Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item, Macrolex Orange R was considered to be non-clastogenic and non-aneugenic to human lymphocytes in an in vitro mammalian cell Micronucleus Test and non-mutagenic in a bacterial reverse mutation test and a HPRT Test in V79 cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Macrolex Orange R Article Number 00204692
batch Number 976-009
Orange powder

Target gene:

His

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

up to 5000 µg per plate; highest dose according to guideline

Vehicle / solvent:

Macrolex Orange R was suspended in DMSO and formed an orange suspension.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: Nitrofurantoin, 4-nitro-1,2-phenylene diamine, Cumene hydroperoxide, 2-aminoanthracene,

Remarks:

The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Rationale for test conditions:

At 158 μg per plate, the substance started to precipitate.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about
twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise,
the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly
with modifications, until a final evaluation is possible.

Statistics:

not applied

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

MACROLEX Orange R was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37° C. Other conditions remained unchanged.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 158 µg per plate and above.

Relevant evidence of mutagenic activity of MACROLEX Orange R was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine, cumene hydroperoxide and 2-amino­anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, MACROLEX Orange R was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/ microsome test.

Conclusions:

MACROLEX Orange R was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella microsome test.

Executive summary:

MACROLEX Orange R was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37° C. Other conditions remained unchanged.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 158 µg per plate and above.

Relevant evidence of mutagenic activity of MACROLEX Orange R was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine, cumene hydroperoxide and 2-amino­anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, MACROLEX Orange R was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella microsome test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 09 November 2017 Experimental completion date 13 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian forward mutation assay

Specific details on test material used for the study:

Appearance: Solid powder, red
Storage Conditions: At room temperature
Expiry Date: 18 February 2018
Stability in Solvent: Stable in DMSO over 4 and 24 hours at room temperature in the light at concentrations of 0.1 mg/mL and 200 mg/mL based on Envigo Study no. HM74TS.

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×106 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system.

Test concentrations with justification for top dose:

Pre-experiment toxicity test
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 15.8 μg/mL and 2024.0 μg/mL were used. The highest concentration was chosen with respect to the OECD guideline 476 regarding the purity of the test item (98.8%).
In the pre-experiment no relevant toxic effects were observed after 4 hours in the presence and absence of metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 63.3 μg/mL and above with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item

Main experiment:
The concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment. The individual concentrations were spaced by a factor of 2.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.
4 hour exposure period with and without S9 mix - 1.0, 2.0, 4.1, 8.1, 16.3, 32.5, 65.0 and 130.0 µg/ml
The cultures at the three highest concentrations without metabolic activation and at the highest concentration with metabolic activation were not continued to avoid analysis of too many concentrations showing precipitation. The cultures at the two lowest concentrations with metabolic activation were not continued as a minimum of only four concentrations is required by the guidelines.

Vehicle / solvent:

The vehicle was DMSO. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Final concentration: 300 μg/mL = 2.4 mM

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Final concentration: 2.3 μg/mL = 8.9 μM

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Pre-Test on Toxicity
A pre-test was performed in order to determine the toxicity of the test item. In addition the pH and osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

Experimental Performance

Seeding
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline.
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0×10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item Macrolex Orange R was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.

The main experiment was evaluated at the following concentrations:
without S9 mix: 1.0; 2.0; 4.1; 8.1; and 16.3 μg/mL
with S9 mix: 4.1; 8.1; 16.3; 32.5; and 65.0 μg/mL

Precipitation visible at the end of treatment was noted at 16.3 μg/mL without metabolic activation, and at 65.0 μg/mL with metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation.
No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentrations scored for gene mutations.
The mean mutant frequency obtained in the solvent controls was 25.4 mutants per 10^6 cells with and without S9 mix. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mean mutant frequencies of the groups treated with the test item was from 11.1 up to 25.5 mutants per 10^6 cells.
The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Macrolex Orange R is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Macrolex Orange R to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The main experiment was performed with a treatment period of 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (2024 μg/mL) was chosen with respect to the OECD guideline 476 regarding the purity of the test item.

No relevant increase in mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Macrolex Orange R is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 09 May 2017 Experimental completion date: 24 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Macrolex Orange R
Physical state/Appearance: Red powder
Chemical Name: 1H-Indole-5-carboxylic acid, 2-[2-(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-2,3-dihydro-1,3,3-trimethyl-, methyl ester
Expiry Date: 18 February 2018
Storage Conditions: Room temperature in the dark

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: whole blood

Details on mammalian cell type (if applicable):

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: female, aged 26 years
Main Experiment: male, aged 29 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).

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test:
All exposure groups: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL.

Main experiment:
4-hour exposure group (-S9): 0, 0.5, 1, 2, 4, 8, 16 and 32 µg/mL
4-hour exposure group (+S9): 0, 1, 2, 4, 8, 16, 32 and 64 µg/mL
24-hour exposure group (-S9): 0, 0.5, 1, 2, 4, 8, 16 and 32 µg/mL

Vehicle / solvent:

DMSO was used as the vehicle because the test item was suspendable in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/mL for 4-hour exposure

Positive control substance:

mitomycin C

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.075 µg/mL for 24-hour continuous exposure

Positive control substance:

other: Demecolcine

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

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 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 dose range of test item used was 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 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, 0.5, 1, 2, 4, 8, 16 and 32 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, 1, 2, 4, 8, 16, 32 and 64 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, 0.5, 1, 2, 4, 8, 16 and 32 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.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.

A test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.

In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions (i.e. S9 concentration or S9 origin)) could be useful.

Statistics:

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.

Species / strain:

primary culture, other: whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/mL. The maximum dose was the maximum achievable dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 15.63 µg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 62.5 µg/mL in the 4-hour exposure group in the presence of S9-mix. Precipitate also persisted after exposure on the slide at 1000 µg/mL in the 24-hour continuous exposure group only.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 1000 µg/mL in the all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for all exposure groups.


Micronucleus Test – Main Experiment
The dose levels of the controls and the test item are given in the table in section 'any other information on results'.
The qualitative assessment of the slides determined that the precipitate was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item in all three exposure groups.
Precipitate of test item was noted in the parallel blood-free cultures at the end of the exposure at 32 µg/mL in the 4-hour exposure group without S9, at 64 µg/mL in the 4-hour exposure group with S9 and at and above 8 µg/mL in the 24-hour continuous exposure group. Therefore, these dose levels were chosen as the maximum concentration selected for micronucleus frequency analysis because they were the lowest precipitating dose levels.

They confirm the qualitative observations in that no dose related inhibition of CBPI was observed.
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 binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

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

Group	Final concentration of test itemMacrolex Orange R(µg/mL)
4-hour without S9	0*, 0.5, 1, 2, 4, 8*, 16*, 32*, MMC0.2*
4-hour with S9 (2%)	0*, 1, 2, 4, 8, 16*, 32*, 64*, CP5*
24-hour without S9	0*, 0.5, 1, 2*, 4*, 8*, 16, 32,DC0.075*

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

MMC = Mitomycin C

CP = Cyclophosphamide

DC = Demecolcine

Conclusions:

The test item, Macrolex Orange R, 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:

Introduction

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 on precipitate. The dose levels selected for the Main Test were as follows:

Group	Final concentration of test itemMacrolex Orange R(µg/mL)
4-hour without S9	0, 0.5, 1, 2, 4, 8, 16, 32
4-hour with S9 (2%)	0, 1, 2, 4, 8, 16, 32, 64
24-hour without S9	0, 0.5, 1, 2, 4, 8, 16, 32

Formulation analysis was carried out in the main experiment to determine the concentration of the test item concentration (maximum dose).

Results

All vehicle (dimethyl sulphoxide (DMSO)) 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 non-toxic to human lymphocytes 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 was the lowest precipitating dose level.

The dose formulation analysis demonstrated accurate formulation of the test item at 6.4 mg/mL.

 

Conclusion

The test item, Macrolex Orange R was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

no study available

Justification for classification or non-classification

The test item, Macrolex Orange R was considered to be non-clastogenic and non-aneugenic to human lymphocytes in an in vitro mammalian cell Micronucleus Test and non-mutagenic in a bacterial reverse mutation test and a HPRT Test in V79 cells. No in vivo data are available. Classification is not warrated.