[(9-oxo-9H-thioxanthen-2-yl)oxy]acetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies conducted to recognised testing guidelines with GLP certification.

Genetic toxicity in vitro gene mutation study in bacteria - AMES; OECD 471

The objective of this study was to determine the potential of CMTX 2-carboxymethyloxy-thioxanthone and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Although 2.3- to 5.3-fold, dose related increases were observed in two tester strains, both in the absence and presence of S9-mix, the results were not greater than three times the concurrent control and not above the laboratory historical control data range in all occasions. Therefore it is concluded that CMTX 2-carboxymethyloxy-thioxanthone is equivocal mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay.

Genetic toxicity in vitro cytogenicity / chromosome aberration study in mammalian cells; OECD 473

The objective of this study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of CMTX 2-carboxymethyloxy-thioxanthone was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

In conclusion, this test is valid and CMTX 2-carboxymethyloxy-thioxanthone is not clastogenic in human lymphocytes under the experimental conditions described in this report.  CMTX 2-carboxymethyloxy-thioxanthone may have the potential to disturb mitotic processes and cell cycle progression.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Oct 2018 to 30 Jan 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25% (assumed 100%)
Test item storage: At room temperature protected from light desiccated

Test concentrations with justification for top dose:

At a concentration of 1000 µg/mL CMTX 2-carboxymethyloxy-thioxanthone precipitated in the culture medium. In the dose-range finding study, blood cultures were treated with 31.3, 62.5, 125, 250, 500 and 1000 µg CMTX 2-carboxymethyloxy-thioxanthone/mL culture medium with and without S9-mix.

First Cytogenetic Assay:
Based on the results of the dose-range finding test the following dose levels were selected for the cytogenetic assay:
With and without S9-mix: 50, 200, 400, 500, 600 and 750 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Table 2 (Appendix 1) shows the mitotic index of cultures treated with various test item concentrations or with the positive or negative control items.
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix: 50, 200, 400 and 600 µg/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix: 50, 200 and 500 µg/mL culture medium (3 h exposure time, 24 h fixation time).

To obtain more information about the possible clastogenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to CMTX 2-carboxymethyloxy-thioxanthone in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 10, 50, 75, 100, 125, 150, 175 and 200 µg/mL culture medium (24 h or 48 h exposure time, 24 h or 48 h fixation time).

Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 10, 50 and 100 µg/mL culture medium (24 h or 48 h exposure time, 24 h or 48 h fixation time).

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Dose-range Finding Test:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose-range finding test. CMTX 2-carboxymethyloxy-thioxanthone was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.

Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of CMTX 2-carboxymethyloxy-thioxanthone for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.

The highest tested concentration was determined by the solubility of the test item in the culture medium.

After 3 h exposure to CMTX 2-carboxymethyloxy-thioxanthone in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).
Cytotoxicity of CMTX 2-carboxymethyloxy-thioxanthone in the lymphocyte cultures was determined using the mitotic index.

Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

Cytogenetic Assay:
The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications. CMTX 2-carboxymethyloxy-thioxanthone was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.

First cytogenetic assay:
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of CMTX 2-carboxymethyloxy-thioxanthone for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 to 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay. Initially, in the presence of S9-mix, the positive control did not give an appropriate response. Therefore this part of the first cytogenetic assay had to be repeated.
Based on the mitotic index of the dose-range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary. The follow up experiment was performed with the following modifications of experimental conditions.

Second cytogenetic assay:
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of CMTX 2-carboxymethyloxy-thioxanthone for 24 h and 48 h in the absence of S9-mix.
The cells were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

Chromosome Preparation:
During the last 2.5 to 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

Preparation of Slides:
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Mitotic Index/Dose Selection for Scoring of the Cytogenetic Assay:
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analyzable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analyzed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of Slides for Chromosome Aberrations:
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Species / strain:

lymphocytes: Human

Remarks:

Dose-Range Finding Test; 3 h exposure time, 24 h fixation time; Concentrations in µg/mL: Control, 31.3, 62.5, 125, 250, 500, 1000

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed from concentrations of 31.3 µg/mL upwards

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: Human

Remarks:

Dose-Range Finding Test; 24 h exposure time, 24 h fixation time; Concentrations in µg/mL: Control, 31.3, 62.5, 125, 250, 500, 1000

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed from concentrations of 31.3 µg/mL upwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: Human

Remarks:

Dose-Range Finding Test; 48 h exposure time, 48 h fixation time; Concentrations in µg/mL: Control, 31.3, 62.5, 125, 250, 500, 1000

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed from concentrations of 31.3 µg/mL upwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: Human

Remarks:

Dose-Range Finding Test; 3 h exposure time, 24 h fixation time; Concentrations in µg/mL: Control, 31.3, 62.5, 125, 250, 500, 1000

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed from concentrations of 125 µg/mL upwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

First Cytogenetic Assay; 3 h exposure time, 24 h fixation time; Concentrations in µg/mL: Control, 50, 200, 400, 500. 600, 750, MMC-C; 0.5 µg/mL, MMC-C; 0.75 µg/mL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed from concentrations of 200 µg/mL upwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

First Cytogenetic Assay; 3 h exposure time, 24 h fixation time; Concentrations in µg/mL: Control, 50, 200, 400, 500. 600, 750, CP; 10 µg/mL

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed from concentrations of 200 µg/mL upwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

Second Cytogenetic Assay; 24 h exposure time, 24 h fixation time; Concentrations in µg/mL: Control, 10, 50, 75, 100, 125, 150, 175, 200, MMC-C; 0.2 µg/mL, MMC-C; 0.3 µg/mL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed from concentrations of 10 µg/mL upwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

Second Cytogenetic Assay; 48 h exposure time, 48 h fixation time; Concentrations in µg/mL: Control, 10, 50, 75, 100, 125, 150, 175, 200, MMC-C; 0.1 µg/mL, MMC-C; 0.15 µg/mL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed from concentrations of 10 µg/mL upwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Dose-range Finding Test

At a concentration of 1000 µg/mL CMTX 2-carboxymethyloxy-thioxanthone precipitated in the culture medium.  In the dose-range finding study, blood cultures were treated with 31.3, 62.5, 125, 250, 500 and 1000 µg CMTX 2-carboxymethyloxy-thioxanthone/mL culture medium with and without S9-mix.  

The pH and osmolarity of a concentration of 500 µg/mL were 7.6 and 396 mOsm/kg respectively (compared to 7.8 and 408 mOsm/kg in the solvent control).

Table 1 (Appendix 1) shows the mitotic index of cultures treated with various test item concentrations or with the negative control item.

First Cytogenetic Assay

Based on the results of the dose-range finding test the following dose levels were selected for the cytogenetic assay:

With and without S9-mix: 50, 200, 400, 500, 600 and 750 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Table 2 (Appendix 1) shows the mitotic index of cultures treated with various test item concentrations or with the positive or negative control items.

The following dose levels were selected for scoring of chromosome aberrations:  

Without S9-mix: 50, 200, 400 and 600 µg/mL culture medium (3 h exposure time, 24 h fixation time).

With S9-mix: 50, 200 and 500 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Both in the absence and presence of S9-mix, CMTX 2-carboxymethyloxy-thioxanthone did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Appendix 1:  Table 3, Table 4).

CMTX 2-carboxymethyloxy-thioxanthone increased the number of polyploid cells and cells with endoreduplicated chromosomes both in the absence and presence of S9-mix at the highest dose levels (600 and 500 µg/mL respectively).

Second Cytogenetic Assay

To obtain more information about the possible clastogenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to CMTX 2-carboxymethyloxy-thioxanthone in the absence of S9-mix for 24 or 48 hours.  The following dose levels were selected for the second cytogenetic assay:  

Without S9-mix: 10, 50, 75, 100, 125, 150, 175 and 200 µg/mL culture medium (24 h or 48 h exposure time, 24 h or 48 h fixation time).

Table 5 (Appendix 1) shows the mitotic index of cultures treated with various test item concentrations or with the positive or negative control items.  

Based on these observations the following doses were selected for scoring of chromosome aberrations:

Without S9-mix: 10, 50 and 100 µg/mL culture medium (24 h or 48 h exposure time, 24 h or 48 h fixation time).

CMTX 2-carboxymethyloxy-thioxanthone did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Appendix 1; Table 6 - 7).

CMTX 2-carboxymethyloxy-thioxanthone increased the number of polyploid cells, both at the 24 h and 48 h exposure times.

Evaluation of the Results

The ability of CMTX 2-carboxymethyloxy-thioxanthone to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments.  The highest concentration analyzed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater.

The mitotic indices of cultures treated with various CMTX 2-carboxymethyloxy-thioxanthone concentrations or with the negative control items are presented in Table 1, Table 2 and Table 5 (Appendix 1).  The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of CMTX 2-carboxymethyloxy-thioxanthone are presented in Table 3, Table 4 and Table 6 – 7 (Appendix 1).  Duplicate cultures are indicated by A and B.  The criteria according to which the aberrations were classified are outlined in Appendix 2.  Appendix 3 presents the statistical evaluations of the test results.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database (see Appendix 4).  The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database (see Appendix 6).  The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells (see Appendix 3).  In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.  It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix CMTX 2-carboxymethyloxy-thioxanthone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

It was noted that after 3h exposure CMTX 2-carboxymethyloxy-thioxanthone increased the number of polyploid cells and the number of cells with endoreduplicated chromosomes both in the absence and presence of S9-mix, at the highest concentrations tested.  Additionally, after 24 h and 48 h exposure the test item increased the number of polyploid cells.  This may indicate that CMTX 2-carboxymethyloxy-thioxanthone has the potential to disturb mitotic processes and cell cycle progression.

Conclusions:

In conclusion, this test is valid and CMTX 2-carboxymethyloxy-thioxanthone is not clastogenic in human lymphocytes under the experimental conditions described in this report. CMTX 2-carboxymethyloxy-thioxanthone may have the potential to disturb mitotic processes and cell cycle progression.

Executive summary:

The objective of this study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of CMTX 2-carboxymethyloxy-thioxanthone was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

The CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder.  The vehicle of the test item was dimethyl sulfoxide.

In the first cytogenetic assay, CMTX 2-carboxymethyloxy-thioxanthone was tested up to 600 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence of 1.8% (v/v)

S9-fraction, and up to 500 µg/mL in the presence of 1.8% (v/v) S9-fraction.  Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, CMTX 2-carboxymethyloxy-thioxanthone was tested up to 100 µg/mL for both a 24 h continuous exposure time with a 24 h fixation time and for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix.  Appropriate toxicity was reached at this dose level.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.  Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations.  In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.  

It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

CMTX 2-carboxymethyloxy-thioxanthone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

It was noted that after 3h exposure CMTX 2-carboxymethyloxy-thioxanthone increased the number of polyploid cells and the number of cells with endoreduplicated chromosomes both in the absence and presence of S9-mix, at the highest concentrations tested.  Additionally, after 24 h and 48 h exposure the test item increased the number of polyploid cells.  This may indicate that CMTX 2-carboxymethyloxy-thioxanthone has the potential to disturb mitotic processes and cell cycle progression.

In conclusion, this test is valid and CMTX 2-carboxymethyloxy-thioxanthone is not clastogenic in human lymphocytes under the experimental conditions described in this report.  CMTX 2-carboxymethyloxy-thioxanthone may have the potential to disturb mitotic processes and cell cycle progression.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Oct 2018 to 08 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”.

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25% (assumed 100% for testing)
Test item storage: At room temperature protected from light desiccated

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:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
The results are shown in Table 1 and Table 2.
The individual data are presented in Appendix 3.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).

Untreated negative controls:

yes

Remarks:

Solvent control, DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

Salmonella typhimurium bacteria and Escherichia coli bacteria

Rationale for test conditions:

Recommended test system in international guidelines

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

No formal hypothesis testing was done.

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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:

First Experiment: Direct Plate Assay:
CMTX 2-carboxymethyloxy-thioxanthone was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate. The results are shown in Table 1 and Table 2. The individual data are presented in Appendix 3.

Precipitate:
Dose range finding test: Precipitation of CMTX 2-carboxymethyloxy-thioxanthone on the plates was not observed at the start of the incubation period. Precipitation of the test item on the plates was observed at the end of the incubation period at the concentration of 5000 µg/plate in both tester strains.
First mutation experiment: Precipitation of CMTX 2-carboxymethyloxy-thioxanthone on the plates was not observed at the start of the incubation period. Precipitation of the test item on the plates was observed at the end of the incubation period at the concentration of 5000 µg/plate in tester TA1535 in the absence of S9-mix only.

Toxicity:
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. The definitions are stated in Appendix 2.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity:
In tester strain TA98, CMTX 2-carboxymethyloxy-thioxanthone induced up to 2.3- and 3.2-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In the other tester strains, no increase in the number of revertants was observed upon treatment with the test item.

Second Experiment: Pre-Incubation Assay:
To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, CMTX 2-carboxymethyloxy-thioxanthone was tested up to the dose level of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The results are shown in Table 3, the individual data are presented in Appendix 3.

Precipitate:
Precipitation of CMTX 2-carboxymethyloxy-thioxanthone on the plates was observed at the start of the incubation period at the concentration of 5000 µg/plate and no precipitate was observed at the end of the incubation period.
 
Toxicity:
Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the presence of S9-mix. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed in the other tester strains.

Mutagenicity:
In tester strain TA98, CMTX 2-carboxymethyloxy-thioxanthone induced up to 3.4- and 2.4-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.
In tester strain TA1537, CMTX 2-carboxymethyloxy-thioxanthone induced up to 5.3- and 3.7-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.
In the other tester strains, no increase in the number of revertants was observed upon treatment with the test item.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In the absence of S9-mix, the test item induced dose related increases in two tester strains (TA1537 and TA98).  The increase observed in tester strain TA1537 was greater than three times the concurrent control (5.3-fold).  However the increase was within the laboratory historical control data range and was only observed in the second experiment. The increases observed in tester strain TA98 were greater than three times the concurrent (up to 3.4-fold) in the second experiment only and above the laboratory historical control data range in the first experiment only.

In the presence of S9-mix, the test item induced dose related increases in two tester strains (TA1537 and TA98).  The increase observed in tester strain TA1537 was greater than three times the concurrent control (3.7-fold).  However the increase was within the laboratory historical control data range and was only observed in the second experiment. The increases observed in tester strain TA98 were greater than three times the concurrent (up to 3.2-fold) in the first experiment only and within the laboratory historical control data range in both experiments.

All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

Conclusions:

Although 2.3- to 5.3-fold, dose related increases were observed in two tester strains, both in the absence and presence of S9-mix, the results were not greater than three times the concurrent control and not above the laboratory historical control data range in all occasions. Therefore it is concluded that CMTX 2-carboxymethyloxy-thioxanthone is equivocal mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of CMTX 2-carboxymethyloxy-thioxanthone and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder.  The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay.  The test item precipitated on the plates at the dose level of 5000 μg/plate.  The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.   Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98.  CMTX 2-carboxymethyloxy-thioxanthone precipitated on the plates at the dose level of 5000 μg/plate in tester strain TA1535 in the absence of S9-mix only.  The bacterial background lawn was not reduced at any of the concentrations tested and npreo biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the

pre-incubation assay.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the presence of S9-mix.  The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed in the other tester strains.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In the absence of S9-mix, the test item induced dose related increases in two tester strains (TA1537 and TA98). The increase observed in tester strain TA1537 was greater than three times the concurrent control (5.3-fold).  However the increase was within the laboratory historical control data range and was only observed in the second experiment. The increases observed in tester strain TA98 were greater than three times the concurrent (up to 3.4-fold) in the second experiment only and above the laboratory historical control data range in the first experiment only.

In the presence of S9-mix, the test item induced dose related increases in two tester strains (TA1537 and TA98). The increase observed in tester strain TA1537 was greater than three times the concurrent control (3.7-fold).  However the increase was within the laboratory historical control data range and was only observed in the second experiment. The increases observed in tester strain TA98 was greater than three times the concurrent (up to 3.2-fold) in the first experiment only and within the laboratory historical control data range in both experiments.

All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

Although 2.3- to 5.3-fold, dose related increases were observed in two tester strains, both in the absence and presence of S9-mix, the results were not greater than three times the concurrent control and not above the laboratory historical control data range in all occasions. Therefore it is concluded that CMTX 2-carboxymethyloxy-thioxanthone is equivocal mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Substance is not clastogenic according to OECD 473 test, and not mutagenic according to OECD 471.