Registration Dossier

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the Reverse Mutation Assay 'Ames Test' using Salmonella typhimurium and Escherichia coli the test item was considered to be non-mutagenic under the conditions of this test.

In the Micronucleus Test in Human Lymphocytes in vitro the test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

In the V79 HPRT Gene Mutation Assay the test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October 2017 - 21 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Physical State / Appearance: Red Powder
Batch: 021340
Storage Conditions: Room temperature, in the dark
Target gene:
hypoxanthine-guanine phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Harlan CCR
- Suitability of cells: The high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The maximum recommended dose level was 5000 μg/mL however the maximum achievable dose level was 2500 μg/mL due to formulation issues at the MRD.
0.02 to 2 μg/mL in the absence of metabolic activation and 0.03 to 3 μg/mL in the presence of metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 1 x 10e7 cells/225 cm2 flask approximately 24 hours before dosing

DURATION
- Preincubation period: 24 hoiurs
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Rationale for test conditions:
prliminary experiment results
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
Statistics:
Student’s t-test
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at 2 μg/mL in the absence of metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The vehicle control values were all considered to be within an acceptable range, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.
- Precipitation: No precipitate of the test item was observed throughout.

RANGE-FINDING/SCREENING STUDIES: No precipitate of the test item was observed in either of the exposure groups. The maximum concentration selected for the main mutagenicity experiment was therefore limited by the onset of item-induced toxicity in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines.

Conclusions:
Lumière Pink S.M. 8135N did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 21 July 2017 and 22 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification: Lumière Pink S.M. 8135N
Physical state/Appearance: Red powder
Batch: 021340
Purity: Preparation containing ≥90% UVCB (treat as 100%)
Expiry Date: 28 April 2022
Storage Conditions: Room temperature in the dark
Intended use/Application: Dyestuff/ pigment
No correction for purity was required.

Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fractions
Test concentrations with justification for top dose:
The test item was considered to be a UVCB* and therefore the maximum recommended dose was initially set at 5000 µg/mL. However, due to formulation difficulties the maximum dose level that could be achieved was 2500 µg/mL. The test item was treated as 100% pure and and no purity adjustment was required in the formulations
Vehicle / solvent:
The test item was insoluble in culture media at 50 and 25 mg/mL and dimethyl sulphoxide (DMSO) at 500 mg/mL but was soluble in DMSO at 250 mg/mL in solubility checks performed in house. Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Identity: Dimethyl sulphoxide (DMSO) Supplier: Fisher Batch number: 1684307 Purity: >99% Expiry date (5 years after opening): Preliminary Toxicity test: 20/07/2022 Preliminary Toxicity Test Repeat: 25/08/2022 Main Experiment: 19/09/2022
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Identity: Demecolcine (DC) CAS No.: 477-30-5 Supplier: Sigma Aldrich Batch Number: BCBR3135V Purity: >98% Expiry Date: 13 January 2022 Solvent: Sterile distilled water Concentration: 0.075 µg/mL for 24-hour continuous exposure
Details on test system and experimental conditions:
Test System and Supporting Information
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 26 years
Preliminary Toxicity Test Repeat: male, aged 28
Main Experiment: male, aged 27 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Batch Nos. PB/NF S9 30/6/17 and 20/8/17 were used in this study. Prior to use each batch of S9 is tested for its capability to activate known mutagens in the Ames test.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.


Experimental Design and Study Conduct

Test Item Preparation
The test item was considered to be a UVCB* and therefore the maximum recommended dose was initially set at 5000 µg/mL. However, due to formulation difficulties the maximum dose level that could be achieved was 2500 µg/mL. The test item was treated as 100% pure and therefore no purity correction was required.
The test item was insoluble in culture media at 50 and 25 mg/mL and dimethyl sulphoxide (DMSO) at 500 mg/mL but was soluble in DMSO at 250 mg/mL in solubility checks performed in house. Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

The pH and osmolality readings are presented in the following table:

Dose Concentration (µg/mL) 0 9.77 19.53 39.06 78.13 156.25 312.5 625 1250 2500
pH 7.33 7.35 7.36 7.37 7.36 7.38 7.37 7.35 7.35 7.35
Osmolality mOsm 448 454 476 458 459 447 458 439 424 405


The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 µg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.

The preliminary toxicity test was initially performed with a test item dose range of 9.77 to 2500 µg/mL. However, due to excessive toxicity and no surviving dose levels at the end of exposure the preliminary toxicity test was repeated with the dose range of 0.125 to 16 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0.125, 0.25, 0.50, 0.75, 1, 1.5 and 2 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0.5, 1, 2, 3, 4, 5 and 6 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0.0625, 0.125, 0.25, 0.5, 0.75, 1 and 1.5 µg/mL.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Assessments
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

Coding
The slides were coded before analysis using a computerized random number generator.

Cytokinesis Block Proliferation Index (CBPI)
A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.

Scoring of Micronuclei
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
The criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
Evaluation criteria:
Acceptability Criteria
The following criteria were used to determine a valid assay:
• The concurrent negative control was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9 mix.
• Cell proliferation criteria in the solvent control were considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations was analyzed.
Statistics:
Statistical Analysis
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible
Key result
Species / strain:
lymphocytes: human lymphocytes
Remarks:
human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test item was excessively toxic
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 9.77 to 2500 µg/mL. The maximum dose was the maximum achievable dose level. However, due to excessive toxicity and no surviving dose levels at the end of exposure the preliminary toxicity test was repeated with the dose range of 0.125, 0.25, 0.5, 1, 2, 4, 8, 12 and 16 µg/mL.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of exposure in any of the three exposure groups of the repeat preliminary toxicity test.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2 µg/mL in the 4-hour exposure in the absence of metabolic activation (S9), up to 4 µg/mL in the presence of S9 and up to 1 µg/mL in the 24-hour exposure group. The test item induced marked evidence of toxicity in all three exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 2 µg/mL for the 4-hour exposure group in the absence of S9, 6 µg/mL in the presence of S9 and was 1.5 µg/mL for the 24-hour exposure group.



 Micronucleus Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Exposure Group

Final concentration of test itemLumière Pink S.M. 8135N(µg/mL)

4-hour without S9

0*, 0.125, 0.25, 0.50*, 0.75*, 1*, 1.5, 2, MMC0.2*

4-hour with S9 (2%)

0*, 0.5, 1, 2*, 3*, 4*, 5, 6, CP5*

24-hour without S9

0*, 0.0625, 0.125, 0.25, 0.5*, 0.75*, 1*, 1.5,DC0.075*

*             = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC= Mitomycin C

CP           = Cyclophosphamide

DC          = Demecolcine

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring up to 2 µg/mL in the 4-hour exposure in the absence of S9, up to 5 µg/mL in the presence of S9 and up to 1 µg/mL in the presence of S9.   

The CBPI data for the short exposure groups and for the 24-hour exposure group confirm the qualitative observations in that there was a dose-related increase in toxicity in all three exposure groups. In the 4-hour exposure group in the absence of S9, 33%, 55% and 59% cytostasis was achieved at 0.5, 0.75 and 1 µg/mL, respectively. The dose level of 1.5 µg/mL exceeded acceptable toxicity with 69% cytostasis. In the presence of S9, 17%, 44% and 57% cytostasis was achieved at 2, 3 and 4 µg/mL, respectively. The 24-hour exposure group achieved 19%, 48% and 56% cytostasis at 0.5, 0.75 and 1 µg/mL, respectively. The maximum dose level selected for analysis of binucleate cells was based on toxicity and was 1 µg/mL in the absence of S9 and 4 µg/mL in the presence of S9.

The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced optimum toxicity (55±5% cytostasis) in all three exposure groups.

Conclusions:
The test item, Lumière Pink S.M. 8135N, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Test were as follows:

Exposure Group

Final concentration of test itemLumière Pink S.M. 8135N(µg/mL)

4-hour without S9

0, 0.125, 0.25, 0.50, 0.75, 1, 1.5, 2

4-hour with S9 (2%)

0, 0.5, 1, 2, 3, 4, 5, 6

24-hour without S9

0, 0.0625, 0.125, 0.25, 0.5, 0.75, 1, 1.5

 Results

All vehicle (Dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was excessively toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced optimum toxicity (55±5% cytostasis) in all three exposure groups.

 Conclusion

The test item,LumièrePink S.M. 8135Nwas considered to be non-clastogenic and non-aneugenic to human lymphocytesin vitro.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Jun - 14 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification: Lumière Pink S.M. 8135N
Physical state/Appearance: Red powder
Batch: 021340
Purity: Preparation containing ≥90% UVCB (treated as 100%)
Expiry Date: 28 April 2022
Storage Conditions: Room temperature in the dark

The vehicle control used was as follows:
Identity: Dimethyl sulphoxide
Batch number (purity): 1690734 (>99%), Expiry: 03/2022

Direct acting compounds in the absence of S9-mix:
Identity: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)
Batch number: 67F-3700
Expiry date: 18 September 2017
Solvent: DMSO
Concentration: 2 μg/plate for WP2uvrA
3 μg/plate for TA100
5 μg/plate for TA1535

Identity: 9-Aminoacridine (9AA)
Batch number: S32398-438
Purity: 99.9%
Expiry date: 01 October 2017
Solvent: DMSO
Concentration: 80 μg/plate for TA1537

Identity: 4-Nitroquinoline-1-oxide (4NQO)
Batch number: 030M1206
Purity: 100%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 0.2 μg/plate for TA98

Direct acting compounds in the presence of S9-mix:
Identity: 2-Aminoanthracene (2AA)
Batch number: STBB1901M9
Purity: 97.5%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 1 μg/plate for TA100
2 μg/plate for TA1535 and TA1537
10 μg/plate for WP2uvrA

Identity: Benzo(a)pyrene (BP)
Batch number: 090M1400V
Purity: 96%
Expiry date: 12 October 2017
Solvent: DMSO
Concentration: 5 μg/plate for TA98
Target gene:
histidine or tryptophan synthesis
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
no
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48 h (Experiment 1 & 2)

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
The test material may be considered positive in this test system if it induces a reproducible, dose-related
and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnetts Regression Analysis (* = p < 0.05)
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A pink test item induced colouration was noted from 50 μg/plate becoming red at and above 1500 μg/plate. A test item precipitate (powdery in appearance) was also observed from 1500 μg/plate. These observations did not prevent the scoring of revertant colonies.

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Plate Incorporation)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

130

27

8

31

13

116 (127)

9 (18)

10 (9)

20 (26)

13 (14)

134

18

8

28

15

 

Experiment 2 (Pre-Incubation)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

76

18

35

23

14

96 (85)

19 (15)

41 (31)

13 (21)

8 (10)

82

9

31

26

9

 

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Negatives results were obtained in three different in vitro genotoxicity tests.