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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of Chromium diboride was investigated in a bacterial reverse gene mutation assay conducted according to OECD 471 with and without metabolic activation. There was no increase of mutant colonies compared to the negative control observed in all strains. Consequently, the target substance Chromium diboride is considered to be non-mutagenic.

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:
2016-08-05 to 2016-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED:
- Source of cells: MOLTOX, INC., NC 28607, USA (TA 98, 1535 and 102), Xenometrix AG, Switzerland (TA 100 and 1537)
MEDIA USED
- Type and identity of media: Nutrient medium: 8 g Nutrient Broth and 5 g NaCl per litre, plus 125 µL ampicillin for TA 98, TA 100, TA 102); Agar Plates: Vogel-Bonner Medium E agar plates contain per litre 15 g Agar Agar, 20 mL Vogel-Bonner salts and 50 mL glucose solution (40%); Overlay Agar: The overlay agar contains per litre: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H20 and 12.2 mg biotin
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (Conditions: see "Any other information on materials and methods"; Results: see "Any other information on results" Table 2). Due to strong precipitation 2500 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
10.0, 31.6, 100, 316, 1000 and 2500 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (AppliChem; Lot No. 0000803731)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535, without S9, 10 µg/plate
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
TA 98 (10 µg/plate) and TA 1537 (40 µg/plate), without S9
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102, 1 µL/plate, without S9
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
All strains, with S9, 2.5 µg/plate (10 µg/plate for TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, Experiment I), preincubation (Experiment II)
- Cell density at seeding (if applicable): approx. 10^9 cells/mL, 100µL/plate

EXPERIMENTAL PERFORMANCE
- Experiment I :
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain), 2000 μL Overlay agar.
- Experiment II:
For the pre-incubation method the following materials were mixed and pre-incubated for 60 min at 37 °C: 100 μL of the test item preparation, 100 µL of the tester strain, 500 µL sterile buffer or the metabolic activation system. After adding the overlay agar (2000 μL), the mixture was poured onto the surface of a minimal agar plate.

DURATION
- Preincubation period (Experiment II): 60 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/dose level including the controls (for TA 1537 (1000 µg/plate, without S9) only two plates were evaluated)

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation). A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control
Evaluation criteria:
A test is considered acceptable if for each strain:
- The bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- The negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2013 -2015) (see “Any other information on material and methods” Table 1)
- Corresponding background growth on negative control, solvent control and test plates is observed
- The positive controls show a distinct enhancement of revertant rates over the control plate
- At least five different concentrations of each tester strain are analysable.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate with S9 in TA 98 and TA 100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see "Additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In experiment II toxic effects of the test item were noted in tester strains TA 98 and TA 1537 at a concentration of 2500 μg/plate (without metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in the following tester strains was regarded as not biologically relevant due to lack of a dose-response relationship: TA 98 (at a concentration of 10.0 μg/plate (without metabolic activation)), TA 1535 (at a concentration of 31.6 μg/plate (without metabolic activation)), TA 1537 (at concentrations of 10.0 and 1000 μg/plate (with metabolic activation)).
Remarks on result:
other:
Remarks:
Experiment I, precipitation was observed in all tester strains used in the experiment at a concentration of 2500 µg/plate.

Results of the pre-experiment:

Table 2: Results of the pre-experiment

Substance Dose (µg/plate) TA 98
Mutation Factor [toxicity]*
TA 100
 Mutation Factor [toxicity]*
without S9 with S9 without S9 with S9

Solvent Control (DMSO)

1.0 1.0 1.0 1.0
4-NOPD 10.0 18.6 - - -
NaN3 10.0 - - 4.3 -
2-AA 2.50 - 28.8 - 10.0
Test Item Chromium diboride 3.16 0.8 1.2 0.8 0.8
10.0 0.9 1.0 0.9 0.9
31.6 0.8 0.9 0.9 0.8
100 0.7 0.9 1.0 0.8
316 0.6 1.0 1.0 0.9
1000 0.9 0.9 0.9 0.8
2500 0.4 [P] 0.2 [P] 0.2 [P] 0.2 [P]
5000 0.4 [P] 0.3 [P] 0.3 [P] 0.3 [P]

* [toxicity parameter]: B = Background lawn reduced, N = No background lawn, P = Precipitation, P* = not evaluable, precipitation interferes with scoring

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Chromium diboride did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Chromium diboride is considered to be non-mutagenic according to CLP criteria in this bacterial reverse mutation assay .
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 of S. typhimurium were exposed to Chromium diboride (>99.4 % purity) in water at concentrations of 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate in the presence and absence of mammalian metabolic activation.

Chromium diboride was tested up to the limit concentration (5000 µg/plate) in a pre-experiment. Due to precipitation at the high doses, the maximum dose was reduced to 2500 µg/plate for the main experiment. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains and both experiments. Therefore Chromium diboride is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The mutagenic potential of Chromium diboride (target substance) was investigated in a bacterial reverse gene mutation assay conducted according to OECD 471 with and without metabolic activation using five different tester strains of Salmonella typhimurium. There was no increase of mutant colonies compared to the negative control observed in all strains. Consequently, the test item Chromium diboride is considered to be non-mutagenic.

Justification for classification or non-classification

Based on the available data, Chromium diboride does not warrant classification for mutagenicity.