Reaction mass of (disodium 4-[[4-(acetylmethylamino)-2-sulphonatophenyl]amino]1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium sulphate and sodium chloride)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The bacterial mutagenicity assay (Ames) is positive, but in neither the mammalian cell HGPRT assay nor in a chromosomal aberration assay mutagenic activity was detected up to the limit dose.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP regulation; full report available

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat Liver S-9

Test concentrations with justification for top dose:

0 - 312,5 - 625 - 1250 - 2500 - 5000 µg/plate

Vehicle / solvent:

water

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-anthramine (2AM)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 60 min.
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

Evaluation criteria:

Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle), are presented in a table.

Acceptance criteria
This study would be considered valid since the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the range of our historical data (appendix 2),
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the range of our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: 100g/L
- Precipitation: No

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxicity was noted towards all the strains used, both with and without S9 mix.

Remarks on result:

other: strain/cell type: TA 100

Remarks:

Migrated from field 'Test system'.

First experiment with S9

Strain	Dose (µg/plate)	Revertants / Plate (Mean)	Standard eviation	Ratio
TA 100	0	95	16	1,00
	312,5	92	5	0,97
	625	111	11	1,17
	1250	112	8	1,18
	2500	105	9	1,11
	5000	101	22	1,07
	2AM	2286	94	24,06
TA102	0	343	33	1,00
	312,5	352	13	1,03
	625	351	19	1,02
	1250	348	8	1,02
	2500	359	25	1,05
	5000	338	35	0,99
	2AM	2139	443	6,24
TA 98	0	39	8	1,00
	312,5	47	2	1,21
	625	45	2	1,15
	1250	39	4	0,99
	2500	46	5	1,17
	5000	54	3	1,39
	2AM	737	57	18,9
TA 1535	0	18	6	1,00
	312,5	21	5	1,21
	625	26	2	1,45
	1250	28	10	1,58
	2500	19	3	1,06
	5000	19	7	1,09
	2AM	278	65	15,72
TA 1537	0	19	8	1,00
	312,5	24	4	1,26
	625	19	5	0,98
	1250	15	5	0,79
	2500	24	5	1,22
	5000	21	5	1,07
	2AM	95	11	4,90

 

Second experiment with S9

Strain	Dose (µg/plate)	Revertants / Plate (Mean)	Standard Deviation	Ratio
TA 100	0	94	10	1,00
	312,5	144	9	1,54
	625	178	24	1,90
	1250	200	15	2,13
	2500	271	32	2,90
	5000	168	4	1,79
	2AM	1153	32	12,31
TA102	0	365	27	1,00
	312,5	423	19	1,16
	625	403	5	1,10
	1250	403	16	1,10
	2500	397	9	1,09
	5000	374	10	1,02
	2AM	2670	123	7,31
TA 98	0	39	4	1,00
	312,5	49	8	1,26
	625	54	6	1,38
	1250	57	5	1,45
	2500	56	9	1,44
	5000	44	13	1,13
	2AM	1098	240	28,16
TA 1535	0	20	4	1,00
	500	20	3	0,98
	1000	19	5	0,95
	2000	17	1	0,85
	2500	21	3	1,02
	5000	18	2	0,90
	2AM	214	9	10,54
TA 1537	0	16	2	1,00
	312,5	17	3	1,02
	625	20	8	1,24
	1250	16	1	1,00
	2500	14	6	0,86
	5000	10	2	0,63
	2AM	82	15	5,02

Third Experiment with S9

Strain	Dose (µg/plate)	Revertants / Plate (Mean)	Standard Deviation	Ratio
TA 100	0	146	8	1,00
	625	244	7	1,67
	1250	305	11	2,09
	2500	341	6	2,34
	3000	341	32	2,34
	5000	309	46	2,12
	2AM	1117	144	7,65

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation only in strain TA 100 after preincubation

The test item showed mutagenic activity in a bacterial reverse mutation test on Salmonella typhimuriumTA 100 strain, with metabolic activation and preincubation method.

Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce reverse mutation in Salmonella typhimurium.

The test item was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

A third experiment was performed with S9 mix.

The first experiment was performed according to the direct plate incorporation method. The second and the third with S9 mix were performed according to the preincubation method (60 minutes, 37C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used.

Each strain was exposed to five dose-levels of the test substance (three plates/dose-level).

After 48 to 72 hours of incubation at 37C, the revertant colonies were scored.

The test substance was dissolved in distilled water.

Results

The test substance was freely soluble in the vehicle at 54.348 mg/ml, corresponding to 50 mg of the active material/ml (purity of the commercial product: 92%). Since the test substance was freely soluble and non-toxic in the preliminary toxicity test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the guidelines.

The selected treatment-levels for the mutagenicity experiments were: 312.5, 625, 1250, 2500 and 5000 µg/plate, for all strains in the first and second experiments, except for the TA 1535 strain in the second experiment with S9 mix where the treatment-levels were: 500, 1000,2000,2500 and 5000 µg/plate.

In the third experiment, the dose-levels were: 625, 1250, 2500, 3000 and 5000 µg/plate.

Slight to strong blue coloration was observed in the Petri plates when scoring the revertants at dose-levels. This coloration did not impede the automatic counting of the bacterial colonies. No precipitate was observed at any dose-level.

No relevant toxicity was noted towards all the strains used, both with and without S9 mix.

The test substance did not induce any significant increase in the number of revertants, with S9 mix and without S9 mix, in any of the five strains using the direct plate incorporation method.

With S9 mix and the preincubation method, a significant increase in the number of revertants, in comparison to the vehicle control, was noted in the TA 100 strain, at the dose-levels of 1250 and 2500 µg/plate. In addition, a dose-related increase was observed in this strain, at all dose-levels tested, except at the highest dose-level of 5000 µg/plate where a slight toxicity could have masked the increase in the number of revertants.

A third experiment was performed under the same conditions, in order to check the reproducibility of the significant increase observed. In this third experiment, the significant induction of the number of revertants was confirmed.

The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria.

The study was therefore considered as valid.

Conclusion

Under our experimental conditions, the test substance showed mutagenic activity in this bacterial reverse mutation test on Salmonella typhimuriumTA 100 strain, with metabolic activation and preincubation method.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

2000

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: very poor sample characterisation

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (Aroclor)

Test concentrations with justification for top dose:

0 - 1250 - 2500 - 5000 µg/ml

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h & 20 h
- Expression time (cells in growth medium): 24 h
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method:relative total growth (viability by trypan blue exclusion)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication:Yes

Evaluation criteria:

The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell was calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p=0.05). Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative. Negative results with metabolic activation may need to be confirmed on a case-by-case basis. In those cases where continuation of negative results is not necessary, justification will be provided.

Criteria for a Valid Test
The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p =0.05, Fisher's exact test) relative to the solvent control

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Full report according to Guideline and GLP regulations

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:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 30.0; 100.0*; 300.0, 1000.0, 3000.0 and 5000.0 µg/ml
with S9 mix: 30.0; 100.0*; 300.0, 1000.0, 3000.0 and 5000.0 µg/m
Experiment II:
without S9 mix: 30.0*; 300.0; 1000.0, 3000.0 and 5000.0 µg/m
with S9 mix: 30.0*; 300.0; 1000.0, 3000.0 and 5000.0 µg/ml
* not evaluated, culture not continued;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none /medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Seeding
Exponentially growing stock cultures (more than 50% confluent) were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2% in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free salt solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400mg
Glucose 1000 mg
NaHC03 350mg

Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/1 EDTA (ethylenediamine tetraacetic acid).
The cell suspension was seeded into plastic culture flasks (Greiner, D-72632 Frickenhausen).
Approximately 1. 5 x 1E06 (single culture) and 5 x 1E02 cells (in duplicate) were seeded in MEM with 10% FCS (complete medium) for the determination of mutation rate and toxicity, respectively (see experimental scheme).

Treatment
After 24 h the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 µl/ml S9 mix. After 4 h this medium was replaced with complete medium following two washing steps with "saline G" .
The Saline G solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400mg
Glucose 1100 mg
Na2HP04x1H20 290mg
KH2P04 150mg

the pH was adjusted to 7.2.

Experimental Scheme:
Segment a): Procedure for determination of toxicity
Segment b): Procedure for determination ofmutation rates
Day 1: Subculturing of a log-phase culture which showed an initial spontaneous mutation rate at the beginning of the experiment of 1.7 (experiment I) and 6.6 (experiment II) mutants per 10E6 cells.
a) About 500 cells in 5 ml medium/25 cm2 -plastic-flask for cloning efficiency; in duplicate per experimental point
b) About 1.5xl06 cells in 30 ml medium/175 cm2-plastic-flask for the mutagenicity test, 1 flask per experimental point

Day 2: Treatment of a) and b)
experiment I
Day 5: Subculturing ofb) in 175 cm2-plastic-flasks 1.5x106 cells in 30 ml medium/175 cm2 -plastic-flasks

experiment II
Day 6: see day 5
Day 8: Fixation and staining of colonies in a)-flasks determination of concentration-related cloning efficiency
Day 9: Subculturing ofb) in five 80 cm2-plastic-flasks containing selective medium:
mutant selection (about 3-5x105 cells/flask);
subculturing ofb) in two 25 cm2-flasks for cloning efficiency (about 500 cells/flask)
Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (cloning efficiency).
Day 1 7: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (mutant selection).

The cultures were incubated at 37° C in a humidified atmosphere with 4.5 % C02. The colonies were stained with 10% methylene blue in 0.01 % KOH solution (E. MERCK, D-64293 Darmstadt, F.R.G.).
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope (Nikon, D-40407 Dusseldorf, F.R.G.).

Evaluation criteria:

Acceptability of the Assay

The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 106 cells found in the negative and/or solvent controls fall within the laboratory historical control data range: 1 - 32 mutants/10E6 cells.
b) the positive control substances must produce a significant increase in mutant colony frequencies.
c) the cloning efficiency (absolute value) of the negative and/or solvent controls must exceed s0 %.

The data of this study comply with the above mentioned criteria


Evaluation of Results

A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test article producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not obsenred.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (1 - 32 mutants per 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce gene mutations in the HPRT in V79 cells in vitro with and without metabolic activation.

Executive summary:

The test article was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments using identical procedures, both with and without liver microsomal activation.

The test article was tested with the following concentrations:

Experiment I:

without S9 mix: 30.0; 100.0*; 300.0, 1000.0, 3000.0 and 5000.0 µg/mL

with S9 mix: 30.0; 100.0*; 300.0, 1000.0, 3000.0 and 5000.0 µg/mL

Experiment II:

without S9 mix: 30.0*; 300.0; 1000.0, 3000.0 and 5000.0 µg/mL

with S9 mix: 30.0*; 300.0; 1000.0, 3000.0 and 5000.0 µg/mL

* not evaluated, culture not continued;

 

Up to the highest test concentration no visible precipitation occurred.

 

Strong toxic effects, evident as a reduction in the cloning efficiency of the cells occurred in both experiments at 5000 µg/mL without metabolic activation. In the presence of metabolic activation only slight toxic effects were observed at the maximal concentration. Up to the highest investigated concentration no relevant and reproducible increase in mutant colony numbers was observed, neither in the presence nor in the absence of metabolic activation.

In the first experiment without metabolic activation unusually low numbers of spontaneous mutant colonies occurred in the solvent control due to statistical fluctuations at such small numbers. This effects results in a colony count above 3 times the number of mutant colonies of the corresponding solvent control at almost all concentrations throughout the experiment. This increase was judged to be biologically irrelevant since it is based upon the unusually low colony count of the solvent control. Furthermore, the absolute numbers of colonies are low at all concentrations of the test article and remain well within the range of the solvent controls of the experiments.

In this study in both experiments (with and without S9 mix) the range of the solvent controls was from 1.3 up to 11.8 mutants per 10E6 cells; the range of the groups treated with the test article was from 2.1up to 17.5 mutants per l0E6 cells.

EMS (0.6 mg/ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

 

In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo micronucleus assay no mutagenic activity was detected up to the limit dose.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

in polychromatic erythrocytes in the bone marrow

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP regulation

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

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; Switzerland
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 40.0 g (SD ± 3.4 g); females mean value 31.5 g (SD± 1.6 g)
- Housing: Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 24 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water is a known inocciousstandard vehicle
- Concentration of test material in vehicle: 50 to 200 mg/ mL
- Amount of vehicle: 10 ml/kg b.w

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

single

Post exposure period:

none

Remarks:

Doses / Concentrations:
500 mg/mL
Basis:
other: actual injected

Remarks:

Doses / Concentrations:
1000 mg /mL
Basis:
other: actual injected

Remarks:

Doses / Concentrations:
2000 mg/ mL
Basis:
other: actual injected

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): usual standard
- Route of administration: ip
- Doses / concentrations: 40 mg/kg bw.

Tissues and cell types examined:

bone marrow, polychromatic arythrocytes

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut 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-Grunwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover 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. 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 the PCEs. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.03 - 0.15 %; mean = 0.086 ± 0.027 PCEs with micronuclei).
- the positive controls are in the range of our historical control data (1.0 - 2.71 %; mean = 1.653 ± 0.409 PCEs with micronuclei).
- at least 80 % of animals are evaluable

A test item is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test item producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 2000 mg/kg: reduction of spontaneous activity, apathy and closure of eyes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that the test item had no cytotoxic properties in the bone marrow. However, the analytics with the bone marrow samples showed that the item was bioavailable. As shown in the analytics report the bone marrow samples of the male mice contained 5.27, 5.12 and 3.75 µg and the samples of the female mice had 3.56, 6.65 and 6.27 µg of the test item one hour post treatment. The test item concentrations were comparatively lower in the bone marrow samples taken 4 hours after the treatment (test item concentrations in the bone marrow samples were 1.40, 1.30 and 0.847 µg/sample for the male mice and 0.525, 1.37 and 2.59

µg/sample in the female mice), indicating the excretion or metabolism of the test item.

Conclusions:

Interpretation of results (migrated information): negative
The test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in deionised water. Deionised water was used as vehicle control. The volume administered intraperitoneally 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 animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 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.

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 control thus indicating that the test item had no cytotoxic effectiveness in the bone marrow.

In comparison to the corresponding vehicle controls there was no 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 intraperitoneally was used as positive control which showed a substantial increase of induced micronucleus frequency.

CONCLUSION

In 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, the test item is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

No findings indicating mutagenic activity of the test item were detected an in vivo mouse micronucleus assay after exposure up to the limit dose.

Justification for selection of genetic toxicity endpoint
In vivo study selected

Justification for classification or non-classification

no classification

All in vitro and in vivo assays were negative except for the bacterial mutagenicity assay (Ames). The positive result of the Ames test was not considered a key outcome as this test is a screening assay for mutations and more failure-prone compared to the other tests performed. As a weight-of-evidence the negative results of the in-vitro HPRT and chromosome aberration assay as well as the in-vivo micronucleus test were regarded more reliable.