BRUGGOLITE® FF6

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is considered to be non-mutagenic in the bacterial reverse mutation test.

The chromosome aberration test (in-vitro) shows chromosomal aberrations in cultured cells at a concentration of 5,000 μg/ml.

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:

May 1998 - July 1998

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:

OECD 471

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Strains TA 98, TA 100, TA 1535, TA 1537 were used.

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

- Type and identity of media: The nutrient medium contained 15 g per litre nutrient broth (SIFIN GmbH, Berlin)
- Properly maintained: yes
- Periodically checked for histidine dependence
- Periodically checked for cell membrane permeability
- Periodically checked for ampicillin resistance

Additional strain / cell type characteristics:

other: hisD3052, rfa, uvrB, pKM101

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: The nutrient medium contained 15 g per litre nutrient broth (SIFIN GmbH, Berlin)
- Properly maintained: yes
- Periodically checked for histidine dependence
- Periodically checked for cell membrane permeability
- Periodically checked for ampicillin resistance

Additional strain / cell type characteristics:

other: hisG46, rfa, uvrB, pKM101

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

- Type and identity of media: The nutrient medium contained 15 g per litre nutrient broth (SIFIN GmbH, Berlin)
- Properly maintained: yes
- Periodically checked for histidine dependence
- Periodically checked for cell membrane permeability
- Periodically checked for ampicillin resistance

Additional strain / cell type characteristics:

other: HisG46, uvrB, rfa

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

- Type and identity of media: The nutrient medium contained 15 g per litre nutrient broth (SIFIN GmbH, Berlin)
- Properly maintained: yes
- Periodically checked for histidine dependence
- Periodically checked for cell membrane permeability
- Periodically checked for ampicillin resistance

Additional strain / cell type characteristics:

other: hisC3076, rfa, uvrB

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 metabolic activation

Test concentrations with justification for top dose:

Experiment I:
Concentration range (with metabolic activation/with S9): 0.05, 0.1, 0.5, 1.0, and 5.0 mg/plate Concentration range (without metabolic activation/without S9): 0.05, 0.1, 0.5, 1.0, and 5.0 mg/plate
Experiment II:
Concentration range (with metabolic activation/with S9): 0.05, 0.1, 0.5, 1.0, and 5.0 mg/plate
Concentration range (without metabolic activation/without S9): 0.05, 0.1, 0.5, 1.0 and 5.0 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation system (without S9 mix) Migrated to IUCLID6: TA 1535 and TA 100

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation system (without S9 mix) Migrated to IUCLID6: TA 1537

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation system (without S9 mix) Migrated to IUCLID6: TA 98

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

yes

Positive controls:

yes

Remarks:

TA 1535, TA 100, TA 1537, TA 98

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

The test substance was tested at five concentration levels of 0.05, 0.1, 0.5, 1.0 and 5.0 mg/plate.

The following were added to sterile disposable tubes containing 2 ml of top agar:
- 0.1 ml test article dilution or
- 0.1 ml of solvent (negative control) or
- 0.1 ml positive control solution
- 0.1 ml of appropriate bacterial culture
- 0.5 ml of S9 mix or 0.5 ml of 0.2 M phosphate buffer

The contents of each tube were mixed and added to a Petri dish containing minimal agar.
When the top agar had set, the Petri dishes were inverted and incubated at +3 7°C for 48 hours.
Revertant colonies were scored manually at the end of the incubation period.

NUMBER OF REPLICATIONS:
Each test variant was performed as threefold replicate.

OTHER:
All used bacterial strains are defective in DNA-repair (L\uvrB). The strains also have a defective lipopolysaccharide barrier on the cell wall (rfa). These properties confer extra sensitivity to DNA damage and also greater permeability to large molecules. The strains TA 98 and TA 100 also contain a plasmid (pKM101) which enhances the effor prone repair and confers ampicillin resistance. The strains are routinely tested for histidine dependence, cell membrane permeability and ampicillin resistance.

Evaluation criteria:

POSITIVE RESULT:
A test is considered to be positive if the test article induces dose related increases in numbers of revertants scored in two separate experiments and these increases are deemed to be of biological relevance. Reproducible increases at one experimental point may also indicate a positive response. For a biologically relevant response the number of revertants is expected to be at least double the spontaneous reversion rate in the S. typhimurium strains TA 98, TA 100. In the S. typhimurium strains TA 1535 and TA 1537 the number ofrevertants is expected to be at least the tripie of the spontaneous reversion rate.

NEGATIVE RESULT:
A test article producing neither a dose related and reproducible increase in the number of revertants nor a reproducible positive response at any experimental point is considered to be non-mutagenic in this test system.

Statistics:

STATISTICAL METHDODS USED:
The results are presented as individual plate counts. In addition mean values and standard deviations are calculated for each strain and experimental point.

PARAMETERS THAT WERE ANALYSED:
- dose related increase in revertant numbers (with and without S9 metabolic activation)
- reproducible increase at a single experimental point (with and without S9 metabolic activation)

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.05, 0.1, 0.5, 1.0, 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.05, 0.1, 0.5, 1.0, 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
The revertant frequencies of the vehicle control were within the expected range and the positive control chemieals induced marked increases in revertant colonies. No biologically relevant increases in revertant numbers were obtained after treatment with Brüggolit FF6 in any bacterial strain and at any concentration tested. This applies to both, the presence and absence of metabolie activation.
See attached PDF-File "1998_07_03_02_FF6_Reverse Mutation Test_Results" (Table 2 -5)

TEST-SPECIFIC CONFOUNDING FACTORS:
none

RANGE-FINDING/SCREENING STUDIES:
The response of the untreated tester strains and the responses of the tester strains to the vehicle control, the test article at specified concentrations and the positive controls are shown in the attached PDF-File "1998_07_03_02_FF6_Reverse Mutation Test_Results" (Table 1).
At the chosen concentrations (5.0 to 0.01 mg/plate) no influence was observed to the number of spontaneous revertants and to the bacterial
backround lawn.

COMPARISON WITH HISTORICAL CONTROL DATA: In both independet experiments controls gave counts of spontaneous revertants within the normalranges obtained in this laboratory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Toxicity rangefinder (preleminary test)

Main experiment:

The responses of the untreated tester strains and the responses of the tester strains to the vehicle control, the test article at specified concentrations and the positive controls are presented in Tables 2 to 5 (two independent experiments without and with metabolic activation). All vehicle controls gave counts of spontaneous revertants within the expected ranges.

The positive controls markedly increased the number of revertant colonies in all bacterial strains with and without metabolie activation. No increase in revertant numbers which might indicate a mutagenic response was observed at any concentration of the test article and in any bacterial strain.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

the test item is considered to ben non-mutagenic in the bacterial reverse mutation test.

Executive summary:

The test item was tested for a possible potential to induee gene mutation in bacteria.

As test organisms the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 were used. The bacteria were exposed to the test item both, in the presence and absence of rat Iiver 89 metabolic activation. Two independent experiments were performed according to the standard plate incorporation method.

Toxicity Rangefinder: At the chosen concentrations of 5.0 to 0.01 mg/plate no influence was observed to the number of spontaneaous revertants and to the bacterial background lawn. Based on these results concentrations of 0.05, 0.1, 0.5, 1.0 and 5.0 mg/plate were chosen for the mutation experiment.

Mutation Experiment: No increase in revertant numbers which might indicate a mutagenic response was observed at

any concentration of the test article and in any bacterial strain. After treatment with the test article neither a dose related increase in revertant numbers nor a reproducible increase at a single experimental point was observed for any bacterial strain

tested. This applies to both, the presence and absence of rat liver S9 metabolic activation.

Based on the results of the reported study it is concluded that the test item does not induce gene mutation in Salmonella typhimurium under the experimental conditions described. Therefore, the test item is considered to be non-mutagenic in the bacterial reverse mutation test.

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:

key study

Study period:

November 1998 - March 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Planning and conduct of the study were based on the guidance provided by the following guideline: - Appendix to Directive 92/69/EEC of the commission of July 31, 1992, relating to the seventeenth adjustment of the Directive 67/548/EEC of the Council for the adaptation to teclmical progress of legal and administrative regulations for the classification, packaging and designation ofhazardous materials. Part B.10 In vitra Mammalian Cytogenetic Test Amtsblatt der Europäischen Gemeinschaften Nr. L 383 A, p. 148 - 150, 29.12.1992 - OECD Guidelines far the Testing ofChemicals (473 adopted July 21 , 1997)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chinese Hamster V79 cells

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster V79 cells

Metabolic activation system:

S9 liver microsomal fraction (RCC Cytotest Cell Research GmbH)

Test concentrations with justification for top dose:

Toxicity Range Finding Experiment: 439 - 5000 µg/ml
First Experiment:
with metabolic activation: Concentration range in: 1250 - 5000 µg/ml
without metabolic activation: Concentration range: 1250 - 5000 µg/ml
Second Experiment
without metabolic activation: Concentration range: 625 - 5000 µg/ml
with metabolic activation: Concentration range: 1250 - 5000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: destillated water

Details on test system and experimental conditions:

METHOD OF APPLICATION:
First and second Experiment:
V 79 cells in logarithmic growth were trypsinised and used to seed fresh cultures in 75 cm2 flasks with a density of about 2 x 106 cell per flask. These were incubated overnight, to reestablish logarithmic growth. The medium was then removed and replaced by fresh medium and solutions of the test articIe at the appropriate concentrations or positive control chemical. S 9 was added to the appropriate flasks. Treatment in the presence of S 9 was carried out in serum-free medium and treatment in the absence of metabolie activation was carried out in culture medium containing
FCS. At the end of the 4 hours exposure period the cells were washed using phosphate buffered saline (PBS) and fresh culture medium was added.

DURATION
- Preincubation period: overnight
- Exposure duration:
with metabolic activation: 4 hours
without metabolic activation: 4 hours
- Fixation time: 18 (first experiment) and 18 and 26 hours (second experiment)

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:
two cultures were prepared for each experimental point

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

Marked cytotoxicity - as expressed by a reduction in degree of confluency versus the controls

Statistics:

A statistical analysis was carried out to analyse the percentage of cells with struetural chromosome aberrations, excluding gaps. Comparisons weremade between the chromosome aberration frequencies in control cells and at each dose level using the Fisher' s Exact test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
not determined

RANGE-FINDING/SCREENING STUDIES:
Following 4 hours treatment a slight depression in eell growth was observed at eoneentration exeeeding 1481 µg/ml. After 18 and 26 hours
continuous treatment, however, all cultures treated with the test article showed a dose depending eytotoxie effeet. Inhibition in cell growth of at least 50 % was observed at eoneentrations exeeeding 1481 µg/ml (18 hours treatment) and µg/ml (26 hours treatment).

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: main test (1st Experiment)

First Experiment:

Marked cytotoxicity - as expressed by a reduction in degree of confluency versus the controls - was observed at concentration of 5,000 µg/ml both with and without metabolic activation. The number of aberrant metaphases in cultures treated with the test article, except the top concentration with metabolic activation, did not exceed 3.5 % (table 2 and 3). At a concentration of 5,000 µg/ml in the presence of metabolic activation the number of aberrant metaphases was 13.0 %.

Second Experiment:

Cultures treated with the test article continuously in the absence of metabolic activation at concentrations exceeding 1250

µg/ml (18 hours sampling time) and 625 µg/ml (26 hours sampling time) showed marked reduction in degree of confluency and damage of cell integrity.

For this reason at a coneentration of 5000 µg/ml less than 200 metaphases were evaluated at 18 hours sampling time and the cultures were not scorable at the late sampling time. Following 4 hours treatment (with metabolic activation) a slight depression in eonflueney was observed at coneentration of 5000 µg/ml.

In the absence of metabolie activation in one of the duplicate eultures and in both cultures in the presenee of metabolie activation a slight inerease in the aberration rate was found at a eoneentration of 5000 µg/mI at the 18 hours sampling time.

At the 26 hour sampling time the maximum aberration score determined in negative controls and cultures treated with the test article was 2 % (table 6 and 7).

At concentrations of 5000 and 2500 µg/ml the incidence of endoreduplications was recorded.

Marked inereases in the frequencies of aberrant eells eompared with untreated controls were seen in cultures treated with the positive control chemicals ethyl methanesulfonate and cyclophosphamide both in the Ist and 2nd experiment.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

It is concluded that the test item induces chromosomal aberrations in cultured chinese hamster V79 cells at a concentration of 5000 µg/ml when tested under the experimental conditions reported.

Executive summary:

The test item was examined for the ability to cause chromosomal damage in cultured chinese hamster V79 cells following in vitro treatment in the presence and absence of S9 metabolic activation.

Two independent assays for chromosomal aberrations were perfarmed and two parallel cultures were set up for each experimental point.

Range finder experiment: marked cytotoxicity (as expressed by an inhibition of cell growth - was found at concentrations exceeding 1481 µg/ml (4 hours treatment). After continuous treatment reduction in cell growth of at least 50 % was observed at concentrations exceeding 1481 µg/ml (18 hours treatment) and 658 µg/ml (26 hours treatment) respectively.

On the basis of these findings concentrations of 1250,2500 and 5000 µg/ml were evaluated in the 1st experiment, both in the

in the absence ar presence of S9 mix. In this experiment cells were treated far 4 hours and harvested 18 hours after the start of treatment.

In the 2nd experiment the cells were continuously treated in the absence of S9 metabolism until cell sampling after 18 or 26 hours. In the presence of S9 metabolism, the treatment duration was 4 hours again and cells were harvested after a subsequent 14 or 22 hours recovery period. For the 2nd experiment concentrations of 625, 1250, 2500 and 5000 µg/ml (without S9 mix) or 1250, 2500 and 5000 µg/ml (with S9 mix) were evaluated for early harvest time. For the late

harvest time only the highest scorable concentration was analysed.

200 metaphases spread from 2 cultures per experimental point (100 per culture) were selected for chromosomal aberration analysis.

In the cytogenetic experiments a reduced degree of confluency indicated Cytotocity at a concentration of 5000 µg/ml after 4 hours treatment. After continuous treatment cytotoxicity was observed at concentrations exceeding 1250 µg/ml (18 hours sampling time) and 625 µg/ml (26 hours sampling time).

In the st experiment the test article induced an increased and statistically significant incidence of structural chromosomal aberrations at the concentration of 5000 µg/ml in the presence of metabolic activation. At the same concentration with and without metabolic activation a slight, however, statistically significant increase in aberration frequency was observed in the

2nd experiment.

The positive substances induced sufficient aberrations to confirm effectiveness of the test procedures.

It is concluded that the test item induces chromosomal aberrations in cultured chinese hamster V79 cells at a concentration of 5000 µg/ml when tested under the experimental conditions reported. Therefore, a mutagenic effect of the test article can not be excluded completely. As in the AMES-Test (ToxLabs/1998/6994 RM) no evidence of a mutagenic effect to bacteria occurred, the mutagenic potential of the test article should be investigated by in vivo mutagenicity testing.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test item is considered to be non-mutagenic in the mouse bone marrow micronucleus test.

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:

May 2001 - November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: Charles River Wiga GmbH, Sulzfeld
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 32.3 g +/- 1.6 g; females: 26.4 +/-1.5 g
- Assigned to test groups randomly: yes, under following basis: 25 male and female animals each were assigned to 5 experimental groups to 5 animals per group and sex using random-charts. The cages were labelIed with coloured cage cards showing the study number, dose group, time
of sacrifice, animals' sex and numbers and date of dosing.41
- Fasting period before study: Overnight fasting before oral administration and until 3 hours after administration food was available ad libitum.
- Housing: Altromin Type S8/15, granulated soft wooed bedding, batch no 060801
- Water (e.g. ad libitum): tap water (municipal supply), Makrolon bottles, changed daily
- Acclimation period: 7 days before start of dosing (pre-experiment for toxicity)
13 days before start of dosing (main experiment)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-24°C
- Humidity (%): 45-65%
- Air changes (per hr): air conditioned
- Photoperiod (hrs dark / hrs light): artificial light was set to give a cycle of 12 hours light and 12 hours dark; Light from 6.30 a.m. until 6.30 p.m.

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: The test item was solved in deionised water shortly before administration
- Concentration of test material in vehicle: 2000 mg/kg body weight
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
test item: limit dose of 2000 mg/kg body weight
vehicle control : Deionised water (10 mI/kg body weight)
positive control : 40 mg/kg body weight CPA (Cyclophosphamide)

Duration of treatment / exposure:

Bone marrow smears were prepared at 12 hours (dose group), at 24 hours (vehicle contral, positive control, dose graup) and at 48 hours (dose group) after dosing.

Frequency of treatment:

single treatment with limit dose of 2000 mg/kg body weight

Post exposure period:

After administration the animals were monitored at approximately 1, 6, 24 and 48 hours for any symptoms of acute toxicity.

Remarks:

Doses / Concentrations:
2000 mg/kg body weight
Basis:
nominal in water

No. of animals per sex per dose:

25 mala and female animals each were assigned to 5 experimental groups to 5 animals per group and sex.
2 animals per sex were treated.

Control animals:

yes

Positive control(s):

Cyclophosphamide (CPA), dissolved in deionised water
- Route of administration: oral administration using a metal catheter
- frequency of administration: single dose
- dose: 40 mg/kg body weight
- volume administered: 10 ml/kg body weight

Tissues and cell types examined:

bone marrow removed from the femurs.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the results of the pre-experiment the limit dose of 2000 mg/kg body weight could be chosen

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
sampling intervals of 12, 24 and 48 hours after treatment. Only the sampling interval of 24 hours was chosen for the vehicle and the positive
contral group.

DETAILS OF SLIDE PREPARATION:
Bone marrow was removed from the femurs and smears prepared on microscope slides. The smears were aged for approximately 24 hours before staining with May-Grünwald/Giemsa solution.

METHOD OF ANALYSIS:
All slides were coded before scoring and scored blind. A minimum of 2000 polychromatic erythrocytes (PCE) was scored for the presence of micronuclei (= MPCE = micronucleated polychromatic erythrocytes) for each animal. The proportion o fPCEs among total erythrocytes
(PCEs + normochromatic erythrocytes [NCE]) was determined for each animal on the basis of 200 erythrocytes.

Evaluation criteria:

The test item is classified mutagenic, if it induces a statistically significant increase at the sampling times of 12, 24 or 48 hours with biological relevance. A statistically significant increase might require further confirmation by the demonstration of a dose response relationship at the respective sampling time.

Statistics:

Micronucleus scores (MCPE) and the proportion of PCEs among total erythrocytes are presented as individual values. In addition group means and standard deviations are calculated for each sex and experimental group. The statistical significance compared to the vehicle control were proved by means the Welch t-test (Rasch et al., Verfahrensbibliothek Versuchsplanung und -auswertung, Berlin 1981).
Statistical significance is declared at the 5 % level (one-sided).

Sex:

male/female

Genotoxicity:

negative

Remarks:

2000 mg/kg b.w.

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg body weight
A preliminary study on acute toxicity was performed with a small group of animals under identical conditions to these in the mutagenicity study with respect to animals, vehicle, route, frequency and volume of administration to assess the approximate MTD (maximum tolerated dose).
Two animals per sex were treated with a single oral dose of 2000 mg/kg body weight. This dose corresponds to the limit dose in accordance with the OECD Guideline. After administration the animals were monitored at approximately 1, 6, 24 and 48 hours far any symptoms of acute toxicity.
None ofthe animals died.

RESULTS OF DEFINITIVE STUDY
Mean values of micronuc1eated polychromatic erythrocytes (MPCEs) of 0.20 % (males) and 0.22 % (females) were found for the vehic1e control group. The range ofthe historical negative controls in the testing facility (since 1997) was 0.11 to 0.24 % in males and 0.12 to 0.27 % in females.
In BRÜGGOLIT FF6 treated groups mean MPCE values were in the range from 0.19 to 0.26 % in males and from 0.18 to 0.27 % in females.
The statistical analysis of the MPCE counts did not show any statistically significant difference in comparison to the vehicle control.
Treatment with the positive control chemical (CycJophospharnide, 40 mglkg body weight) induced statistically significant increases in the incidence of MPCEs with a group mean value of 2.79 % in males and 2.49 % in females.

Conclusions:

Interpretation of results (migrated information): negative
not to be classified

Executive summary:

Groups of 5 male and 5 female NMRI-mice were exposed to the test item at the limit dose of 2000 mglkg body weight. The test item was administered orally using a metal catheter. Deionised water (10 mI/kg body weight) served as vehicle control and Cyclophosphamide (CPA) at a dose of 40 mglkg body weight - also administered orally - was used as positive control.

A dose of 2000 mg/kg body weight was chosen as the maximum dose in accardance with the guideline because no of the animals died at this dose in the pre-experiment.

Bone marrow smears were prepared at 12 hours (dose group), at 24 hours (vehicle contral, positive control, dose graup) and at 48 hours (dose group) after dosing.

Two thousand polychromatic erythrocytes per animal were analysed far the presence of mieranuclei. To investigate bone marrow toxieity the proportion of polyehromatie erythrocytes among total erythrocytes was evaluated on the basis of 200 erythrocytes.

The frequency of mieronucleated polychromatie erythroeytes (MPCEs) in the vehicle control group was within the physiologieal range. Treatment with CPA induced statistieally significant inereases in the ineidenee of MPCEs.

In none ofthe experimental groups treated with the test item an increase in MPCEs was observed.

The treatments induced statistically significant decreases of the proportion of polychromatic erythrocytes among total erythrocytes in all cases in female animals and for the last sacrifice time in the male animals. But these values are in the range of the historical control data in the testing facility. Therefore it is deemed these changes are ineidental and not eaused by the administration ofthe test item.

Based on the results of the study reported here it is concluded that the test item does not induce micronuclei in polychromatic erythrocytes of NMRI-mice under the described experimental conditions. The test item is therefore considered to be non-mutagenic in the mouse bone marrow micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substance is considered to be non-mutagenic in the bacterial reverse mutation test.

The bacterial reverse mutation test conducted with Salmonella typhimurium strains supports a conclusion that the substance is non-mutagenic at concentration from 0.01 to 5.0 mg/plate

The chromosome aberration test (in-vitro) shows chromosomal aberrations in cultured cells at a concentration of 5,000 µg/ml.

In the in-vivo mammalian erythrocyte micronucleus test none of the experimental groups treated with the test item showed an increase in MPCEs (micronucleated polychromatie erythroeytes). The test item is therefore considered to be non-mutagenic in the mouse bone marrow micronucleus test.

Based on these results, it can be concluded that the positive in vitro findings for chromosomal aberration are overruled by the overall weight of evidence of negative in vivo and other in vitro test for this endpoint.

Short description of key information:
Bacterial reverse mutation test (in-vitro): non-mutagenic
Chromosome Aberration Test (in-vitro): test item induces chromosomal aberrations in cultured cells at a concentration of 5000µg/ml
Mammalian Erythrocyte Micronucleus Test (in-vivo): non-mutagenic

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test item needs not to be classified since the positive in vitro findings for chromosomal aberration are overruled by the overall weight of evidence of negative in vivo and other in vitro tests.