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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March 2018 - 29 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Method

Species / strain
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:
S9 fraction prepared from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
(a) 50, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 3200 and (h) 5000 ug/plate The test item did not show toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates, both in the presence and absence of metabolic activation.

Based on these observations, a top dose of 5000 ug/plate was tested in the mutation assay, as recommended by the OECD 471 guideline.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoantharacene
Details on test system and experimental conditions:
In the initial mutation assay, which was a plate incorporation mode of exposure, the bacterial suspensions were exposed to the test item, vehicle and the positive controls in the presence and absence of an exogenous metabolic activation system. These bacterial suspensions were then mixed with overlay agar and plated immediately onto minimal medium viz., his- for Salmonella typhimurium and trp- for Escherichia coli, respectively.

In the confirmatory assay, which was a pre-incubation mode of exposure, the test constituents were mixed with the bacteria inside a tube, incubated in an incubator shaker, mixed with overlay agar and plated immediately onto minimal medium his- for Salmonella typhimurium and trp- for Escherichia coli, respectively.

After 67 hours of incubation, the revertant colonies were counted and compared with the number of spontaneous revertants in the vehicle control plates.


DURATION
- Preincubation period: 20 min
- Exposure duration: 67h
Evaluation criteria:
To determine a positive result, there should be a dose related increrase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the testi item either in the presence or absence of metabolic activation system. The test will be judged postive if the increase in mean revertants at the peak of the dose respomse is equal to or greater than two times the mean vehicle control values for strains TA98, TA100 or WP2uvrA(pKM101) or equal to or greater than three times the mean vehicle control values for strains TA1535 and TA1537. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respecitive threshold cited above or a non-dose responsive increase that is equal to or greated than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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 applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test item tetrakis(2-ethylhexane-1,3-diolato)titanium was not mutagenic in the bacterial reverse mutation test up to the OECD 471-recommended top dose of 5000 µg/plate under the conditions of testing employed.
Executive summary:

Both the Salmonella typhimurium and Escherichia coli tester strains were found to be reliable and responsive to the different genotypic characterization tests like the amino acid requirement, rfa mutation, uvr mutation and the R-factor plasmids. Similarly, the spontaneous revertant counts of the vehicle control groups of these tester strains were in the ranges of the test facility’s historical control data.  

The positive controls produced a more than 3-fold increase in the mean numbers of revertant colonies when compared to the respective vehicle controls, demonstrating the sensitivity of the assay procedure.  

The test item at doses up to 5000 µg/plate did not cause a two fold increase in the mean numbers of revertant colonies in the strains TA98, TA100 and WP2uvrA (pKM101) or three fold increase in the mean numbers of revertant colonies in the strains TA1535 and TA1537 either in the presence or absence of the metabolic activation system when compared to the respective vehicle control plates.