Indole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, 473 (adopted on 29 July 2016).

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

1

Reference substance name:

Indole

EC Number:

204-420-7

EC Name:

Indole

Cas Number:

120-72-9

Molecular formula:

C8H7N

IUPAC Name:

1H-indole

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Additional strain / cell type characteristics:

other: pooled blood of three male donors

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

585 micorgram/liter

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Appropriate number of whole blood cultures were established in sterile disposable centrifuge tubes by placing 9.4 mL of culture medium containing 90% of complete culture medium RPMI medium with L- Glutamine (Lonza, Lot no: 0000606793, Exp: 09 February 2019) with 20% (v/v) heat inactivated foetal bovine serum (Invitrogen, Lot no.: 42F3371K, Exp: 20 March 2019), 1% L-glutamine (Invitrogen, Lot no. 17225307, Exp. 20 March 2019) and 1% Penstrep (Lonza, Lot no.17C225307, Exp: 20 March 2019)], 8% of pooled blood and 2% of Phytohaemagglutinin (PHA-M, Invitrogen, Lot no. 1788587, Exp: April 2018). The cultures were then incubated at 37 ± 1 °C for approximately 48 h and shaken continuously.

Evaluation criteria:

The test item will be considered to have the potential to induce chromosome aberrations in this assay if:
1. The assay is valid
2. A proportion of cells with structural aberrations at one or more concentrations that exceeds the historical negative control (normal) range is observed in both replicate cultures
3. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) is observed (p  0.05)
4. There is a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Evidence of a concentration-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations. Analysis of additional cells from vehicle / or treated cultures or further experimental work may be deemed necessary to aid evaluation of the data.

Statistics:

not specified

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Any other information on results incl. tables

Analysis of Chromosomal aberration data:

The details of chromosomal aberrations are presented in Appendix 3, Appendix 4 and Appendix 5. Positive controls from both experiments induced a clear increase in chromosomal aberrations compared to the concurrent solvent control cultures. The chromosomal aberration frequencies in the solvent controls fell within the historical negative control ranges (Appendix 6). A total of 300 cells were scored from each culture. The assay was therefore considered valid.

Structural aberrations:

Treatment of cultures with the test item in the absence and presence of S-9 (both experiments) resulted in frequencies of cells with structural aberrations which were similar to those in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within historical negative control ranges.

Numerical aberrations:

No increases in the frequency of cells with numerical aberrations, which exceeded the historical negative control range, were observed in cultures treated with the test item in the absence and presence of S-9 (both experiments).

Applicant's summary and conclusion

Conclusions:

Based upon the results obtained in this study and in line with OECD Guideline for Testing of Chemicals, 473 (adopted on 29 July 2016) it is concluded that, the given test item INDOLE, supplied by Moraya Global Ltd., did not induce chromosome aberrations in cultured human peripheral blood lymphocytes.

Executive summary:

INDOLE was tested in an in vitro chromosomal aberration assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments. Treatments covering a broad range of doses were performed both in the absence and presence of metabolic activation (S-9). The test item was dissolved in dimethyl Sulphoxide (DMSO) and the highest dose level used in the main experiments, 585 µg/mL, was determined following a solubility trial and subsequent preliminary cytotoxicity range-finding experiment.

A dose range-finding experiment was conducted with a 3-hours exposure to the test item with 17-hours recovery in the absence and presence of the S-9 metabolic activation system. In addition, a 20-hour exposure in the absence of S-9 was also conducted. 49, 98, 195, 390, 585, and 1170 µg/mL concentrations were analysed.

In Experiment 1, treatment in the absence and presence of S-9 was for 3-hours followed by a 17-hours recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test item dose levels for chromosome analysis were selected by evaluating the effect of test item on mitotic index. Chromosome aberrations were analysed at three dose levels  (see below). The highest concentrations chosen for analysis, 585 µg/mL both in the presence and absence of S-9, induced approximately 63.6% and 57.2% cytotoxicity (reduction in mitotic index), respectively.

In Experiment 2, treatment in the presence of S-9 for 3-hours followed by a 17-hours recovery period and in the absence of S-9 was continuous for 20-hours. Chromosome aberrations were analysed at three dose levels (see below) and the highest concentrations chosen for analysis, 585 µg/mL in the presence (3+17) and absence (20+0) of S-9, induced approximately 55.3% and 63.5% cytotoxicity (reduction in mitotic index), respectively.

Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. Mitomycin C and Cyclophosphamide were employed as positive control chemicals in the absence and presence of S-9, respectively. Cells receiving these were sampled in each experiment,  20-hours after the start of treatment; both compounds shows increase in the proportion of cells with structural aberrations.

Treatment of cultures with test item in the absence and the presence of S-9 resulted in frequencies of cells with structural aberrations which were similar to those in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within historical negative control ranges.

No increases in the frequency of cells with numerical aberrations, which exceeded the historical negative control range, were observed in cultures treated with the test item in the absence and presence of S-9.

Based upon the results obtained in this study and in line with OECD Guideline for Testing of Chemicals, 473 (adopted on 29 July 2016) it is concluded that, the given test item INDOLE, supplied by Moraya Global Ltd., did not induce chromosome aberrations in cultured human peripheral blood lymphocytes.

Based upon the results obtained in this study and in line with OECD Guideline for Testing of Chemicals, 473 (adopted on 29 July 2016) it is concluded that, the given test item INDOLE, supplied by Moraya Global Ltd., did not induce chromosome aberrations in cultured human peripheral blood lymphocytes.