Chromium trichloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

chromium trichloride and chromium(III) compounds are considered non-mutagenic

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented, contributing to assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Human leukocytes

Species / strain / cell type:

other: Human leukocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

32×10E-6M

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

Peripheral blood was obtained from a normal healthy man. Human leukocytes were cultured for 72 h according to the method already described.
CrCl3 was added to the leukocyte culture 24 h before the 4-h prefixation treatment with colchicine. Chromosome preparations were made by the standard flame-drying method.

Species / strain:

other: Human leukocytes

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

Leukocyte cultures were treated with chromium compounds at random throughout the cell cycle; the frequency of structural chromosomal aberrations was given. When cultures were treated by the same concentration (32 × 10E-6 M) of each chromium compound, the karyotype of chromosome was not observed after treatment with compounds such as K2Cr207 and K2CrO4. In cultures treated with higher concentrations of chromium, a significant number of structural aberrations was observed compared with those in the control culture.
As chromium compounds had strong cytotoxiclty and comparatively high activity for chromosome damage, leukocytes were next exposed to lower concentrations near the natural level of chromium.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under this experimental conditions provide in this report, chromium trichloride did not induce positive clastogenic effects.

Executive summary:

This report was published to investigate chromosomal aberrations in human leukocyte cultures with compounds containing Cr 3+ including Cr(NO3)3, Cr(OOCCH3)3 and CrCl3, but also with K2CrO4 and K2Cr2O7. Peripheral blood was obtained from a normal healthy man. Human leukocytes were cultured for 72 h. Chromium compounds were added to the leukocyte culture 24 h before the 4-h prefixation treatment with colchicine. There was no significant difference between test and control cultures in the aberrations with breaks and exchanges or total aberrations, when CrCl3 was tested. In a comparison of the total aberration yield on an equimolar basis, the efficiency in inducing chromosomal aberrations was in the order K2Cr2O7 > K2CrO4 > > Cr(CH3COO)3 > Cr(NO3)3, CrCl3. The last three compounds containing trivalent chromium were significantly less efficient as compared with the first two hexavalent chromium compounds.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A wide variety of chromium(III) mutagenicity studies are reported in public peer-reviewed journals, the most reliable ones having been assessed here.

Nakamuro (1978) published an investigation for chromosomal aberrations in human leukocyte cultures with compounds containing Cr 3+ including Cr(NO3)3, Cr(OOCCH3)3 and CrCl3, but also with K2CrO4 and K2Cr2O7. Peripheral blood was obtained from a normal healthy man. Human leukocytes were cultured for 72 h. Chromium compounds were added to the leukocyte culture 24 h before the 4-h prefixation treatment with colchicine. There was no significant difference between test and control cultures in the aberrations with breaks and exchanges or total aberrations, when CrCl3 was tested. In a comparison of the total aberration yield on an equimolar basis, the efficiency in inducing chromosomal aberrations was in the order K2Cr2O7 > K2CrO4 > > Cr(CH3COO)3 > Cr(NO3)3, CrCl3. The last three compounds containing trivalent chromium were significantly less efficient as compared with the first two hexavalent chromium compounds and considered negative in this assay.

Bianchi (1980) tested the ratio of sister chromatid exchanges (SCE) and of chromosome aberrations (CA) in treated/control CHO cells when observed at subtoxic concentrations of Cr(III) (20 - 150 µg/ml). SCE increase (1.0) and CA increase for Cr(III) (1.8) were not statistically significant. Available data indicated that chromium trichloride cannot produce a clastogenic effects under the conditions of this report. Chromium trichloride does not induce SCEs and failed to increases the frequency of chromosome aberrations.

Fernando (1977) in this publication reported about four chromium(VI) compounds and two chromium(III) compounds that were investigated for mutagenicity in an "Ames"-test-system (TA1537, TA1535, TA98 and TA100). Whereas all chromium(VI) compounds showed clear mutagenicity the trivalent chromium compounds did not, neither in presence or absence of metabolic activation with S-9 -mix. Trivalent chromium, tested as chromium potassium sulfate and chromic chloride, did not show any toxic or mutagenic effect on the same salmonella strains, even at doses of 20 mg/plate. This is consistent with the current view that trivalent chromium is considerably less toxic than hexavalent chromium (Underwood, E. J. 1971. Chromium, p. 253-266. In E. J. Underwood (ed.), Trace elements in human and animal nutrition. Academic Press Inc., New York.)

Vernier (1982) used CHO cell cultures which were treated for two division cycles (30 h) with Cr compounds in the presence of 3 x 10E-5 M bromodeoxyuridine. Chromosomal preparations were obtained and scored following the procedures, similar to the OECD guideline 473 and 479. Chromosomal aberrations and SCEs were scored in the same 2nd division metaphases. In particular, when pure Cr(III) compounds are tested, they result devoid of cytotoxic activity even at concentrations near their solubility limit, and do not produce SCEs in mammalian cells in vitro.

Lewis & Majone (1981) in their publication assessed on sister chromatid exchange assay in mammalian cells using Chinese hamster ovary cells (CHO) of different chromium (III) and chromium (VI) species. Whereas chromium (VI) compounds showed positive results the chromium (III) compounds except of chromite (Cr2O3) all were negative. The positive result observed for chromite was attributed to an impurity of chromium (VI) in the test material showing that mutagenicity test results of chromium compounds are very sensitive to chromium (VI) impurities. Chromium trichloride hexahydrate was shown being absent of mutagenicity and cytotoxicity in this assay (without metabolic activation) and did not induce sister chromatid exchanges significantly different from controls.

DeFlora (1981) found that chromium trichloride hexahydrate was negative in all five salmonella typhimurium tester strains (TA98, 100, 1535, 1537 and 1538), with and without metabolic activation.

In a review article by DeFlora (1990) mutagenicity data from public literature was assessed. Although Cr(III) appears to be capable of producing genetic effects when directly challenged with purified nucleic acids or subcellular targets (e.g., cell nuclei), such a potential genotoxic capacity was generally lost when Cr(III) compounds were assayed in cellular systems. Positivity in various short-term tests of Cr(III) compounds contaminated with Cr(VI) traces further confirms the differential activity of these chromium species, which is generally ascribed to their selective ability to cross cell membranes. Positive results with Cr(III) compounds may be ascribed to a variety of factors including, e.g., unchecked contamination with Cr(VI) traces, non-specific effects at very high doses, penetration of Cr(III) by endocytosis following long-lasting in vitro exposures or under special treatment conditions, such as exposure of cells to detergents or to subtoxic phosphate concentrations. In addition, a technical artefact may result from the interaction of Cr(III) with DNA during extraction procedures (Levis and Bianchi, 1982).

It was also stressed that, in positive in vitro studies, the potency of Cr(III) compounds was several orders of magnitude lower than that of Cr(VI) compounds tested in the same systems.

In total 209 studies on chromium(III) mutagenicity were assessed resulting in

48 (23.0%) positives,

19 (9.1%) positives, contaminated with chromium(VI),

141 (67.5%) negatives and

1 (0.5%) with unclear results

In vivo studies on highly soluble chromium(III) substances were all negative.

Thus, the weight of evidence approach, shows chromium trichloride being negative for genotoxicity by the literature review performed in 1990 by DeFlora et al.

Whereas chromium trichloride is very well soluble, but only poorly absorbed in the gastrointestinal tract, other chromium(III) compounds have been developed to serve as nutritional supplement to diet (chromium(III) is an essential trace element). Such compounds are typically organic complexes, although having slightly lower solubility than chromium trichloride they do provide the benefit of better absorption in the GI tract and thus higher bioavailability for chromium(III).

Shara (2005) investigated chromium(3+) tri(pyridine-3-carboxylate) (NBC); results of this test according to OECD 471 indicate that the revertant frequencies at all concentrations of NBC in strains TA 1535, TA 97a, TA 98, TA 100, and TA 102, with and without the presence of a metabolic activation system, were found comparable to those observed in the negative (vehicle) control groups. Plate counts for the spontaneous histidine revertant colonies in the negative control groups were found to be within the frequency ranges expected. Under the conditions of this study, it is concluded that NBC is non-mutagenic in S. typhimurium strains TA 1535, TA 97a, TA 98, TA 100, and TA 102. Furthermore he tested NBC in a mutagenicity test - mouse lymphoma assay according to OECD 476 for its potential to induce mutagenicity in the 3.7.2 C clone of the L5178Y mouse lymphoma cell line. The study was performed with and without metabolic activation up to precipitating concentrations in DMSO. No cytotoxicity was observed and no mutagenicity was seen in any of the experiments. Thus, it is concluded that NBC is considered to be negative for the induction of mutagenicity in this assay.

The National Institutes of Health Public Health Service U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES in 2010 reported about chromium picolinate monohydrate (CPM, CAS No. 27882-76-4). In the standard 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 or TA98 or E. coli strain WP2 uvrA/pKM101 when chromium picolinate monohydrate was tested with or without exogenous metabolic activation (S9). No evidence of mutagenicity was observed in a second and third assays, where testing was performed with chromium picolinate as reported by Zeiger et al. (1992), using strains TA97, TA98, TA100, TA102, TA104, and TA1535, tested with and without 10% and 30% hamster and rat liver S9 mix. Chromium picolinate was tested with or without exogenous metabolic activation (S9). All strains were found being negative (with and without S9).

Finally, in vivo no increase in the frequency of micronucleated NCEs was observed in male B6C3F1 mice administered chromium picolinate monohydrate (80 to 50,000 ppm) in feed for 3 months, indicating no potential for chromium picolinate monohydrate to induce chromosomal alterations in dividing cell populations in this test system. In female mice, however, the small increase in micronucleated NCEs noted in the highest exposure concentration group (50,000 ppm) was not significant at P = 0.0396, but it resulted in a significant trend test (P = 0.005) and, therefore, the test with chromium picolinate monohydrate was judged to be equivocal in female mice. No significant alterations in the percentage of PCEs among total erythrocytes was observed in exposed mice, indicating that these exposure concentrations of chromium picolinate monohydrate did not induce bone marrow toxicity. Also no induction of micronucleated PCEs 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 PCEs among total erythrocytes was observed in dosed rats, indicating that these doses of chromium picolinate did not induce bone marrow toxicity. Thus, chromium(III) picolinate was considered negative in this in vivo bone marrow micronucleus test with rats.

In conclusion chromium trichloride and in general chromium(III) compounds are considered non-mutagenic in vitro and in vivo in different test systems. Although in public literature some positive non-standard test findings were reported, mainly when directly challenged with purified nucleic acids or subcellular targets (e.g., cell nuclei or DNA), in classical test systems chromium(III) compounds were found negative for mutagenicity. S. Carlos B. Oliveira & A. M. Oliveira-Brett (In situ evaluation of chromium – DNA damage using a DNA-electrochemical biosensor, Anal Bioanal Chem (2010) 398, p. 1633 – 1641) concluded that chromium(III) compounds cannot enter into cells but chromium(VI) compounds (known to be mutagenic) can easily do. Within the cells then redox-reactions leading to chromium(IV) and chromium(V) species may be responsible to oxidative DNA damage. This could explain the equivocal and positive in vitro findings in DNA and nucleic acid test systems with chromium(III) reported in literature. Another important aspect is contamination of chromium(III) with chromium(VI) that might have led to positive findings with “reported” chromium(III) compounds in public literature as demonstrated by Lewis & Majone (1981). Especially in older publications, chromium(VI) impurities in chromium(III) test substances were often not measured or reported.

Justification for selection of genetic toxicity endpoint
Study with human cells, supported by several in vitro and in vivo studies

Justification for classification or non-classification

Chromium(III) compounds such as chromium trichloride are considered non-mutagenic in vitro and in vivo and thus do not require classification for mutagenicity according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC).