Reaction products of tetraisopropyl titanate (4+), hydroxyalkaloic acid and substituted alkanol

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:

From January 26th, 2017 to January 30th, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Study carried out with a 57.7 % water solution of the test item. In addition, only three strains were used

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 tissue fraction

Test concentrations with justification for top dose:

1250, 625, 313, 156, 78.1, 39.1, 19.5, and 9.77 µg/plate
Since 25.0 µL of test item solution is used in the preparation of each plate, this permitted a maximum dose level of 1250 µg/plate to be tested in the mutation experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water for injection

- Justification for choice of solvent/vehicle: sterile water for injection is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50.0 mg/ mL. This result permitted a maximum concentration of 1250 µg/plate to be tested in the mutation experiment.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

sterile water for injection

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Absence of S9

Controls 2

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

sterile water for injection

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION
A single experiment, using the pre-incubation method, was performed.

DURATION
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were held at 4°C for 24 hours.

NUMBER OF REPLICATES: Three replicate wells were used at each test point.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight reduction of revertant colonies was observed at the highest dose level in the absence of S9 metabolism

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Solubility

Solubility of the test item was evaluated in a preliminary trial using sterile water for injection. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50.0 mg/mL. Since 25.0 µL of test item solution is used in the preparation of each plate, this permitted a maximum dose level of 1250 µg/plate to be tested in the mutation experiment. This concentration corresponds to 5000 µg/plate using the standard method (OECD guideline No. 471).

Main Assay

A single experiment was performed. The test item was assayed at a maximum dose level of 1250 µg/plate and at seven lower dose levels: 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/plate. No precipitation of the test item was noted at the end of the incubation period at any concentration, in the absence or presence of S9 metabolism. Slight reduction of revertant colonies was observed with TA100 all tester strains at the highest dose level in the absence of S9 metabolism indicating a possible toxic effect. No relevant increase in revertant numbers was observed after treatment with test item at any dose level, in any tester strain, in the absence or presence of S9 metabolism. Sterility of the S9 mix and test item solutions was confirmed by the absence of colonies on additional agar plates separately spread with these solutions. Marked increases in revertant numbers were obtained in this test following treatment with the positive control items, indicating that the assay system was functioning correctly.

Analysis of results

Acceptance criteria

The assay was considered valid if the following criteria were met:

1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.

2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.

3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing

increasing numbers of mutant colonies with increasing dose levels.

Evaluation

Results show that mean plate counts for untreated and positive control plates fell within testing facility acceptance criteria based on historical control data (confidence interval: mean value ± 2 standard deviations). The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid. The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:

Not genotoxic

Executive summary:

Method

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy, according to OECD guideline 471. The three tester strains TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The modified 6 well bacterial mutation method was used. The test item was used as a solution in sterile water for injection.

Observations

In the Main Assay, using the preincubation method, the test item was assayed at a maximum dose level of 1250 µg/plate, corresponding to 5000 µg/plate when the standard method (OECD guideline No. 471) is used, and at seven lower dose levels: 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/plate.

No precipitation of the test item was noted at the end of the incubation period at any concentration, in the absence or presence of S9 metabolism. A slight reduction of revertant colonies was observed with TA100 tester strain at the highest dose level in the absence of S9 metabolism, indicating a possible toxic effect. No relevant increase in revertant numbers was observed after treatment with MTC 2874 at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusion

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.