Chromium tin calcium silicon sphene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro genetic toxicity of chromium tin calcium silicon sphene has been evaluated in a bacterial reverse mutation assay. This study was performed according to the OECD TG 471 under GLP and is considered to be reliable without restrictions. 

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-09-02 to 2019-10-04

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 1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed 2017-10-06

Type of assay:

bacterial reverse mutation assay

Target gene:

hisD (TA 98), hisG (TA 100, TA 1535), hisC (TA 1537), and trpE (WP2 uvr A pKM101)

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Post-mitochondrial fraction from livers of rodents treated with Aroclor. Supplied by Moltox (Trinova Biochem, Germany).
- quality controls of S9: metabolic capability, sterility, and enzymatic activity

Test concentrations with justification for top dose:

- 0.02, 0.06, 0.19, 0.56, and 1.67 mg/plate
- The top concentration was based on the solubility profile of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two experiments (plate incorporation and preincubation)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Bacteria were growing in the late exponential or early stationary phase of growth.
- Test substance added in agar (plate incorporation; first experiment) and preincubation (second experiment)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): Colonies were counted immediately after the 48-hour exposure period.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytotoxicity evaluation of the test item was based on the decrease in the number of revertant colonies, or a clearing or diminution of the background lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Methods: The number of revertant colonies per plate was counted and recorded by an automatic colony counter.

Rationale for test conditions:

- The test item was soluble in DMSO at a concentration of 16.7 mg/mL, with no precipitation signs being observed in the assay final mixture with PBS. Nevertheless, precipitation signs were observed in the assay final mixture with PBS at concentrations of 16.7 mg/mL. Therefore, the concentration selected for the cytotoxicity assay was 16.7 mg/mL (1.67 mg/plate).
- No test item related cytotoxic activity was observed in the bacterial system (TA 100) over the concentration range tested between 0.02-1.67 mg/plate.

Evaluation criteria:

- The criteria used for determining a positive result take into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
- A result is considered positive whenever the number of revertants of the test item-treated plates is increased when compared to the solvent-treated plates according to the following criteria: Species Strain Mutagenic Ratio (R) cut-off point: Fold increase in revertant colony number above vehicle control: 2-fold (TA 98, TA 100, and WP2 uvr A pKM101), and 3-fold (TA 1535 and TA 1537)

Statistics:

Not mandatory for this assay.

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating 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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating 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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating 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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating 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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

BACTERIAL REVERSE MUTATION ASSAY
- No test item related cytotoxic activity was observed at any of the concentrations tested.
- Precipitation signs were observed in the assay final mixture with PBS at concentrations of 16.7 mg/mL. Therefore, the top concentration selected for the main experiment was 16.7 mg/mL (1.67 mg/plate).
- None of the concentrations assayed for the test item showed an increase in the R value (number of revertant colonies in treatment vs. vehicle control) either with or without S9 metabolic activation regardless of the procedure.
- No dose response exceeding the threshold for the test item was observed in any of the tested bacterial strains.
- The revertant colonies in the vehicle control cultures were well within the historical control data range demonstrating the validity of the assay.
- The positive control mutagens induced distinct increases in the revertant colony number above the threshold values demonstrating the sensitivity of the assay and the metabolic activity of the S9 mix.

Conclusions:

The test item Chromium tin calcium silicon sphene (Pigment Red 233) does not induce point mutations or frameshifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure over the concentration range tested.

Therefore, the test item Chromium tin calcium silicon sphene (Pigment Red 233) is considered to be non-mutagenic under the experimental conditions assayed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacteria reverse mutation assay

Gürel (2019) investigated the mutagenic potential of chromium tin calcium silicon sphene in a bacterial reverse mutation assay according to OECD TG 471 (1997) under GLP. The test was performed in S. typhimurium TA 98, TA 100, TA 1535, and TA 1537 as well as E. coli WP2 uvrA (pKM101) in two independent experiments using triplicate cultures. The cell cultures were exposed to the test material, both in absence and presence of metabolic activation, up to a concentration level of 1.67 mg/plate due to the presence of precipitates in the assay final mixture with PBS at a concentration of 16.7 mg/mL. The two independent experiments were performed according to the plate incorporation and pre-incubation procedure. Treatment with chromium tin calcium silicon sphene did not result in a mutagenic response at all concentrations tested both in absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system.

In a supporting study (Engelhardt, 1992), the gene mutation potential of chromium tin calcium silicon sphene was evaluated in the bacterial reverse mutation test according OECD TG 471 and under GLP. The S. typhimurium tester strains TA 98, and TA 100 were exposed using the plate incorporation and pre-incubation method up to the maximum recommended concentration of 5 mg/plate. No positive mutagenic responses were observed at any concentration level or with any tester strain in either the absence or presence of S9 metabolic activation. Appropriate positive controls demonstrated the sensitivity of the test system.

Conclusion:

Chromium tin calcium silicon sphene is not expected to cause gene mutations due to the absence of mutagenic effects in bacterial reverse mutation assays.

Justification for classification or non-classification

It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days. Either no or only marginal increases in Cr and Sn plasma concentrations were observed, and only a minor fraction (<0.002%) of the total administered dose of Cr and Sn was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

Moreover, chromium tin calcium silicon sphene is not expected to cause gene mutations due to the absence of mutagenic effects in bacterial reverse mutation assays.

The classification criteria acc. to regulation (EC) 1272/2008 and its subsequent amendments as germ cell mutagen are not met, thus no classification is required.