19 ± 1	83 ±4*	3
^aResults averaged from two experiments. * P < 0.05, Student's / test. For all other values, P > 0.05.

Table 4 Mutagenicity of chromium compounds to human fibroblasts
Treatment (aim)	surviving fraction	6-TG^rMutants/ 1 x 10^6survivors
		Average^a	Range^b
Control	1.00	1	(<0.5-12)
MNNG
1.7	0.52	36^c
3.0	0.10	99^c	(30-171)
5.0	0.01	1187^c	(303-1500)
Chromium(III) compounds
Soluble
CrCl36H20
750.0	0.90	5	(2-7)
1000.0	0.84	4	(1-8)
Insoluble
CrCl3
50.0	0.71	<1	(<1 - <4)
100.0	0.69	2	(<1 - 4)
250.0	0.66	3	(3 - 3)
750.0	0.81	17^c	(15 - 20)
^aThe average number of 6-TG^rcolonies, calculated from the 0 term of the Poisson series, P0= e^-m, from 2-3 experiments.
^bNumbers in parentheses, range of 6-TG^rmutants per 1 x 10^6survivors from 2-3 experiments.
^cP < 0.01, χ^2, using a 2 x 2 table. For all other values, P > 0.01.

Conclusions:

Of the trivalent compounds studied, soluble CrCl3-6H2O was not mutagenic, while insoluble CrCl3 induced up to 4-fold increases in the mutation frequency.

Executive summary:

A HPRT assay was performed with the soluble and insoluble Cr (III) compounds CrCl3-6H2O and CrCl3. The insoluble CrCl3 induced up to 4-fold increases in the mutation frequency to 6-thioguanine resistance at cytotoxic concentrations in primary human fibroblast cells (HFC). The soluble CrCl3-6H2O, on the other hand, did not increase the mutation frequency.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Comet assay, rat, diet, negative

Micronucleus assay, mouse, ip, OECD 474, GLP, negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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:

no guideline followed

Principles of method if other than guideline:

The study was designed to assess the genotoxicty of the test substance in rat peripheral blood lymphocytes using the comet assay

GLP compliance:

not specified

Remarks:

Data published, GLP not specified

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Chromium (III) propionate cation (CrProp) in the form of its nitrate salt (chemical formula [Cr3O(O2CCH2CH3)6(H2O)3]+(NO3)- was synthesized in the laboratory of Department of Product Ecology, Poznan University of Economics. The contents of elemental Cr (20.5%) was determined by the AAS method (spectrometer AAS-3 with BC correction, Zeiss, Germany).

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Licensed Laboratory Animals Breeding Center (Poznan, Poland)
- Age at study initiation: 12 weeks
- Weight at study initiation: ca. 196 g
- Assigned to test groups randomly: no, according to similar mean body mass
- Housing: single cages
- Diet (e.g. ad libitum): ad libitum (Labofeed H)
- Water (e.g. ad libitum): ad libitum (deionized water)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 55 - 60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

- Vehicle(s)/solvent(s) used: none

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): commercial diet for maintenance of adult rodents (Labofeed H)

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

Post exposure period:

No post exposure period; rats were sacrificed 12 hours after the end of the exposure period.

Dose / conc.:

1 000 other: mg Cr(III)/kg diet

Remarks:

Cr(III) given as [Cr3O(O2CCH2CH3)6(H2O)3]NO3, equivalent of 100 mg Cr/kg body mass/day

No. of animals per sex per dose:

Six female rats

Control animals:

yes, plain diet

Positive control(s):

Cr(VI) as K2Cr2O7 at a dose of 10 mg Cr(VI)/kg diet

Tissues and cell types examined:

At the end of the study after a 12 hour starvation period, rats were sacrifced by carbon dioxide asphyxiation, blood was collected into Li-heparinised tubes. Rat peripheral blood lymphocytes (PBL) were obtained. The liver, kidneys, heart, spleen, pancreas and ovaries were harvested and weighed.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 4 week exposure via food, sampling after 12 h starvation period

DETAILS OF SLIDE PREPARATION: Rat peripheral blood lymphocytes (PBL) were separated by the standard method. The cells were suspended in the RPMI 1640 medium without L-glutamine and centrifuged over Gradisol L at 1200 rpm for 15 min. Next, centrifugation was performed twice at 700 rpm for 8 min. The PBL suspension (30 µl) was mixed with 70 µl of 1% low melting point agarose in the RPMI 1640 medium at 37 °C. The mixture was pipetted onto microscope slides previously pre-coated with
a layer of 1% normal agarose. The slides were immersed in lysis solution (2.5 M NaCl, 0.1 M Na2EDTA, 10 mM Tris, 1% of freshly added Triton X-100, pH 10) for 1 h to remove proteins. Slides were prepared in duplicate.

METHOD OF ANALYSIS: The slides were then placed in a horizontal electrophoretic tank in cold buffer (4°C, 3 M NaOH, 1 mM Na2EDTA, pH 13) for 40 min to allow DNA unwinding. The electrophoresis was carried out in the same solution for 30 min (at 300 mA, 0.56 V/cm). Afterwards electrophoresis slides were removed from the tank, immersed in neutralization buffer (0.4 M Tris, pH 7.5), and stained with DAPI (2 µg/mL in distilled water).

Evaluation criteria:

Slides were examined with an Axiophot fluoresence microscope. The spontaneous strand breaks were measured as total comet length (increase in DNA migration). Average values were calculated for 50 comets per slide.

Statistics:

One-way ANOVA and Tukey's t-test.

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Average feed intake was similar across all 3 groups. Body weight gain was significantly lower (by 30%) in the Cr(VI) group compared to the CrProp and control groups. Feeding efficiency ratio (body weight gain (g) per 100 g diet) was lower in the Cr(VI) group compared to the control and CrProp groups but not significantly so. Results are presented in Table 1.
The Cr(VI) rats had significantly lower spleen and pancreas weight (by 30.6% and 54.5%, respectively), increased heart weight (by 65.2%) as compared to the control group. CrProp did not affect organ weights.
There were no signs of toxicity with CrProp at a dose of 1000 mg Cr/kg diet.
Histological analyses did not show deleterious changes in liver and kidney tissue.
The mean comet length obtained from lymphocytes of rats exposed to Cr(VI) was significantly longer (by 27%) than the control group and the CrProp group (Table 1).

Table 1. Mean comet length and nutritional indices in rats fed Cr(III) or Cr(VI) in the diet for 4 weeks.

Index		Control group	Positive control Cr(VI) group	Test group Cr(III)
Comet length	Mean ± SD	57.76±0.51^a	73.50±2.19^b	59.08±1.09^a
	Median	57.25	73.92	58.58
Feed intake (g/day/rat)	Mean ± SD	17.6±0.5	17.7±0.63	18.5±0.5
Body weight gain (g/28 days)	Mean ± SD	9.5±3.0^b	7.5±2.7^a	107±2.8^b
Feeding efficiency ratio (g/bw/100 g of diet)	Mean ± SD	1.90±0.97	1.51±0.92	2.11±0.69

Letter subscripts indicate significance at p < 0.05.

Conclusions:

Chromium(III) propionate complex was not genotoxic in the comet assay.

Executive summary:

The genotoxicity of a chromium(III) propionate complex was assessed in rat peripheral blood lymphocytes using the comet assay. Female adult Wistar rats (6 per group) were administered Cr(III) for 4 weeks in standard diet, provided ad libitum. Cr(III) was given at a dose of 1000 mg Cr(III)/kg diet (as [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]NO₃equivalent to 100 mg Cr/kg bw/day). Cr(III) treatment did not affect body weight gain, feeding efficiency or organ weights as compared to rats fed standard diet alone. Cr(III) treatment did not produce a positive result in the comet assay with lymphocytes, suggesting the compound is not genotoxic in rats.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 SEP 1991 - 28 FEB 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

strain Bor:NMRI (SPF Han)

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
- Fasting period before study: not stated
- Housing: females in groups of up to 3, males were kept singly in type I cages, bedding of soft wood granules spot checked for contaminants at regular intervals
- Diet: ad libitum (Altromin 1324 Standard diet)
- Water: ad libitum (tap water)
- Acclimation period: 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:

corn oil (suspension); 20 ml/kg bw

Details on exposure:

Animals were administered single intraperitoneal doses of the test substance at a dose level of 10000 mg/kg bw. Additional groups were administered vehicle or cyclophosphamide.

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 48 hours.

Frequency of treatment:

Single ip 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 48 hours.

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Positive control(s):

deionized water
- Justification for choice of positive control(s):
- Route of administration: ip
- Doses / concentrations: 20 mg/kg bw, volume 10 mL/kg

Tissues and cell types examined:

Femoral bone marrow cells

Details of tissue and slide preparation:

Femors were flushed with foetal bovine serum. Smears of femoral bone marrow cells were stained and fixed (Schmid's method).

CRITERIA FOR DOSE SELECTION:
Dose levels were based on the results of a sighting study perfomed using dose levels of 5000 and 1000 mg/kg bw. At 5000 mg/kg bw/d signs of systemic toxicity (apathy, spasm, staggering gait, roughened and stretching of the body) were observed in the animals administered the test material. No deaths occurred.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): sampling after 16, 24 and 48 hours fotr the test item treated groups. Negative and positive control groups were sampled at 48 hours.

DETAILS OF SLIDE PREPARATION: At least one intact femur was prepared from each sacrificed animal (not pre-treated with a spindle inhibitor). The bone marrow was suspended in FCS, centrifuged and the supernatant was removed. The sediment was mixed to produce a homogeneous suspension. One drop of the suspension was placed on a well-cleaned slide and spread with a suitable object. The smears were dried and stained. The slides were then 'destained' with methanol, rinsed with deionized water and left to dry.

METHOD OF ANALYSIS: Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes.

OTHER:

Evaluation criteria:

1000 polychromatic erythrocytes (PCEs) per animal were counted and the incidence of cells with micronuclei counted manually. The ratio of PCEs to normochromatic erythrocytes (NCEs) was also noted; the incidence of micronucleated NCEs was also noted. A test was considered negative if there was no significant increase in the proportion of micronucleated PCEs at any time point, or if a significant increase was seen but was within the historical control range.

Statistics:

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

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

(systemic toxicity and cytotoxicity)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Signs of systemic toxicity (apathy, abnormal gait, rough fur, spasm and dyspnoea) were observed in the animals administered the test material. No deaths occurred. The feeding behaviour was normal.

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 and 5000 mg/kg bw
- Clinical signs of toxicity in test animals: At 5000 mg/kg bw/d signs of systemic toxicity (apathy, spasm, staggering gait, roughened and stretching of the body) were observed in the animals administered the test material. No deaths occurred.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): A significantly increased incidence of micronucleated PCEs was only seen in the positive control group.
- Ratio of PCE/NCE (for Micronucleus assay): 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 between all groups. The proportion of micronucleated PCEs was comparable between the vehicle control and all test substance groups; a significantly higher proportion of micronucleated PCEs was seen in the positive control group.
- Appropriateness of dose levels and route: A significantly increased incidence of micronucleated PCEs was seen in the positive control group.

Summary of results:

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:

The results of this study are clearly negative.

Executive summary:

The genotoxicity of chromium (III) oxide was investigated in a GLP-compliant mouse micronucleus assay according to OECD Test Guideline 474. 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

in vitro

Bacterial reverse mutation test

A bacterial reverse mutation assay similar to OECD Guideline 471 was performed using the plate incorporation method. In this study chromium (III) chloride was tested in the S. typhimurium plate incorporation assay with tester strains TA100, TA98, TA1535 and TA1537 at dose levels up to and including 10,000 µg/plate without metabolic activation and with liver S9 preparations from Sprague- Dawley rats and Syrian golden hamsters. As a result, chromium chloride did not induce a mutagenic response in any of the tested strains of S. typhimurium (Whittaker et al. 2005).

Chromium picolinate monohydrate was tested in a bacterial reverse mutation test in in Salmonella typhimurium strains TA98, TA100 and Escherichia colistrain WP2 uvrA/pKM101 at a concentration range of 100 to 10,000 μg/plate with and without exogenous metabolic activation (S9). The substance was negative in all tested strains with and without metabolic activation (NTP, 2010).

Gene mutation in mammalian cells

A HPRT assay was performed with the soluble and insoluble Cr (III) compounds CrCl3-6H2O and CrCl3. The insoluble CrCl3 induced up to 4-fold increases in the mutation frequency to 6-thioguanine resistance at cytotoxic concentrations in primary human fibroblast cells (HFC). The soluble CrCl3-6H2O, on the other hand, did not increase the mutation frequency (Biedermann and Landolph 1990).

Chromosomal aberration

The frequencies of chromosome aberrations and SCE have been determined in CHO cells after incubation for 2 division cycles with different concentrations of Cr compounds added directly to the growth medium. The frequency of chromosome aberrations is increased after treatment with Cr(III) compounds (i.e. chromium hydroxide sulphate), being about doubled after exposure to 25-150 µg/ml. On the other hand, the frequencies of SCE are not significantly increased.

in vivo

Comet assay

The genotoxicity of a chromium(III) propionate complex was assessed in rat peripheral blood lymphocytes using the comet assay. Female adult Wistar rats (6 per group) were administered Cr(III) for 4 weeks in standard diet, provided ad libitum. Cr(III) was given at a dose of 1000 mg Cr(III)/kg diet (as [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]NO₃equivalent to 100 mg Cr/kg bw/day). Cr(III) treatment did not affect body weight gain, feeding efficiency or organ weights as compared to rats fed standard diet alone. Cr(III) treatment did not produce a positive result in the comet assay with lymphocytes, suggesting the compound is not genotoxic in rats (Staniek et al., 2009).

Micronucleus assay

The genotoxicity of chromium (III) oxide was investigated in a GLP-compliant mouse micronucleus assay according to OECD Test Guideline 474. 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 (Herbold et al., 1992).

Trivalent chromium is an essential trace element and highly water-soluble and bioavailable complexes of chromium (III) are used as dietary supplements. In clinical cases, the administration of chromium (III) salts has been shown to improve the efficacy of insulin in diabetic patients. Although some data generated using acellular systems indicate that chromium (III) has the potential to bind to DNA and cause DNA damage in vitro genotoxicity studies were generally negative in bacteria (NTP 2010, Whittaker et at. 2005). Studies of in vitro genotoxicity in mammalian cells gave mixed results; weakly positive results were obtained with water soluble chromium (III) compounds that are taken up by cells (Biedermann and Landolph 1990). The results therefore suggest that the inability of chromium (III) to cross the cell membrane effectively reduces activity in in vitro systems.

In contrast to the ambiguous results in in vitro studies, the in vivo studies show clear negative results for Cr (III) compounds. Chromium oxide did not induce micronucleated polychromatic erythrocytes in mice in a guideline-compliant study using intraperitoneal dosing (Herbold, 1992). Chromium(III) propionate was also negative in an in vivo Comet assay (Staniek et al. 2009), clearly demonstrating the lack of cytogenic and gene muation effects of Cr (III) in vivo.

Further in vivo genotoxicity studies summarized in four literature reviews also showed that chromium (III) compounds did not produce DNA strand breaks/crosslinks or chromosomal aberrations in rats and were negative for somatic mutations and recombination in Drosophila (Eastmond et al, 2008; FIOH, 2006; ATSDR, 2000; HSE, 1989).

Thus, all in all, the registered substance is negative for 'Genetic toxicity'.

Justification for classification or non-classification

Based on the results of these studies no classification is proposed according to Regulation (EC) No 1272/2008.