[No public or meaningful name is available]

Genetic toxicity: in vitro

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:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

µ2-(2-oxido-2-acetato- κ1O-1,3-dicarboxylate- κ2O)-bis[(2-oxidopropane-2-carboxylato-1,3-dicarboxylate)(2-oxidopropane-1,2,3-tricarboxylate- κO)titanium](4+)

Molecular formula:

[{Ti2(C6H7O7)(C6H6O7)}(µ2-C6H6O7)]

IUPAC Name:

µ2-(2-oxido-2-acetato- κ1O-1,3-dicarboxylate- κ2O)-bis[(2-oxidopropane-2-carboxylato-1,3-dicarboxylate)(2-oxidopropane-1,2,3-tricarboxylate- κO)titanium](4+)

Test material form:

liquid

Specific details on test material used for the study:

Test Item Name TYZOR AC 436
IUPAC Name Citric acid, titanium salt
C.A. Name Information not provided by Sponsor
CAS Number (EC # 923-927-0)
Batch/Lot Number 334-301300/000005
Analysed Purity As Ti: 4.9% w/w (Refer Certificate of Analysis in APPENDIX 2)
Appearance Yellow to brownish clear liquid
Manufactured by Dorf Ketal Speciality Catalyst Private Limited
Supplied to JRF by Dorf Ketal Speciality Catalyst Private Limited
Date of Manufacture September 2017
Date of Expiry September 2019
Storage Condition (at JRF) As per the instruction received from the Sponsor on storage of the test item, the test was stored:
Storage Condition : Protected from light, air and moisture. Once opened container was kept under nitrogen blanket to prevent composition .

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Following doses were selected for the pre-experiment: 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate. In treated concentrations there was Moderate Inhibition was observed at 1.582 and 5 mg/plate.
There was no reduction in colony count as well as background lawn at concentrations (0.002, 0.005, 0.016 0.050, 0.158 and 0.501 mg/plate) both in absence and presence of metabolic activation, when compared to that of the negative control group.
Based on the results, 0.501 mg/plate was selected as the highest dose for the main study trials both in the absence and as well as in the presence of metabolic activation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoantharacene; 4-Nitro-o-phenylenediamine

Details on test system and experimental conditions:

Pre-experiment for toxicity:
To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Dose selection for main study:
In the pre-experiment, the concentration range of the test item was 0.002 - 5 mg/plate based on the solubility and precipitation test.
In TA 98 and TA 100, Moderate Inhibition was observed at treated concentrations 1.582 and 5 mg/plate, there was no reduction in colony count as well as background lawn at concentrations (0.005, 0.016 0.050, 0.158 and 0.501 mg/plate) both in absence and presence of metabolic activation, when compared to that of the negative control group.

Based on the results of pre-experiment, following doses were selected for the main study trials:
0.002, 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate, both in the absence (-S9) as well as in the presence (+S9) of metabolic activation.

Concentrations used in the experiments (Pre-experiment, Trial-I, Trial-II) were spaced with (√10) half log intervals.

Experimental performance:
For each strain and dose level, including the controls three plates (triplicate) were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, negative control and reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacterial suspension,
2000 µL Overlay agar

In the pre-incubation assay 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and incubated at 37 ± 2°C for 60 mi-nutes. After pre-incubation 2.0 mL overlay agar (47 ± 2°C) was added to each tube. The mixture was poured on minimal glucose agar plates.
After solidification the plates were incubated in inverted position for 48 hours at 37 ± 2°C.

Evaluation criteria:

The Bacterial reverse mutation assay is considered acceptable as, it met the following criteria:
- Regular background growth in the negative control.
- The positive control substances produced a significant increase in mutant colony frequencies.
- The spontaneous reversion rates in the negative control are in the range of in-house historical data.


A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate Inhibition was observed at 1.582 and 5 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate Inhibition was observed at 1.582 and 5 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate Inhibition was observed at 1.582 and 5 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate Inhibition was observed at 1.582 and 5 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate Inhibition was observed at 1.582 and 5 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item AC 436 did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used under the experimental conditions reported.

Executive summary:

This study was performed to investigate the potential of AC 436 to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1537, TA 1535, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrationsviz.,0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations: 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with AC 436 at any dose level in both the trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The spontaneous reversion rates in the negative and positive controls are within the range of in-house historical data.

The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i..e. Plate incorporation method and Pre-incubation method.