Chromium tris((2-ethylhexanoate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria:

An Ames bacterial reverse mutation assay (Savineau, 2017) was performed on the substance Chromium 2 -ethylhexanoate according to OECD guideline 471. Doses (5000, 1500, 500, 150 and 50 µg/plate) performed from solutions of the test item do not induce any mutagenic change in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Eschirichia coli WP2(urA-)(pKM101) without, or with metabolic activation. The test produced negative results, and thus provide evidence for an absence of concern.

In vitro gene mutation in mammalian cells:

Chromium picolinate (source substance) and its component compounds, chromium (III) chloride and picolinic acid, were evaluated in Salmonella typhimurium and L5178Y mouse lymphoma cells (Whittaker, 2005). Neither chromium picolinate nor chromium chloride induced a mutagenic response in S. typhimurium. However, in the L5178Y mouse lymphoma mutation assay, chromium picolinate induced mutagenic responses without and with the addition of S9.

A mouse lymphoma assay with Niacin-bound chromium is also available (Shara et al., 2005). Assay was negative with lower tested concentrations compared to Whittaker publication.

In vitro chromosomal aberration:

Under the test conditions of an OECD 473 guideline compliant test (Gudi et al., 2005), Chromium picolinate (source substance) did not induce structural or numerical chromosome aberration in Chinese hamster ovary cells up to doses that were insoluble in the culture medium.

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

Study period:

2016-12-15 to 2017-04-14

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:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on 2 November 2016

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation by a microsomal fraction of rat liver (S9-mix 10%v/v)

Test concentrations with justification for top dose:

- Concentrations for assay n°1: 50, 150, 500, 1500 and 5000 µg/plate
- Concentrations for assay n°2: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Solutions were prepared in ethanol.

Untreated negative controls:

yes

True negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Remarks:

Test item was completely soluble in ethanol at all concentrations tested.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Stock solution was formulated in DMSO.

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.15 M

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Stock solution was formulated in NaCl 0.15 M.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Stock solution was formulated in DMSO.

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.15 M.

Positive controls:

yes

Positive control substance:

other: cis-Platinium (II) Diammine Dichloride: for E. Coli without S9

Remarks:

Stock solution was formulated in NaCl 0.15M.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-anthramine: for TA1535, TA1537, TA98 and TA100 with S9

Remarks:

Stock solution was formulated in DMSO.

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Stock solution was formulated in acetone.

Details on test system and experimental conditions:

PREPARATION OF STOCK SOLUTION
-Test item is completely soluble in ethanol at all concentrations tested.
- Stock solution for each assay was prepared at 100 mg/mL in ethanol.
- Test item is tested at 5000, 1500, 500, 150 and 50 µg/plate.
- 0.1 mL volume additions of test item solution were used for all treatments.

BACTERIA
- Strains of Salmonella typhimurium and Eschericia coli are purchased from Moltox. They are maintained in LEMI laboratory.
- The genotype of bacterial strains was checked.

EXPERIMENTS
- Triplicate plates test material.
- Negative, positive and vehicle controls were included in triplicate with and without metabolic activation.
- S9 fraction, microsome fraction, prepared from Sprague Dawley rat liver homogenate, was provided by Moltox USA.
- For experiments without metabolic activiation: 0.1 mL bacterial culture, 0.1 mL test article solution or control and 0.5 mL of buffer solution were added to 2 mL overlay agar at 45°C containing 10%v/v of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains and 5%v/v of nutrient broth n°2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/L for Escherichia coli strain. Plates are incubated at 37°C over 48-72 hour period.The pre-incubation phase is performed for the second assay if the first assay is negative.
- For experiments with metabolic activation: protocol similar to that described above, except that 500 µL of S9-mix fraction is quickly added before pouring the mixture onto the plates. Since the first assay was negative, the pre-incubation test was performed for the second assay.

COLONY COUNTING
- After a 48-72 hour incubation period at 37°C, revertant colonies are counted in each plate.

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and E. coli WP2 (uvrA) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependant relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA98, TA100 and E. coli WP2 (uvrA) (pKM 101) and three fold for TA1535 and TA1537.
All results must be confirmed in an independant experiment.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

not valid

Additional information on results:

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for positive controls (with and without metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (5000, 1500, 500, 150 and 50 µg/plate) without and with metabolic activaiton in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2(uvrA)(pKM101).
Results are confirmed in an independant experiment.

Conclusions:

Interpretation of results: negative
Doses (5000, 1500, 500, 150 and 50 µg/plate) performed from solutions of the test item do not induce any mutagenic change in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Eschirichia coli WP2(urA-)(pKM101) without, or with metabolic activation, according to the OECD guidelines n°471.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOG APPROACH
The read across follows Scenario 2 - Different compounds have qualitatively similar properties: properties of the target substance predicted to be quantitatively equal to those of the source substance or prediction based on a worst-case approach (as described in the 2017 Read-Across Assessment Framework document).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Chromium tris(2-ethylhexanoate) (EC 222-357-3, CAS 3444-17-5)
SOURCE: Chromium (3+) picolinate (CAS n° 14639-25-9, EC n° 477-680-4)

3. ANALOG APPROACH JUSTIFICATION
The target substance and the source substance are organic salts of Chromium (3+). The organic counter ion of the target and source substances are small (6-8 carbon) carboxylic acids of similar molecular weight and polarity. The biologically-reactive center of all the molecules would be predicted to be the Cr (3+).
4. DATA MATRIX
See Read Across document attached to CSR.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian cell gene mutation test

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cells were originally obtaines from MS; PAtricia Poorman-Allen, Glaxo Welcome Inc., Research Triangle Park, NC, USA. The cells were grown in Fisher's medium for leukemic cells of mice (Irvine Scientific, Irvine, CA, USA) supplemented with 10% horse serum ( Hyclone Laboratories, Logan, UT, USA) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI, USA). Cells were screened for the presenceof mycoplasma contamination before and after cryopreservation.

Additional strain / cell type characteristics:

other: 3.7.C

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

50, 150, 500 and 1000 µg/mL. The doses were within the range yielding approximately 0-90% cytoxicity.

Vehicle / solvent:

Distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Method of applicaiton: in suspension
Temperature: 37 +/- 1°C
Replicates: duplicate cultures, three plates
Exposure duration: 4H
Expression time: 48H
Incubation time: 10 - 12 days
Selection agent: trifluorothymidine (TFT) - final concentration = 3 µg/L
Determination of cytotoxicity: cloning efficiency
Additional determination: size of colony

Evaluation criteria:

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 1000000 surviving cells above the concurrent solvent control value.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Mutagenicity and cytotoxicity of chromium picolinate:

	Dose [µg chromium picolinate/mL]	Rat S9	Relative total growth [% of control]	Mutant frequency per 1000000 survivors*
Solvent control	0	-	100.0	85
	50	-	98.5	122
	150	-	84.5	87
	500	-	76.0	380
	1000	-	58.5	400
Positive control (MMS)	10	-	43.0	401
Solvent control	0	+	100.0	60
	50	+	114.5	61
	150	+	94.5	94
	500	+	86.5	237
	1000	+	31.5	387
Positive control (DMBA)	4	+	33.0	392

* 1000000 cells in a measured volume volume were plated in each of three plates/culture in the presence of trifluorothymidine (3 µL/mL) after 2 days of expression. The values are for duplicate cultures.

Conclusions:

Under the test conditions of an assay in L5178Y mouse lymphoma calls, Chromium picolinate induced positive responses both in the presence and absence of metabolic activation. At a level of 124.0 µg Cr/mL, the highest level tested, the mutant frequency per 1000000 survivors (minus background) was 315 and 327 without activation and with S9, respectively.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOG APPROACH
The read across follows Scenario 2 - Different compounds have qualitatively similar properties: properties of the target substance predicted to be quantitatively equal to those of the source substance or prediction based on a worst-case approach (as described in the 2017 Read-Across Assessment Framework document).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Chromium tris(2-ethylhexanoate) (EC 222-357-3, CAS 3444-17-5)
SOURCE: Niacin bound chromium (III) complex (CAS 64452-96-6)

3. ANALOG APPROACH JUSTIFICATION
The target substance and the source substance are organic salts of Chromium (3+). The organic counter ion of the target and source substances are small (6-8 carbon) carboxylic acids of similar molecular weight and polarity. The biologically-reactive center of all the molecules would be predicted to be the Cr (3+).
4. DATA MATRIX
See Read Across document attached to CSR.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation test

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsomal enzymes

Test concentrations with justification for top dose:

mammalian microsomal1, 2.5, 5, 10, 25, 50, 100 and 200 µg/mL

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

Method of applicaiton: in medium
Temperature: 37 +/- 2°C
Replicates: duplicate cultures for negative and positive controls, sinlge cultures for each dose
Exposure duration: 3H (confirmatory treatment: 24H)
Expression time: 48H
Incubation time: 10 - 14 days
Determination of cytotoxicity: relative total growth and plating efficiency

Evaluation criteria:

Relative total growth was assessed. Colonies were counted. The mutant frequency was expressed as the number of mutant colonies per million surviving cells at the time of mutant selection.

Statistics:

The data were analyzed using ANOVA and Scheffe's S method. Statistical significance was set at p < 0.05.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

There was evidence of substance-related precipitate at concentration levels 50 (without activation only), 100 and 200 µg/ml.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental conditions of an OECD 476 test, Niacin-bound chromium did not induce mutagenic effects in the mammalian cell gene mutation test in L5178Y mouse lymphoma cells TK (+/-), either with or without metabolic activation. Niacin-bound chromium is concluded to be negative for the induction
of mutagenicity in this assay.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOG APPROACH
The read across follows Scenario 2 - Different compounds have qualitatively similar properties: properties of the target substance predicted to be quantitatively equal to those of the source substance or prediction based on a worst-case approach (as described in the 2017 Read-Across Assessment Framework document).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Chromium tris(2-ethylhexanoate) (EC 222-357-3, CAS 3444-17-5)
SOURCE: Chromium (3+) picolinate (CAS n° 14639-25-9, EC n° 477-680-4)

3. ANALOG APPROACH JUSTIFICATION
The target substance and the source substance are organic salts of Chromium (3+). The organic counter ion of the target and source substances are small (6-8 carbon) carboxylic acids of similar molecular weight and polarity. The biologically-reactive center of all the molecules would be predicted to be the Cr (3+).
4. DATA MATRIX
See Read Across document attached to CSR.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

100 metaphases were scored instead of 200.

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO K1 cells (American Type Culture Collection Repository No. 61) were grown routinely
in McCoy’s 5A culture medium supplemented with 10% fetal bovine serum, 100 units penicillin/ml, 100 µg streptomycin/mL and 2mM d-glutamine.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 from Arcolor 1254-induced rat liver

Test concentrations with justification for top dose:

96.25, 192.5, 385, 770 µg/mL. The doses were selected based on preliminary toxicity assay, 770 µg/mL being the maximum observable solubility in solvent.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Method of applicaiton: in medium
Temperature: 37 +/- 1°C
Replicates: no information
Exposure duration: 4H and 20H without S9, 4H with S9
Fixation time: approximately 20h after initiation of the treatment.
The post-treatment harvest time represents approximately 1.5 normal cell cycles and was selected to ensure that the cells were analyzed in the first division metaphase after initiation of treatment. Two hours prior to cell harvest, Colcemid was added to the cultures at a final concentration of 0.1 µg/mL.
Determination of cytotoxicity: mitotic index, percentage of cells viability

Rationale for test conditions:

Based on the results of the toxicity study, the dose range selected for testing in the chromosome aberration assay spanned a soluble to insoluble range of CrPic from 96.25 to 770 µg/mL.

Evaluation criteria:

Determinations of toxic effects of treatment were based upon the determinations of the percentage of cell viability (Trypan blue exclusion), growth inhibition (cell counts) and observations of mitotic inhibition (number. mitoses/total cells counted). The number and types of aberrations, the percentage of structural and numerical aberrations (% aberrant cells) in the total population of cells, and the mean number of aberrations per cell were calculated.

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher’s exact test to compare pair-wise percent of aberrant cells of each treatment group with that of the findings in control cells treated with the solvent. In the event of a positive Fisher’s exact test (p≤0.05) at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

slight cytotoxicity in the highest precipitating dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A 4-h exposure to various concentrations of Chromium picolinate did not produce any significant increase in chromosomal aberrations in the presence or absence of S9 activation. Similarly, a 20-h treatment with Chromium picolinate did not elicit increases in chromosomal aberrations relative to the control culture.
No substantial toxicity of Chromium picolinate (i.e., >50% cell growth inhibition relative to the solvent control) was observed at any dose level in the non-activated 20-h exposure group. However, due to excessive reduction (87%) in mitotic index at 770 µg/mL, dose levels of 385, 192.5 and 96.25 µg/mL were the
highest test doses that could be evaluated for chromosome aberrations. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p > 0.05, Fisher’s exact test). The percentage of structurally damaged cells in the positive control treatment group was statistically increased compared to the solvent control data indicating the responsiveness of the cells in this test system.

Conclusions:

Under the test conditions of an OECD 473 guideline compliant test, Chromium picolinate diid not induce structural or numerical chromosome aberration up to doses that were insoluble in the culture medium.

Genetic toxicity in vivo

Description of key information

In vivo chromosomal aberration; DNA damage and/or repair:

The potential genotoxicty of Chromium picolinate (source substance) was investigated using the flow cytometer-based micronucleus assay similar to OECD 474 test (Andersson et al., 2007) to evaluate chromosome aberrations. There was no evidence of clastogenic, aneugenic or cytotoxic effects in the erythrocytes isolated from whole blood from mice given a single injection of up to 3 mg/Kg bw 42 hours before sacrifice.

In addition, the potential genotoxicty of Chromium picolinate (source substance) was also investigated using the alkaline version of the Comet assay to evaluate primary DNA damage (Andersson et al, 2007). There was no evidence of Chromium picolinate-induced DNA damage in the peripheral lymphocytes or hepatocytes isolated from mice given a single intraperitoneal injection of 3 mg/kg b.wt. 16 h before sacrifice.

The overall conclusion is that Chromium picolinate (source substance) has no genotoxic potential in vivo. These 2 negative results in vivo overrule the only positive result in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOG APPROACH
The read across follows Scenario 2 - Different compounds have qualitatively similar properties: properties of the target substance predicted to be quantitatively equal to those of the source substance or prediction based on a worst-case approach (as described in the 2017 Read-Across Assessment Framework document).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Chromium tris(2-ethylhexanoate) (EC 222-357-3, CAS 3444-17-5)
SOURCE: Chromium (3+) picolinate (CAS n° 14639-25-9, EC n° 477-680-4)

3. ANALOG APPROACH JUSTIFICATION
The target substance and the source substance are organic salts of Chromium (3+). The organic counter ion of the target and source substances are small (6-8 carbon) carboxylic acids of similar molecular weight and polarity. The biologically-reactive center of all the molecules would be predicted to be the Cr (3+).
4. DATA MATRIX
See Read Across document attached to CSR.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

one sampling time

GLP compliance:

not specified

Type of assay:

other: micronucleus assay to evaluate chromosomalt aberrations

Species:

mouse

Strain:

other: CBA/Ca

Sex:

male

Details on test animals or test system and environmental conditions:

weight at study initiation: around 20g
age at study initiation: 7-8 weeks
source: Scanbur BK - Sweden
water: free access to tap water
diet: free access to standardized rodent pellet diet (B&K Universal, batch 3386)
acclimatisation: at least 7 days before the start of the study

Route of administration:

intraperitoneal

Vehicle:

Phosphate bufferd saline (PBS)

Details on exposure:

Chromium picolinate and the positive control (colchicine) were dissolved in phosphate buffered saline (PBS) and injected intraperitoneal as freshly prepared solutions (0.01 ml/g b.wt.). Control animals received the same volume of vehicle only.

Duration of treatment / exposure:

one single injection

Frequency of treatment:

one single injection

Post exposure period:

After 42 h, blood samples (50 ll/animal) were collected from the orbital plexus under light anaesthesia. This time point was chosen because it is known that it takes about 20 h before polychromatic erythrocytes formed in the bone marrow will appear in the blood.

Dose / conc.:

0.75 mg/kg bw/day (actual dose received)

Dose / conc.:

1.5 mg/kg bw/day (actual dose received)

Dose / conc.:

3 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

vehcile: 4 male animals
chromium picolinate: 3 male animals per dose
positie control: 3 male animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Colchicine (CAS n° 64-86-8) was obtained from Sigma Aldrich (USA) and administered via intraperitoneal route at 1 mg/ Kg bw.

Tissues and cell types examined:

Polychromatic erythrocytes in the blood

Details of tissue and slide preparation:

Three parallel aliquots of 5 µL from each blood sample was layered on a 65% Percoll gradient (Amersham Pharmacia Biotech, Sweden) and centrifuged at 600 x g for 20 min. The erythrocytes collected in the pellet after the Percoll centrifugation were fixed in a solution of
glutaraldehyde and stored for 2–5 days at 4°C until further processed.
The glutaraldehyded fixed cells were stained in a PBS-buffer containing the fluorescent dyes Hoechst 33342 (Sigma/Aldrich, USA) and thiazole orange (Molecular probes, USA). After staining, the samples were analyzed on a FACSVantege SE flow cytometer (BD Immunocytometry systems, USA) equipped with an argon ion laser (Enterprise II, Coherent, USA).

Evaluation criteria:

The frequencies of immature (polychromatic) erythrocytes from peripheral blood with (fMNPCE) or without (fPCE) chromosomal material (micronuclei) was determined using a flow cytometer-based micronucleus assay. An increase in fMNPCE indicates genotoxicity; a decrease in fPCE indicates cytotoxicity.

Statistics:

The relative frequencies of the polychromatic erythrocytes with micronuclei and of the polychromatic erythrocytes without micronuclei were calculated and statistically analyzed using the 2-tailed Students t-test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There was no evidence of clastogenic, aneugenic or cytotoxic effects in the erythrocytes isolated from whole blood from mice given a single
intraperitoneal injection of up to 3 mg/kg b.wt. of chromium picolinate, 42 h before sacrifice.
In contrast, the positive control colchicine (a well-established aneugen) increased the frequency of micronucleated polychromatic erythrocytes in peripheral blood.

In vivo effect of chromium picolinate on the frequency of polychromatic erythrocytes with micronuclei 42 h after a single exposure:

Treatment	Dose [mg/kg]	Frequency of MNPCE [‰] (mean ± SEM)	Frequency of PCE [%]
Control PBS)	0	1.00 ± 0.05	1.9 ± 0.4
Chromium picolinate	0.75	0.95 ± 0.08	2.4 ± 0.4
	1.5	0.94 ± 0.04	2.1 ± 0.3
	3.0	1.08 ± 0.13	2.2 ± 0.3
Colchicine	1.0	3.78 ± 0.46*	1.0 ± 0.4

MNPCE: polychromatic erythrocytes with micronuclei

PCE: polychromatic erythrocytes without micronuclei

* p < 0.001

Conclusions:

The potential genotoxicty of Chromium picolinate was investigated using the flow cytomemeter-based micronucleus assay to evaluate chromosome aberrations. There was no evidence of clastogenic, aneugenic or cytotoxic effects in the erythrocytes isolated from whole blood from mice given a siingle injection of up to 3 mg/Kg bw 42 hours before sacrifice.

Reference 2

Endpoint:

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOG APPROACH
The read across follows Scenario 2 - Different compounds have qualitatively similar properties: properties of the target substance predicted to be quantitatively equal to those of the source substance or prediction based on a worst-case approach (as described in the 2017 Read-Across Assessment Framework document).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Chromium tris(2-ethylhexanoate) (EC 222-357-3, CAS 3444-17-5)
SOURCE: Chromium (3+) picolinate (CAS n° 14639-25-9, EC n° 477-680-4)

3. ANALOG APPROACH JUSTIFICATION
The target substance and the source substance are organic salts of Chromium (3+). The organic counter ion of the target and source substances are small (6-8 carbon) carboxylic acids of similar molecular weight and polarity. The biologically-reactive center of all the molecules would be predicted to be the Cr (3+).
4. DATA MATRIX
See Read Across document attached to CSR.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Alkaline version of the in vivo Comet assay

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

other: CBA/Ca

Sex:

male

Details on test animals or test system and environmental conditions:

weight at study initiation: around 20g
age at study initiation: 7-8 weeks
source: Scanbur BK - Sweden
water: free access to tap water
diet: free access to standardized rodent pellet diet (B&K Universal, batch 3386)
acclimatisation: at least 7 days before the start of the study

Route of administration:

intraperitoneal

Vehicle:

Phosphate bufferd saline (PBS)

Details on exposure:

Chromium picolinate and the positive control (cyclophosphamide) were dissolved in phosphate buffered saline (PBS) and injected intraperitoneal. as freshly prepared solutions (0.01 ml/g b.wt.). Control animals received the same volume of vehicle only.

Duration of treatment / exposure:

one single injection, 3 individual experiments

Frequency of treatment:

one single injection

Post exposure period:

16 hours

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Remarks:

highest soluble dose

No. of animals per sex per dose:

vehcile: 3*4 male animals
chromium picolinate: 3*4 male animals
positie control: 3*2 male animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CAS n°50-18-0) was obtained from Sigma (USA) and administered via intraperitoneal route at 200 mg/ Kg bw.

Tissues and cell types examined:

Freshly isolated lymphocytes from whole blood and isolated nuclei from liver cells were examined.

Details of tissue and slide preparation:

Immediately after sacrifice, 0.5 ml blood was taken from each mouse by heart puncture using a heparinised syringe, and at the same time a small piece of the liver was cut out in a standardized way. The blood and liver samples were then immediately put on ice until further processed for the Comet assay which was performed the same day.
The slides were prepared from layers of cells mixed with agarose (low melting point grade fromfrom Iternational Biotechnologies, USA). Alkaline solutionwas used to lyse the cells at 4°C during 1 hour. After lysis, the slides were immediately transferred to an electrophoresis unit in which the supercoiled DNA was relaxed in the electrophoresis buffer at 4 °C for 40 min. The electrophoresis was performed at 4°C for 10 min. Immediately after the electrophoresis, the slides were neutralised, dried at room temperature and stored in a sealed box until the day of image analysis.
Ethidium bromide-stained nucleoids were examined at 500 x magnification in a fluorescence microscope. The microscope was attached to a black and white chargecoupled device video camera connected to a computer-based image analysis system. Fifty ‘comets’ per slide were randomly captured, avoiding the edges and damaged parts of the gel. Apparently dead cells (comets without distinct heads) and superimposed comets were not captured and analyzed.

Evaluation criteria:

The tail moment was used as an indicator of DNA damage expressing both the distance of migration and the relative amount of DNA in the tail after electrophoresis.

Statistics:

The 2-tailed Students t-test was used.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There was no evidence of Chromium picolinate-induced DNA damage in the peripheral lymphocytes or hepatocytes isolated from mice given a single intraperitoneal injection of 3 mg/kg b.wt. 16 h before sacrifice. Chromium picolinate did not affect the percentage of clouds on the slides.
The positive control cyclophosphamide increased the level of DNA damage both in the liver cells and in the lymphocytes, and this increase was observed without a concomitant increase in the number of clouds.

In vivo effect of chromium picolinate on the DNA damage migration in lymphocytes and hepatocytes 16hours after a single exposure:

Treatment	Doses (mg/Kg)	Lymphocytes			Hepatocytes
		Tail moment (mean +/- SEM)	Clouds (%)	(n)	Tail moment (mean +/- SEM)	Clouds (%)	(n)
Control (PBS)	0	58 +/- 0.9	1.4 +/- 0.3	24	18.6 +/- 3.3	3.1 +/- 1.0	24
Chromium picolinate	3	4.1 +/- 0.6	1.2 +/- 0.4	24	20.0 +/- 4.2	4.0 +/- 1.1	24
Cyclophosphamide	200	38.0 +/- 8.3 (P < 0.001)	0.7 +/- 03	12	50.2 +/- 14.3	2.2 +/- 06	12

Conclusions:

The potential genotoxicty of Chromium picolinate was investigated using the alkaline version of the Comet assay to evaluate primary DNA damage. There was no evidence of Chromium picolinate-induced DNA damage in the peripheral lymphocytes or hepatocytes isolated from mice given a single intraperitoneal injection of 3 mg/kg b.wt. 16 h before sacrifice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Based on available information on genetic toxicity on Chromium (III) compounds, Chromium 2-ethylhexanoate is considered to have no genetic potential.

Justification for classification or non-classification

No classification according to Regulation (EC) 1272/2008 is required for Chromium 2-ethylhexanoate.