3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The 2 following tests have been performed with the following results:

• OECD Guideline 471 (Bacterial Reverse Mutation Assay): non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay
• OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test): clastogenic in this chromosome aberration test after continuous treatment in the absence of S9 mix.

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:

January 2002: 08-14

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

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Target gene:

S. typhimurium TA1537: hisC3076; rfa-; uvrB-;
S. typhimurium TA 98: hisD3052; rfa-; uvrB-; R-factor
S. typhimurium TA1535: hisG46; rfa-; uvrB-;
S. typhimurium TA100: hisG46; rfa-; uvrB-; R-factor;
E. coli WP2 uvrA: trp-; uvrA-

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB deficient

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA deficient

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal activation (S9 mix)

Test concentrations with justification for top dose:

Experiment I - TA98, TA 1535, TA 1537, TA 100, WP2 uvrA at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment II - TA 1535, TA 1537, TA 100, WP2 uvrA at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment II - TA 98 at 33, 100, 333, 1000, 2500 and 5000 µg/plate with S9 mix
Experiment II - TA 98 at 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: the test item BLUE GS 5664.80 was dissolved in DMSO ((MERCK, D-64293 Darmstadt; purity > 99 %) and neutralised with 1N HCl.
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed.

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment ll was performed as a pre-incubation assay

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37' C in the dark.

NUMBER OF REPLICATIONS: Each concentration and the controls were tested in triplicate.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment l, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Statistics:

No statistical evaluation of the data is required

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia colistrain WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

This study was performed to investigate the potential of BLUE GS 5664.80 to induce gene mutations according to the plate incorporation test (experiment l) and the pre-incubation test (experiment ll) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia colistrain WP2 uvrA following OECD TG 471 according to GLP.
It can be stated that during the mutagenicity test and under the experimental conditions reported, the test ítem did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, BLUE GS 5664.80 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of BLUE GS 5664.80 to induce gene mutations according to the plate incorporation test (experiment l) and the pre-incubation test (experiment ll) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia colistrain WP2 uvrA following OECD TG 471 according to GLP.

The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I - all strains at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix

Experiment II - TA 1535, TA 1537, TA 100, WP2 uvrA at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix

Experiment II - TA 98 at 33, 100, 333, 1000, 2500 and 5000 µg/plate with S9 mix

Experiment II - TA 98 at 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate without S9 mix

The plates incubated with the test item showed normal background growth up to 1000 µg/plate with and without metabolic activation in both independent experiments. Due to the intense colour of the test item the background growth could not be assesed in the concentration range of 2500 and 5000 µg/plate.

Slight toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 98 without 59 mix at 2500 and 5000 µg/plate in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with BLUE GS 5664.80 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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

ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test ítem did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, BLUE GS 5664.80 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001, August 30 to 2002 April, 02

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:

February 1998, adopted July 21, 1997

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4A

Version / remarks:

dated May 19, 2000

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of standardized characteristics of the cells.
Thawed stock cultures were propagated at 37°C in 80 cm2 plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 10E5 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 4.5 % carbon dioxide (95.5 % air).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Main experiment I : 3000 µg/ml with and without S9 mix
Main experiment II: 1500 µg/ml (18h interval) & 1000 µg/ml (28h interval) without S9 mix; 3000 µg/ml with S9 mix

Dose selection was performed in a pre-test in accordance to the current OECD Guideline for in vitro mammalian cytogenetic tests. With respect to the ability to formulate a homogeneous suspension of BLUE GS 5664.80, 4000 µg/ml was chosen as top concentration for treatment of the cultures. However, at the end of the treatment procedure it was observed that the test item was not totally suspended in the stock solution. Therefore the concentrations reported for the pre-experiment are not exactly the treatment concentrations. Test item concentrations between 31.3 and 4000 µg/ml (with and without S9 mix) were applied for the evaluation of cytotoxicity. Precipitation of the test item after 4 hrs treatment was observed at 1000 µg/ml and above in the absence of S9 mix and at 500 µg/ml and abovè In the presence of S9 mix.
Using reduced cell numbers as an indicator for toxicity in the pre-test, no toxic effects were observed after 4 hrs treatment up to the highest test item concentration in the absence of S9 mix. ln addition, 4 hrs treatment with 4000 µg/ml in the presence of S9 mix induced strong toxic effects. Considering the ability to formulate a homogeneous suspension and the toxicity data of the pre-test, 3000 µg/ml (with and without S9 mix) were chosen as top concentrations in the main experiment 1.
Dose selection of experiment Il was aise influenced by test item toxicity. ln the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with 1000 µg/ml and above. Therefore, 1500 µg/ml (18 hrs interval) and 1000 µg/ml (28 hrs interval) were chosen as top treatment concentrations for continuous exposure in the absence of S9 mix. ln the presence of S9 mix 3000 µg/ml were chosen as top treatment concentration with respect to the results obtained in experiment 1.

Vehicle / solvent:

- Solvent used: water
- Justification for choice of solvent: On the day of the experiment (immediately before treatment), the test item was formulated in deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v). The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Seeding of the Cultures

Exponentially growing stock cultures more than 50 % confluent were treated with trypsin at 37 °C for approximately 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free sait solution (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free sait solution was composed as follows (per litre): NaCI (8000 mg); KCI (200 mg); KH2P04 (200 mg); NaHP04 (150 mg)
Proer to the trypsin treatment the cells were rinsed with Ca-Mg-free sait solution containing 200 mg/l EDTA (Ethylene diamine tetraacetic acid).
The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopie slides (at least 2 chambers per dish and test group). ln each chamber 1 x 10E4 - 6 x 10E4 cells were seeded with regard to preparation time. The medium was MEM with 10 % FCS (complete medium).

Treatment

Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) ,or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µI S9 mix per ml culture medium were added. Concurrent negative, solvent, and positive contrais were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution was composed as follows (per litre): NaCI (8000 mg); KCI (400 mg); Glucose (1100 mg); Na2 HP04x7H20 (290 mg); KH2P04 (150 mg). pH was adjusted to 7.2

Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
All cultures were incubated at 37° C in a humidified atmosphere with 4.5 % C0 2 (95.5 % air).

Preparation of the Cultures
15.5 hrs and 25.5 hrs, respectively after the start of the treatment Colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonie solution (0.4 % KCI) for 20 min at 37° C. After incubation in the hypotonie solution the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts + 1 part). Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
Additionally, two cultures per test item and solvent control group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control cultures in experiment I without S9 mix (50 metaphase plates scored). Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. ln addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Acceptability of the Test
The chromosome aberration test performed in our laboratory is considered acceptable if it meets the following criteria:
a)The number of structural aberrations found in the negative and/or solvant controls falls within the range of our historical laboratory control data: 0.0 % - 4.0 %.
b) The positive contrai substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data:
Range of 9.0-39.0 with EMS (600-1000 µg/ml) without S9 mix
Range of 7.5-49.5 with CPA (0.47-0.93 µg/ml) with S9 mix

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations are not in the range of our historical control data (0.0 % - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test.
Ref: Richardson, C., et al (1989). Analysis of data from in vitro cytogenetic tests. Kirkland, D.J. (ed.). "Statistical evaluation of mutagenicity test data", Cambridge University Press, Cambridge, 141-154

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment I (Preparation interval of 18h; exposure period of 4h)

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:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II (Preparation interval of 18h and 28h; exposure period of 18h and 28h)

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

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II (Preparation interval of 28h; exposure period of 4h)

Metabolic activation:

with

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:

Range finding pre-test on toxicity
ln the range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Due to the reduced solubility of the test item in the solvent used the concentrations reported for the pre-experiment are not exactly the treatment concentrations (see Dose Selection, page 18). However, concentrations between 31.3 and 4000 µg/ml were applied. Clear taxie effects were observed after 4 hrs treatment with 4000 µg/ml in the presence of S9 mix. ln addition, 24 hrs continuous treatment with 1000 µg/ml and above in the absence of S9 mix induced strong toxic effects.
Precipitation of the test item in culture medium was observed after 4 hrs treatment with 1000 µg/ml and above in the absence of S9 mix and with 500 µg/ml and above in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvant control 283 mOsm, pH 7.2 versus 302 mOsm and pH 7.3 at 4000 µg/ml).

Main experiments
ln the main experiments, precipitation of the test item 4 hrs after start of treatment was observed with 1500 µg/ml and above after 4 hrs treatment in the absence of S9 mix (exp. I) and with 750 µg/ml and above after 4 hrs treatment in the presence of S9 mix (exp. I & Il).
ln all experimental parts clear toxic effects indicated by reduced cell numbers were observed after treatment with the test item, except after 18 hrs continuous treatment in the absence of S9 mix. ln detail, in the absence of S9 mix strangly reduced cell numbers below 50 % of contrai were observed in experiment I after 4 hrs treatment with 3000 µg/ml (35 % of control) and in experiment Il after 28 hrs continuous treatment with 500 µg/ml (34 % of control). ln addition, in the presence of S9 mix the cell numbers were clearly reduced after 4 hrs treatment with 3000 µg/ml (43 % of contrai) at the 18 hrs interval and with 2250 µg/ml (25 % of contrai) at the 28 hrs interval. However, no clear cytotoxicity indicated by reduced mitotic indices below 50 % of contrai were observed in any of the concentrations evaluated.
ln both experiments, after 4 hrs treatment in the absence and the presence of metabolic activation no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (1.0 % - 4.0 % aberrant cells, exclusive gaps) were near the range of the solvent control values (1.5 % - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 % - 4.0 % aberrant cells, exclusive gaps.
ln experiment Il, after continuous treatment in the absence of S9 mix the aberration rates were statistically significant and biologically relevant increased with 500 and 750 µg/ml (4.5 % and 10.5 % aberrant cells, exclusive gaps, respectively) at the 18 hrs preparation interval, and with 500 µg/ml (26.5 % aberrant cells, exclusive gaps) at the 28 hrs preparation interval as compared to the corresponding solvent contrais (0.5 % and 1.5 % aberrant cells, exclusive gaps, respectively) and historical control data: 0.0 % - 4.0 % aberrant cells, exclusive gaps. Additionally, there was a dose related increase of the aberration rates within the concentration range evaluated (250 to 750 µg/ml) after 18 hrs continuous treatment. These findings give evidence for a clastogenic potential of the test item.
ln both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.0 % - 5.1 %) as compared to the rates of the solvent controls (1.6 % - 3.0 %).
ln both experiments, EMS (100 and 200 µg/ml, respectively) and CPA (0.7 and 1 µg/ml, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

BLUE GS 5664.80 is considered to be clastogenic in this chromosome aberration test after continuous treatment in the absence of S9 mix.

Executive summary:

The test item BLUE GS 5664.80, formulated as a homogeneous suspension in deionised water in the cytogenetic experiments, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments.

In experiment 1,the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp.II) after start of treatment with the test item.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in experiment I,with and without metabolic activation, where only 50 metaphase plates were scored.

The highest applied concentration in the pre-test on toxicity (4000 µg/ml) was chosenwith respect to the current OECD Guideline 473 and with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent (deionised water). Due to inhomogenicity in the test item stock solution in the pre-experiment the highest applicableconcentrationforthecytogeneticexperimentswassetupto3000µg/ml.

Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

Toxic effects indicated by reduced cell numbers below 50 % of control were observed in ail experimental parts, except in the absence of S9 mix in experimentIlat interval 18 hrs.

In experimentIl,in the absence of S9 mix statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after 18 hrs and 28 hrs continuous treatment with the test item.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.05) in cells with structural chromosome aberrations (see page 34).

ln conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line)in vitro.

Therefore, BLUE GS 5664.80 is considered to be clastogenic in this chromosome aberration test after continuous treatment in the absence of S9 mix.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The following has been performed with the following results:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test): no induction of micronuclei with bone marrow cells of the mouse

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002 April, 26 to 2002 August, 09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 21, 1997

GLP compliance:

yes

Type of assay:

other: in vivo mammalian somatic cell / structural and numerical chromosome aberration

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 35.0 g (SD ± 3.1 g); females mean value 26.9 g (SD ± 2.3 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single. Cage type: Makrolon Type 1, with wire mesh top (EHRET GmbH, D-79302 Emmendingen). Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Rof1dorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 40-50
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: corn oil
- Justification for choice of solvent/vehicle:
- Amount of vehicle: 10 ml/kg b.w.

Details on exposure:

Pre-experiment for toxicity
A preliminary study on acute toxicity was performed with two animais per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animais were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Dose Selection
lt is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

Treatment
Approximately 18 hours before treatment the animais received no food but water ad libitum. At the beginning of the treatment the animais (including the contrais) were weighed and the individual volume to be administered was adjusted to the animais body weight. The animais received the test item, the vehicle or the positive contrai substance once. Twelve animais, six males and six females, were treated per dose group and sampling time. The animais of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test item.

Duration of treatment / exposure:

Acute exposure

Frequency of treatment:

1 treatment/animal

Post exposure period:

24 and 48h

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

24h preparation interval

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

24h preparation interval

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24h and 48h preparation intervals

No. of animals per sex per dose:

10 (5 males and 5 females)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPA)
- Justification for choice of positive control: The stability of CPA at room temperature is sufficient. At 25°C only 3.5 % of its potency is lost after 24 hours.
- Route of administration: oral
- Doses / concentrations: 10 ml/kg b.w. & 40 mg/kg b.w.

Tissues and cell types examined:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Details of tissue and slide preparation:

Preparation of the Animais:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were eut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Caver slips were mounted with EUKITI (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides.
Ten animais (5 males, 5 females) per test group were evaluated as described.

Data Recording
The data generated are recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups, vehicle, and positive contrai. The micro­ nucleated cells per 2000 PCEs and the ratio of polychromatic to normochromatic erythrocytes are presented for each animal.

Evaluation criteria:

Acceptance criteria
The study was considered valid as the following criteria are met:
a) The negative contrais are in the range of our historical contrai data (0.02 - 0.15 %; mean = 0.086 ± 0.032 PCEs with micronuclei).
b) The positive contrais are in the range of our historical contrai data (1.0 - 2.71 %; mean = 1.678 ± 0.479 PCEs with micronuclei).
c) At least 80 % of animais are evaluable

Evaluation of results
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test; Krauth J, 1971) will be used as an aid in evaluating the results. However, the primary point of consideratrion is the biological relevance of the results.

Ref.: Krauth, J. (1971). Locally most powerful tied rank test in a Wilcoxon situation Annals of Mathematical Statistics, 42, 1949 - 1956

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

toxic reactions to mouse at dose of 2000 mg/kg b.w. (but no cytotoxic to bone marrow)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg b.w. (maximum guideline-recommended dose)
- Clinical signs of toxicity in test animals: yes

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): significant with positive control; not significant with test item
- Ratio of PCE/NCE (for Micronucleus assay): 2000/1590 for vehicle; 2000/1646 for test item (500 mg/kg b.w.; 24h sampling); 2000/1809 for test item (1000 mg/kg b.w.; 24h sampling); 2000/1809 for test item (1000 mg/kg b.w.; 24h sampling); 2000/1786 for Positive control (40 mg/kg b.w.; 24h sampling); 2000/1873 for test item (2000 mg/kg b.w.; 48h sampling);
- Appropriateness of dose levels and route: yes
- Statistical evaluation: Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Not significant with P = 0.1494 for test item at 2000 mg/kg bw; 24h
Not significant with P = 0.4133 for test item at 2000 mg/kg bw; 48h
Significant with P<0.0001 for positive control at 40 mg/kg bw; 24h

Pre-Experiment forToxicity

ln a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. BLUE GS 5664.80 formulated in corn oil. The volume administered was 10 ml/kg b.w.

The animais treated with 2000 mg/kg b.w. expressed toxic reactions as shown in the table:

 

toxicreactions	hours post-treatment male / female
	1 h	2-4 h	6h	24 h	30 h	48 h
reduction of spontaneous activity	2/2	2/2	1/1	0/0	0/0	0/0
eyelid closure	1/1	1/1	0/0	0/0	0/0	0/0
ruffled fur	2/2	2/2	1/1	0/0	0/0	0/0

 

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

 

Toxic symptoms in the Main Experiment

ln the main experiment for the highest dose group 24 animais (12 males, 12 females) received orally a single dose of 2000 mg /kg b.w. BLUE GS 5664.80 formulated in corn oil. The volume administered was 10 ml/kg b.w.

The animais treated with 2000 mg /kg b.w. expressed toxic reactions as shown in the table:

 

toxicreactions	hours post-treatment male / female
	1 h	2-4 h	6h	24 h
reduction of spontaneous activity	12/12	12/12	12/12	12/12
abdominal position	8/8	8/7	9/8	4/4
eyelid closure	8/8	8/8	8/7	3/3
ruffled fur	8/7	8/6	7/4	4/1
apathy	8/9	8/7	5/3	3/2

 

Summary of Micronucleus Test Results

 

 

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE/	NCE
vehicle	0	24	0.055	0-3	2000/	1590
test item	500	24	0.050	0-2	2000/	1646
test item	1000	24	0.045	0-4	2000/	1809
test item	2000	24	0.090	0-4	2000/	1676
cycle-phosphamide	40	24	1.235	9-54	2000/	1786
test item	2000	48	0.065	0-3	2000/	1873

 

Statistical significance

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle control versus test group	Significance	p
500 mg BLUE GS 5664.80/kg b.w.; 24 h	n.t.	-
1000 mg BLUE GS 5664.80/kg b.w.; 24 h	n.t.	-
2000 mg BLUE GS 5664.80/kg b.w.; 24 h	-	0.1494
40 mg CPA/kg b.w.; 24 h	+	<0.0001
2000 mg BLUE GS 5664.80/kg b.w.; 48 h	-	0.4133

n.t. = not tested as the mean micronucleus frequency was not above the vehicle contrai value

- = not significant

+ = significant

Conclusions:

ln conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, BLUE GS 5664.80 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of BLUE GS 5664.80 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse in a test following OECD 474 and GLP.

The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Ten animais (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

 To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs.

The following dose levels of the test item were investigated:

• 24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
• 48 h preparation interval: 2000 mg/kg b.w. The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle contrai thus indicating that BLUE GS 5664.80 did not exert any cytotoxic effects in the bone marrow.

ln comparison to the corresponding vehicle contrais there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

ln conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, BLUE GS 5664.80 is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

As specified in line 10 of table R.7.7 -5 of ECHA guidance R7a (v6.0 -July 2017), with a negative result in gene mutation test in bacteria (OECD 471), a positive result in cytogenicity assay in mammalian cells (OECD 473) and a negative result in cytogenicity assay in experimental animals (OECD 474), the test item is identified as not genotoxic and no further test are required according to Annexes VII to X of the REACH Regulation (EC) No 1907/2006.