2,3,3-trimethyl-3H-indole-5-carboxylic acid

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:

08.07.2016-20.07.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471:
Bacterial Reverse Mutation Test, adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Commission Regulation (EC) No. 440/2008 B13/14, dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,3,3-trimethyl-3H-indole-5-carboxylic acid

EC Number:

282-162-4

EC Name:

2,3,3-trimethyl-3H-indole-5-carboxylic acid

Cas Number:

84100-84-5

Molecular formula:

C12H13NO2

IUPAC Name:

2,3,3-trimethyl-3H-indole-5-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

brown powder
Batch no. CHA024

Method

Target gene:

Salmonella typhimurium (TA strains): histidine locus
Escherichia coli (WP2 uvrA strain): tryptophan locus

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I (plate incorporation test): 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II (pre-incubation test): 33; 100; 333; 1000; 2500; and 5000 µg/plate

Since no toxic effects were observed in the pre-experiment, 5000 µg/plate were chosen as maximal concentration for experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (purity > 99 %)
- Justification for choice of solvent/vehicle: solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD (without activation), 2-AA (with activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment II: pre-incubation;
DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours
NUMBER OF REPLICATIONS: In both experiments, each concentration, including the controls, was tested in triplicate.
DETERMINATION OF CYTOTOXICITY: reduction in the number of revertants (below the indication factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

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 WP2 uvrA) 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 and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Precipitation: no precipitation of the test item occurred up to the highest investigated dose.
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).

HISTORICAL CONTROL DATA:

Strain without S9 mix with S9 mix
Mean SD Min Max Mean SD Min Max
Solvent control 11 2.15 7 23 12 2.14 7 21
TA 1535 Untreated control 12 2.97 6 24 12 2.71 7 26
Positive control 1090 123.80 334 1372 392 62.85 176 549

Solvent control 10 1.83 6 18 13 3.27 7 27
TA1537 Untreated control 10 2.29 6 20 14 3.72 7 25
Positive control 83 12.28 55 131 175 44.44 82 327

Solvent control 24 3.75 16 36 33 5.55 18 51
TA 98 Untreated control 26 4.72 15 43 36 5.83 17 56
Positive control 344 51.13 211 599 3822 857.83 319 5048

Solvent control 155 24.19 84 194 145 31.81 81 204
TA 100 Untreated control 174 21.92 90 206 170 23.62 93 212
Positive control 1956 279.93 6 58 2528 3606 676.07 722 4940

Solvent control 41 5.72 27 63 51 6.91 37 72
WP2uvrA Untreated control 42 6.01 31 63 53 7.05 38 88
Positive control 732 161.66 322 1066 362 72.26 212 858

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment II, the data in the negative control of strain TA 100 without S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Applicant's summary and conclusion

Conclusions:

The test item Trimethylcarbonsäure was evaluated in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA in the presence and absence of rat liver S9 mix. Under the conditions of the study, the test substance was negative for mutagenic potential.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and Escherichia coli strain WP2 uvrA were exposed to Trimethylcarbonsäure in DMSO in concentrations of 0 (control), 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver S9 mix) in a plate incorporation test and in concentrations of 0 (control), 33, 100, 333, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver S9 mix) in a pre-incubation test.

Each concentration and the controls were tested in triplicates.

No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.  

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.  

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Trimethylcarbonsäure at any concentration level, neither in the presence nor 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.

In experiment II, the data in the negative control of strain TA 100 without S9 mix wereslightly above the laboratory's historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies

There was no evidence of induced mutant colonies over background.

Under the conditions of the study, the test substance was negative for mutagenic potential.