Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In vitro Gene Mutation study in Bacteria - AMES

The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

In vitro mammalian cells Chromosome aberration, OECD473

The test article induced structural chromosome aberrationsin the V79 Chinese hamster cell line.

Therefore, the substance is considered to be mutagenic in this chromosomal aberration test.

 

Link to relevant study records

Referenceopen allclose all

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:
From the 28th of August to the 14th of September, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to internationally accepted guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation, TA1535, TA100
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
Without metabolic activation, TA1537, TA1538, TA98
Positive controls:
yes
Positive control substance:
other: 2-amminoanthracene 2-AA
Remarks:
With metabolic activation, TA1535, TA1537, TA1538, TA98, TA100
Details on test system and experimental conditions:
Characterization of the Salmonella Typhimurium Strains
Salmonella typhimurium
TA 1537
his C 3076; rfa-; uvrB-; frame shift mutations
TA 1538
his D 3052; rfa-; uvrB-: frame shift mutations
TA 98
his D 3052; rfa-; uvrB-;R-factor: frame shift mutations
TA 1535:
his G 46; rfa-; uvrB-; base-pair substitutions
TA 100
his G 46; rfa-; uvrB-; R-factor; base-pair substitutions
Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in C C R according to Ames et al . . In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSQ in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl.
The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.

Selective agar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 mL of this nutrientvmedium. Sterilizations were performed at 121 °C in an autoclave.

Overlay agar
The overlay agar contains per litre:
6.0 g Difco Bacto Agar
6.0 g NaCl
10.5 mg L-histidine x HC1 x H20
12.2 mg biotin
Sterilizations were performed at 121 °C in an autoclave.


PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy is performed with strains TA 98 and TA 100. 8 concentrations are tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment are the same as described below forthe experiment. Toxicity of the test article may be evidenced by a reduction in the nunther of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DOSE SELECTION
According to the results of this pre-experiment the concentrations applied in the main experiments are chosen. The concentration range covers at least two decadic logarithms.
The maximum concentration is 5000.0 µg/plate, unless limited by toxicity or solubility of the test article. The concentration range includes at least two decadic logarithms. In this study at least five adequate spaced concentrations are tested. Two independent experiments are performed.
In case the results of the pre-experiment are in accordance with the criteria described above, these data are reported as a part of the main experiments.

EXPERIMENTAL PERFORMANCE
For each strain and dose level, including the controls, a minimum of three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL: test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control)
500 ul: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation)
100 ul: Bacteria suspension (cf. test system, pre—culture of the strains)
2000 ul: overlay agar

After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

DATA RECORDING
The colonies were counted using the BIOTRAN III counter (BIOTRO- NIK, D-6000 Frankfurt, F.R.G.). The counter was connected to an IBM XT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.

A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factorsor not.
Statistics:
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A decrease in the number of spontaneous revertants indicating bacteriotoxicity was observed
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A decrease in the number of spontaneous revertants indicating bacteriotoxicity was observed
Additional information on results:
PRE-EXPERIMENT
The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively.
According to the dose selection criteria, the test article was tested at the following concentrations: 10.0; 100.0; 333.3; 1000.0 and 5000.0 µg/plate.

MAIN EXPERIMENT
A decrease in the number of spontaneous revertants indicating bacteriotoxicity was observed in the following strains at the highest investigated dose: TA 1537 (exp. I), TA 1538 (exp. I and II), TA 98 (exp. I and II) all with metabolic activation and TA 100 (exp. II) without metabolic activation. In strain TA 98 the number of spontaneous revertants was already reduced at 1000.0 µg/plate with metabolic activation in experiment I.
background growth up to 5000.0 p.g/plate with and without S9 mix in all strains used, except in strain TA 1538 in experiment I without S9 mix at 5000.0 µg/p1Ltes where the background growth was slightly reduced.
Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of theSalmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.
The test article did not induce point mutations by base pair substitutions or frameshifts in the genome of the strains used.
Conclusions:
Negative.
The test article did not induce point mutations by base pair substitutions or frameshifts in the genome of the strains used.
The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

Materials and methods

This study was performed to investigate the potential of the substance to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, according to the OECD 471 and EU Method B.13/14.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0; 100.0; 333.3; 1000.0 and 5000.0 µg/plate.

Observations

Up to the highest investigated doses, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.

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

Results

The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From the 22th of August,1989 to the 9th of May, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to internationally accepted guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
EEC Directive 84/449, L 251, B 10, p. 131-133
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Test concentrations with justification for top dose:
EXPERIMENT I
with and without S9 mix:
7 h: 50.0; 100.0; 200.0; 300.0 ug/mL
18 h: 5.0; 25.0; 50.0; 100.0; 200.0; 300.0 ug/mL
28 h: 50.0; 100.0; 200.0; 300.0 ug/ml

Treatment interval was 4 hours. Per experimental point 4 cultures were used in parallel.
According to the criteria mentioned above one (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases
for cytogenetic damage.
In the pre-experiment for toxicity the colony forming ability of the V79 cells was at least slightly reduced after treatment with 300.0 g/ml in the absence and presence of S9 mix. In experiment I this concentration was chosen as highest dose level and the respective samples could be scored for cytogenetic damage at each fixation interval.

EXPERIMENT II
With S9
18h: 25.0; 100.0; and 200.0 ug/ml
in the presence of S9 mix, samples treated with 300.0 ug/ml could not be evaluated because of severe toxic
effects. Therefore, samples treated with 25.0; 100.0; and 200.0 ig/ml were chosen for scoring
Vehicle / solvent:
culture medium (SEROMED, D—1000 Berlin, F.R.G.) without fetal calf serum.
Untreated negative controls:
yes
Remarks:
Concurrent solvent
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
Cell Cultures
The cells have a stable karyotype with a modal chromosome number of 22.
Large stocks of the V79 cell line (supplied by LMP; D-6100 Darmstadt, F.R.G.) are stored in liquid nitrogen in the cell bank of CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, each batch is screened for myco plasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated at 37 °C in 80 cm^2 plastic flasks (GREINER, D-7440 Nürtingen, F.R.G.). Seeding is done with about 5 x10^5 cells per flask in 15 ml of MEM (minimal essential medium; SEROMED; D-1000 Berlin, F.R.G.) supplemented with 10 % fetal calf serum (FCS; Boehringer Mannheim, D-6800 Mannheim, F.R.G.). The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C and 4.5 % carbon dioxide atmosphere.

PRE-EXPERIMENT FOR TOXICITY
The toxicity of the test article was determined in a pre-experi ment in order to establish a concentration dependent plating efficiency relationship. The experimental conditions in this pre experiment were the same as described below for the experiment.


DOSE SELECTION
According to the results from this pre-experiment six concentrations (18 h interval) to be applied in the chromosomal aber
ration assay were chosen.
The highest dose level used was 10 mM (Collection of notifications related to the pharmaceutical affairs law (IV), Toxicity Test Guideline, Yakugyo Jiho Company, Ltd., Tokyo , Japan, page 43, 1984) unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced plating efficiency and/or partial inhibition of mitosis).
If toxic effects were produced the highest dose level should reduce the plating efficiency to approximately 20 - 50 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found.


EXPERIMENTAL PERFORMANCE
Seeding of the Cultures
Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA).
The cells were seeded into Quadriperm dishes (Heraeus, D-6450 Hanau, F.R.G.) which contained microscopic slides (4 chambers per dish and test group). In each chamber 5 x 10 - 1 x 10 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.

Treatment
After 48 h (7 h, 28 h preparation interval) and 55 h (18 h pre paration interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 ul/ml S9 mix. After 4 h this medium was replaced with normal medium after rinsing twice with ‘saline G”.

All incubations were done at 37 °C in a humidified atmosphere with 4.5 % CO2.

Preparation of the Cultures
5, 155 and 25.5 h after the start of the treatment colcemid was added (0.2 ug/ml culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KC1) for 20 mm at 37 °C. After incubation in the hypotonic solution the cells are fixed with 3 + 1 absolute methanol + glacial acetic acid. All four slides per group were prepared.
After fixation the cells were stained with giemsa (Merck, D-6100 Darmstadt, F.R.G.).

Evaluation criteria:
Analysis of Metaphase Cells
Evaluation of the slides were performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions,
exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ±1 °C were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype) is scored.

The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00 % — 4.00 %.
b) the positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible posi tive response for at least one of the test points.
A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the non-parametric Mann-Whitney test. However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05): evaluated by means of the non-parametric Mann-Whitney test) . Evaluation was performed oniy for cells carrying aberrations exclusive gaps.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mitotic index was clearly suppressed after treatment with 300.0 µg/mL (without S9 mix) and with 50.0 µg/mL and higher concentrations
in the presence of S9 mix (exp. I and II).

In the pre-experiment on toxicity (colony forming ability) after treatment with 10.0 ug/mL and higher (without S9 mix) and 30.0 ug/mL and higher (with S9 mix) the colony forming ability was distinctly reduced as compared to the solvent controls.
In the main experiments, at each fixation interval, except at interval 28 h without S9 mix, the mitotic index was reduced at least after treatment with the highest dose levels in the absence and presence of S9 mix, indicating that the substance had cytotoxic properties.
In the absence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.25 % - 3.25 %) were in the range of the control valu es: 1.25 % - 3.75 %.

However, in the presence of S9 mix, at fixation interval 18 h a statistically significant increase in the aberration rate (9.25 %) was found after treatment with 100.0 µg/mL. To clarify this none dose-dependent result a second experiment was performed.
In this experiment samples only treated with 200.0 ug/ml of the test article could be evaluated as top dose level because of severe toxic effects with 300.0 ug/ml. In the samples treated with 200.0 ug/ml again a slight but statistically significant
increase in the aberration rate (5.75 %) as compared to the solvent control (1.75 %) was observed. As the aberration rate was beyond the range of the control data of this study (1.25 % -3.75 %) and of our historical control data (0.00 % - 4.00 %) this increase is regarded as biologically relevant.

Additional indications for effects of the test article on chromatin arrangement arise from occurrence of abnormal interphase nuclei. There were many multinucleate cells carrying a large number of nuclei of various sizes within a single cell. Some cells in mitosis were observed of which the chromosomes appeared clustered in groups of different sizes. Also, a distinct number of interphase nuclei were observed with an abnormal condensed chromatin pattern (in clusters).

These observations, together with the distinct decreased mitotic index and the slightly increased aberration frequency can lead to the assumption that the test article probably do not only interact directly with the chromatin but also affect the cell cycle and disarranged the development of chromatin condensation.

No relevant deviation from the control data was found after treatment with the test article.
EMS (0.72 mg/mL) and CPA (1.40 ug/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Positive
The test article induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line.
Therefore, is considered to be mutagenic in this chromosomal aberration test.
Executive summary:

Materials and methods

The test article was assessed for its potential induce to structural chromosome aberrations in V79 cells of the Chinese hamster in vitro, according to OECD473 and B.17.

Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment

with the test article. The treatment interval was 4 h.

In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated:

Experiment I, with and without S9 mix:

7 h: 300 µg/L

18 h: 25.0; 100.0; 300.0 µg/mL

28 h: 300.0 µg/ /mL

Experiment II, with S9 mix:

18 h: 25.0; 100.0; 200.0 µg/mL

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment with 200.0 and 300.0 µg/mL reduced clearly the plating efficiency of the V79 cells (derived from the plotted dose re sponse curve).

Also the mitotic index was at least slightly reduced after treatment with 300.0 µg/mL (without S9 mix) and with 50.0 µg/mL and higher in the presence of S9 mix (exp. I and II).

There was a slight but statistically significant increase in cells with structural aberrations after treatment with 100.0 (exp. I) and 200.0 µg/mL (exp. II) at fixation interval 18 h with metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line.

Therefore, is considered to be mutagenic in this chromosomal aberration test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Mammalian Erythrocytes Micronucleous, OECD474

Negative. During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus testwith bone marrow cells of the mouse. Therefore, the substance is considered to ne non-mutagenic in the micronucleus in vivo assay.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From the 29th Aaugust to the 7th November, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the European Guidelines based on the OECD Test Guideline, in GLP
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
EEC Directive 84/449, L 251, B 12, p. 137 - 139
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Strain: NNRI
Source: BRL Tierfarm Füllinsdorf CH- 4414 Füllinsdorf/Basel, Switzerland
Number of Animals: 84 (42 males/42 females)
Initial Age at Start of Acclimatization: minimum 10 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: approximately 30 g
According to the suppliers assurance the animals were in healthy condition.
The animals were under quarantine in the animal house of CCR for one week after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
The animals were distributed into the test groups at random and identified by cage number.

HUSBANDRY
Housing: single
Cage Type: . Makrolon Type I, with wire mesh top (EHRET GmbH, D-7830 Einmendingen)
Bedding: granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
Feed: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
Water: tap water, ad libitum (Gemeindewerke, D61O1 Rossdorf, F .R.G.)
Enviromnent: temperature 21 ± 3 °C
relative humidity 30-70 %
artificial light 6.00 a.m.- 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
The vehicle of the test article was used as negative control.
Carlioxymethylcellulose
Source: SERVA, D-6900 Heidelberg
Batch: 17681
Somministration: orally, singly
Volume: 10 mL/kg b.w.
Details on exposure:
DOSE SELECTION
It is generally recommended to use the maximuiri tolerated dose or the highest dose that can be formulated and administered reproducibility. The volmne to be administered should be compatible with physiological space available.
The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on sur vival within 72 hours.
Duration of treatment / exposure:
24, 48 and 72 hours
Remarks:
Doses / Concentrations:
5000 mg/kg b.w.
Basis:
actual ingested
pre-experiment 1°
Remarks:
Doses / Concentrations:
1500 mg/kg bw
Basis:
actual ingested
pre-experiment 2°
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
pre-experiment 2°
No. of animals per sex per dose:
6 males
6 females
Positive control(s):
CPA; Cyclophosphamide
Source: SERVA, D-6900 Heidelberg, F.R.G.
Batch: 17681
Dissolved in: physiological saline
Somministration: 40 mg/kg li.w., orally, singly
Volume: 10 mL/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 20 °C only 1 % of CPA is hydrolised per day in aqueous solution.
Tissues and cell types examined:
To describe a cytotoxic effect the ratio between polychromatic and normochrcmatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.
Details of tissue and slide preparation:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1.000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polycliromatic erythrocytes nor a
statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
Statistics:
Statistical significance at the five per cent level (p < 0.05)
was evaluated by means of the non-parametric Mann-Whitney test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
pre-experiment 1°(4 males/4 females 5000 mg/Kg bw)
All surviving animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure and apathy. One male and one female died within the first hour after treatment.
pre-experiment 2° (2 males/2 females 1500 mg/Kg bw)
All treated animals expressed toxic reactions: reduction of spontaneous activity. One female expressed abdominal position.
Additionally, one male and one female expressed eyelid closure and apathy.
2000 mg/Kg bw
All treated animals died within 6 hours after treatment.

After treatment with 1500 mg/kg b.w.of the substance one male and one female died at preparation interval 24 hours, one female died at preparation interval 48 hours, and one female died at prepa ration interval 72 hours.
The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating no cytotoxic properties.

In comparison to the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with were in the same range as comparedto the nega tive control groups.
Conclusions:
Interpretation of results (migrated information): negative
During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in Carboxymethylcellulose (1 %). This suspending agent was used as negative control. The volume administered orally was 10 ml/kg b.w.

24 h, 48 h and 72 h after a single application of the test article 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. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 1500 mg/kg b.w.

In pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions.

After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative

controls thus indicating no cytotoxic effects.In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article

did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Studies on bacteria (AMES)

This study was performed to investigate the potential of the substance to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, according to the OECD Guideline 471 and EU Method B.13/14.

The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Studies on in vitro mammalian cells

The test article substance was assessed for its potential induce to structural chromosome aberrations in V79 cells of the Chinese hamster in vitro,

according to OECD473 and B.17.

The test article induced structural chromosome aberrations in the V79 Chinese hamster cell line.

Therefore, the substance is considered to be mutagenic in this chromosomal aberration test.

Studies in vivo

This in vivo study was performed to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, according to OECD474 and B.12.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated:

24 h, 48 h, and 72 h preparation interval: 1500 mg/kg b.w.

In pre-experiments the animals expressed toxic reactions.

After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

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

Therefore, the substance is considered to ne non-mutagenic in this micronucleus in vivo assay.

Three studies are available on the substance itself, and ccording to ECHA Guidance R.7a, Figure R.7.7-1 Flow chart of the mutagenicity testing strategy and Table R.7.7-5, a negative Gene mutation test in bacteria (AMES) could be sufficient to fulfill Annex VII of the REACH Regulation 1907/2006/EC.

However, a positive Chromosome aberration test in vitro requires an in vivo evaluation, to fulfil REACH Annexes VII and VIII requirements.

The 1th in vivo test was negative, therefore the substance is considered not genotoxic.

Justification for classification or non-classification

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny.

Substance that are mutagenic in somatic cells may produce heritable effects if they, or their active metabolites,have the ability to interact wit the genetic material of germ cells. Conversey substances that do not induce mutations in somatic cell in viv would not be expected to b germ cell mutagens.
However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests

The tested substance did not create gene mutations in the strains in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, therefore according to the 3.5. of the CLP Regulation n.1272/2008, it could be classified as not mutagenic for germ cells.

However additioanal studies are available on the substance itself and in an in vitro mammalian cell, the test substance induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line.

According to ECHA Guidance R.7a, Figure R.7.7-1 Flow chart of the mutagenicity testing strategy and Table R.7.7-5, a positive Chromosome aberration test in vitro requires an in vivo evaluation, to fulfil REACH Annexes VII and VIII requirements and for the classification.

In a bone marrow cells micronucleus assay of the mouse the substance is considered to be non-mutagenic.

As conclusion, with the evaluation of the all three available result on the test substance, according to the CLP Regulation n.1272/2008 and the ECHA Guidance R.7a, the substance is not classified as mutagenic.