Chromium

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated 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:

other: see 'Remark'

Remarks:

Read across from trivalent chromium(III) chloride used to indicate possible effects caused by chromium. As chromium(III) chloride is more soluble than Cr metal, the results can be regarded as "worst case". Acceptable well-documented study report which meets basic scientific principles.

Data source

Reference

Reference Type:

publication

Title:

Embryotoxicity hazard assessment of methylmercury and chromium using embryonic stem cells.

Author:

Stummann T.C, Hareng L. and S. Bremer

Year:

2007

Bibliographic source:

Toxicology 242, 130-143

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The Embryonic Stem Cell Test (EST), has been endorsed as a scientifically validated in vitro method for detecting embryotoxicity by ECVAM (European Centre for the the validation of alternative methods).
The EST includes a prediction model based on three endpoints: cytotoxicity assays with mouse embryonic stem cells (ES) and 3T3 fibroblasts, and an ES cell cardiac differentiation inhibition assay.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

10060-12-5

Molecular formula:

CrCl3.6H20

Constituent 2

Reference substance name:

Chromium trichloride hexahydrate

IUPAC Name:

Chromium trichloride hexahydrate

Details on test material:

Chromium trichloride hexahydrate (CrIII)
Purity 99.998%
Manufacturer Alfa Aesar, Ward Hill, MA, USA.

No other data available.

Test animals

Species:

other: Mouse ES cell line

Details on test animals or test system and environmental conditions:

Mouse ES cell line D3, fibroblasts of mouse Balb/3T3 clone A31 cell line. Both obtained from ATCC, USA.
ES cells were cultured in Dulbecco's modified Eagles Medium (Invitrogen, San Giuliano Milanese, Italy), complemented with 20% heat inactivated foetal bovine serum (Cambrex, BG Caravaggio, Italy), 2 mM glutamine (Invitrogen), 50 U/ml penicillin and 50 µg/ml streptomycin (Invitrogen), 1% non-essential amino acids (Invitrogen), 0.1 mM B-mercaptoethanol and 0.01 µ/ml leukemia inhibitory factor (Antigenix America, Huntington Station, NY, USA).
3T3 fibroblast were cultured in Dulbecco's modified Eagles Medium (Invitrogen, San Giuliano Milanese, Italy), complemented with 10% heat inactivated foetal bovine serum (Cambrex, BG Caravaggio, Italy), 4 mM glutamine (Invitrogen), 50 U/ml penicillin and 50 µg/ml streptomycin (Invitrogen).

Cardiac and neuronal differentiation of ES cells was carried out by aggregating the cells and culturing under specific conditions (Spielmann and Scholz 2002, Okabe et al 1996). The number of embryoid bodies with contracting myocardial areas was determined microscopically. The neuronal differentiation was characterised by real-time PCR analysis of marker gene expression and immunohistochemistry.

Administration / exposure

Route of administration:

other: in vitro into cell culture

Details on exposure:

The cells were continuously exposed to the test substance for 10 days. The medium containing the metal was renewed on days 3 and 5.

Cr(III) was dissolved at a concentration of 3.8x10exp-1 M in H2O and diluted 100x in medium in order to prepare the highest final concentration (3.8x10exp-3). All other final concentrations were prepared from stock dilution series based on 0.1 M C(rIII) stocks prepared in H2O.

Duration of treatment / exposure:

10 days

Frequency of treatment:

Continuous in medium

Duration of test:

10 days

No. of animals per sex per dose:

6 samples per dose.

Control animals:

yes

Details on study design:

Cardiac differentiation of ES cells was carried out by aggregating the cells and culturing under specific conditions (Spielmann and Scholz 2002, Okabe et al 1996). The number of embryoid bodies with contracting myocardial areas was determined microscopically.

Examinations

Statistics:

Prism 4.0 (GraphPad Software, San Diego, CA, USA) was used for data plotting, non-linear regression and statistical analysis.
Groups were compared using Student's unpaired t-test for two groups or one-way ANOVA analysis with Dunnett's multiple comparison post-test for more than two groups. p<0.05 was considered as statistically significant.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:not examined

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 other: mM

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Cr(III) exhibited low cytotoxic effects on ES cells and 3T3 fibroblasts. Only the highest concentration (3.16 mM for ES and 1 and 3.16 mM for 3T3 cells) showed a statistically significantly inhibited MTT reduction (p<0.01). In line with this, only the highest Cr(III) concentration 3.16 mM suppressed the occurrence of embryoid bodies with beating activity.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The embryonic stem cell test (EST) classified trivalent chromium (Cr(III)) as non-embryotoxic.

Executive summary:

The Embryonic Stem Cell Test (EST), has been endorsed as a scientifically validated in vitro method for detecting embryotoxicity by ECVAM (European Centre for the validation of alternative methods).

The EST includes a prediction model based on three endpoints: cytotoxicity assays with mouse embryonic stem cells (ES) and 3T3 fibroblasts, and an ES cell cardiac differentiation inhibition assay.The Embryonic Stem Cell Test (EST) was used for detecting embryotoxicity caused by trivalent chromium. The results showed that the cytotoxic effect was not higher in ES cells than in 3T3 fibroblasts, and thus the prediction model classified Cr(III) as non-embryotoxic.