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Genetic toxicity in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
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:
S9
Test concentrations with justification for top dose:
0, 62.5, 125, and 250 ug/mL
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 18 - 21 hours
- Exposure duration: 18 hours without S9, 3 hours in the presence of S9
- Expression time (cells in growth medium): 18 hours


SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.1 ug/mL final concentration)
STAIN (for cytogenetic assays): 2% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 well spread metaphases (100 metaphases per culture)

DETERMINATION OF CYTOTOXICITY
- Method: visual - turbid cells or lack of growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: structural chromosome and chromatid aberrations, interstitial deletions, and multiple aberrations

OTHER: The Verneir readings of all aberrant metaphases scored were recorded
Evaluation criteria:
A test is considered to be positive if the aberration yield at at least one concentration is significantly above the concurrent control frequencies.

A test is considered to be negative if there is no significant increase in aberration frequency at any dose level, above the concurrent control frequencies.

A chemical is designated as a clastogen (chromosome breaking agent) if a positive result is obtained in both of two sets.

A chemical is designated as not-clastogenic if a negative result is obtained in both of two tests, one of which utilises two sampling times.

Gaps are recorded separately and not included in the final assessment of clastogenic activity.

Both statistitcal significance and biological relevance are considered together in the evaluation of the results.
Statistics:
Fisher's exact probability test (two-sided)
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
From the aforementioned findings of the chromosome aberration assay it is concluded that 9,9 -bis(methoxymethyl)fluorene is clastogenic in the presence of S-9 mix under the conditions used in the study.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
December 29 1992
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Rat liver treated with aroclor 1254
Test concentrations with justification for top dose:
First experiment: 0, 62, 185, 556, 1667, 5000 ug/plate
Second experiment: 0, 25, 74, 222, 667, 2000 ug/plate
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: N-ethyl_N-nitrosourea, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days
Evaluation criteria:
A study is considered valid if the mean colony counts of the negative control values of the strains are within the acceptable range and if the results of the positive controls meet the criteria for positive response. A reproducible two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the negative control, or a demonstrable concentration-related effect, is taken to indiciate a positive response in this assay system.
Statistics:
None
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test substance was not mutagenic in a bacterial reversion assay performed with Escherichia coli WP2 uvr A.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
May 26 1983
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29 December 1992
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 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Rat liver treated with aroclor)
Test concentrations with justification for top dose:
First experiment: 0, 62, 185, 556, 1667, 5000 ug/plate
Second experiment: 0, 25, 74, 222, 667, 2000 ug/plate
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
Two independent mutagenicity assays were performed according to the protocol.
Evaluation criteria:
A study is considered valid if the mean colony counts of the control values of the strains are within the historical background range and if the results of the positive controls meet the criteria for a postive response. A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontanieous reversion rate observed with the vehicle, together with evidence of a dose-response. Both numerical significance and biological relevance are considered together in the evaluation.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first and second tests a slight toxic effect of the test substance was observed on the bacterial growth above 62 and 74 ug per plate respectively. A dose dependent precipitation was observed in both tests. As the precipitation in the first test at 5,000 ug per plate hampers counting of the number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose respone.
In both the absence and presence of the S9-mix in all strains the test substance did not cause a two fold greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneious revertant rate.
The positive controls gave the expected increase in the number of revertants in both the absence and presence of S9.
Conclusions:
It is concluded that the results obtained with test substance 9,9 -bis(methoxymethyl)fluorene in S. tyhimurium strains TA 1535, TA 1537, TA 98, and TA 100 in both the absence and the presence of S9 -mix do not indicate mutagenicity under the conditions of the study. Therefore this substance is considered not mutagenic in S. typhimurium.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
April 4, 1984
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Version / remarks:
7-1-91 edition
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT on X-chromosome
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: University of Leiden, Netherlands
- Methods for maintenance in cell culture if applicable: cells stored as frozen stock cultires in liquid nitrogen
- Modal number of chromosomes: 22


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 culture medium (with Glutamax I), supplemented with heat-activated foetal calf serum (10% v/v) and gentamycin (50 mg/L). 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (rat liver)
Test concentrations with justification for top dose:
-S9 mix (experiment 1 & 2): 0, 3.125, 6.25, 12.5, 25, 50, 75, 100, 150, 200 ug/mL
+S9 mix (experiment 1): 0, 2.5, 5, 25, 50, 75, 100, 200, 300, 400, 500 ug/mL
+S9 mix (experiment 2): 0, 5, 10, 25, 50, 75, 100, 200, 300, 400, 500 ug/mL
Vehicle / solvent:
DMSO
Most suitable vehicele for the test item used
Untreated negative controls:
yes
Remarks:
Growth medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1.0 x 106 cells per flask

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 8 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
fixed with 4% phosphate buffered formaldehyde solution for about 20 min, cell colonies were stained with 4% Giemsa solution

DETERMINATION OF CYTOTOXICITY
- Method: initial cell yield and by measuring the colony-forming ability of the CHO cells after approx. 24h after addition of test substance
- Any supplementary information relevant to cytotoxicity: cultures were discarded on the day post-treatment since the test substance dose levels resulted in <10% relative initial cell yield and/or precipitation of the test substance was visible at the end of the treatment
Rationale for test conditions:
in accordance with guideline
Evaluation criteria:
a concentration-related increase in mutant frequency
a reproducible positive response for at least one of the test substance concentrations (e.g. the mean mutant frequency should be more than 20 mutant per 1,000,000 clonable cells (final survival)
both numerical and biological significence were considered together in the evaluation
Statistics:
Not applied
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In both presence and absence of S9 mix, the test substance induced no concentration-related and/or reproducible increase in mutant frequency
Conclusions:
Under the conditions employed in this examination the test substance, 9,9-bis-(methoxymethyl)fluorene is not mutagenic at the HPRT locus of Chinese hamster ovary cells

Genetic toxicity in vivo

Link to relevant study records

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Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charels River Wiga GmbH
- Age at study initiation: young adult mice
- Assigned to test groups randomly: computer randomisation
- Housing: males housed individually, females were housed in groups of 5 per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3C
- Humidity (%): 30 - 72%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 7 November 1995 To: 8,9,10 November 1995
Route of administration:
oral: gavage
Vehicle:
maize oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in maize oil one day prior to use.
Duration of treatment / exposure:
1 gavage dose
Post exposure period:
Signs of reactions to treatment were recorded from ca. 1 - 4 hours after treatment and daily thereafter. At 24, 48, and 72h post-treatment, 10 controls (5m/5f) and 10 test animals (5m/5f) were killed by cervical dislocation. At 24h the 5 positive control animals were sacrificed. Bone marrow was immediately collected into foetal calf serum and processed into glassdrawn smears. Two bone marrow smears per animal were prepared, air-dried, fixed in methanol and stained with May-Brunwald Giemsa.
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
nominal concentration
No. of animals per sex per dose:
5 males/ 5 females
Positive control(s):
Mitomycin C (0.75 mg/kg bw)
Tissues and cell types examined:
Bone marrow smears onto glass slides
Details of tissue and slide preparation:
At sacrifice: Bone marrow was immediately collected into foetal calf serum and processed into glassdrawn smears. Two bone marrow smears per animal were prepared, air-dried, fixed in methanol and stained with May-Brunwald Giemsa.
Evaluation criteria:
The number of polychromatic erythrocytes per 1000 erythrocytes and the number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocutes from the vehicle control and treatment group were analysed using three-way ANOVA with factors sex, group, and time.
Statistics:
Because the three way ANOVA yielded significant effects, the negative control group (A) was compared with the treatment group (B), using two sided asymptotic t-tests for each sex and time point. If the criteria for homogeneity of variances was not met (Leven's test), Brown-Forsythe ANOVA was used followed by separate variance t-tests.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In male mice treated with test compound, the number of PE per 1000 E was not statistically different than those found in vehicle controls, indicating that the treatment did not result in cytotoxicity to bone marrow cells. In female mice only at 48h was the number of PE per 1000 E statistically significantly lower than those found in the vehicle controls, indicating that the treatment resulted in cytotoxicity to bone marrow cells.
Conclusions:
No indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated by gavage with 9,9-bis(methoxymethyl)fluorene at a dose level of 2000 mg/kg (limit dose)
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
Draft guideline at the time study was initiated
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 482 (Genetic toxicology: DNA damage and Repair/Unscheduled DNA synthesis in Mammalian cells in vitro)
Principles of method if other than guideline:
To provide data on the potency of the test material to induce unscheduled DNA-synthesis (UDS) in primary rat hepatocytes after short -term exposure to rats.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat strain selected commonly used for this type of study
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga Germany GmbH
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: Not specified
- Assigned to test groups randomly: yes
- Fasting period before study: 2-16 hours before dosing
- Housing: makrolon cages with grid cover of stainless steel
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12hrs dark/12hrs light
Route of administration:
oral: gavage
Vehicle:
PBS containing 0.5% carboxymethylcellulose
Details on exposure:
Animals were dosed by oral gavage
Duration of treatment / exposure:
one dose
Frequency of treatment:
Once
Post exposure period:
16 hours. Signs of reactions to treatment were recorded from 1-4 hours and 12-16 hours.
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
12 males = 2000 mg/kg
12 males = vehicle control group
Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetylaminofluorene. positive control animals were housed in a special "carcinogenicity unit" just prior to administration until sacrifice.
Tissues and cell types examined:
hepatocytes isolated from rat liver
Details of tissue and slide preparation:
liver of each rat was purfused in situ with Ca2+ and Mg2+-free HEPES buffer (0.01M) whilst under Nembutal anaesthesia, followed by an in vitro perfusion with a HEPES-buffered (0.1M) collagenase solution. After isolation, the dissociated cells were incubated for 10 minutes in a shaking waterbath at 37°C, filtrated over a 100 mesh nylon filter, centrifuged, resuspended in WEH medium [= Williams medium E supplemented with 20mM HEPES buffer and bovine serum albumin (10 mg/mL)] Thereafter, the cells were centrifugated, resuspended in WEC medium [= Williams medium E complete, which consists of Williams medium E supplemented with 10% fetal calf serum, L-glutamine (2 mM) and gentamicin (50 ug/mL)], again centifugated and finally resuspended in WEC medium. Cell counts were made with a haeomocytometer. The viability of the hepatocytes was determined by trypan blue exclusion. Only hepatocytes suspensions with viability >6)% were used for in the repair assay.

Suspensions containing 5 x 10^5 cells/mL were prepared in WEC medium. Aliquots (one mL) were seeded onto Thermanox 25 mm ound plastic coverslips in 35mm 6-well dishes containing 2mL WEC. Cultures incubated at 37°C in a humidified incubator containing ca. 5% CO2 and 95% air to allow cells to attach.
Hepatocyte cultures were incubated for c. 18 hours at 37°C. Cells fixed in three 30 changes of absolute ethanol-glacial acetic acid (3:1), air-dried and mounted on glass slides.

Slides were processed for audiradiography using Kodak NTB-2 emulsion. After 7 -14 days of exposure at ca. -20°C three slides per animal were developed with Kodak D19, fixed in Kodak Fixer and washed with water. Slides were stained with haematoxylin and eosin and embedded in DePeX. Slides were developed after 7 days of exposure appeared to be of good quality to allow subsequent scoring. Of each animal two slides were selected and coded by a qualified person not involved in the study to enable "blind" scoring. Reamining slides for each animal kept as a reserve.
Evaluation criteria:
The response is considerd positive if the test substance yields =/>5 NG and =/>10% of the cells are in repair.

A test substance is considered negative if all dose levels and time points produce =/<0 NG.

Both numerical significance and biological relevance were considered together in the evlauation.
Sex:
male
Genotoxicity:
negative
Remarks:
test substance <-3.87 NG
Toxicity:
no effects
Vehicle controls validity:
valid
Remarks:
induced
Positive controls validity:
valid
Remarks:
induced 20.94 NG (positive)

Results of DNA repair test (summarised results for each dose level)

Group

Treatment

Dose

NUC

SD +/-

CYT

SD +/-

NG

SD +/-

%IR

SD +/-

A (negative)

1-4 hr

0

27.67

1.59

35.98

4.25

-8.31

2.67

5.33

2.52

B (test item)

1-4 hr

2000 mg/kg

21.10

2.62

25.95

1.94

-4.85

0.76

6.67

3.79

A (negative)

12-16 hr

0

24.16

4.39

28.03

4.15

-3.87

2.16

15.33

6.66

B (test item)

12-16 hr

2000 mg/kg

26.62

3.09

31.67

5.77

-5.05

3.07

10.67

4.04

D (positive control)

12 -16 hr

50 mg/kg

60.57

10.48

39.64

6.10

20.94

12.14

81.33

15.82

NUC = Mean nuclear grain count

CYT = Mean cytoplasm grain count (nuclear sized area)

NG = Mean net grains per nuclues

SD = Standard Deviation

%IR = Percent of cells in repair (more or equal to 5 NG)

Conclusions:
It was concluded that the test substance did not induce DNA-repair activities in rat hepatocytes short-term in vivo exposure of rats under the conditions used in the present study.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga Germany
- Age at study initiation: 10 - 12 weeks old
- Weight at study initiation: 270 - 333 g
- Assigned to test groups randomly: yes, computer randomisation
- Fasting period before study: 16 hours
- Housing: 5 rats per cage/ separated by sex - sterilized stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 June 1996 To:12,13,14 June 1996
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
On day 0, before dosing animals were weighed. The test substance was suspended in corn oil a few hours before use and administred to rats once via gavage after the 16 hour fast.
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals/sex
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
Mitomycin C at 2.5 mg/kg-bw
Tissues and cell types examined:
Bone marrow sampling
Details of tissue and slide preparation:
At ca. 6, 24, and 48 hours after administration of the test substance 5 animals/sex of each of the three test groups and the negative control group were sacrificed. Two hours prior to sacrifice, animals were injected i.p. with colchicine at a concentration of 4 mg/kg. After sacrifice femurs were dissected free. Bone marrow cells were removed by flushing with Hanks Balanced Salt Solution and fixed with methanol/glacial acetic acid. Slides were stained with 2% Giemsa solution. Two slides per animal were prepared. One thousand cells, 500 cells per slide, were examined to determine the percentage of cells in mitosis. On each slide 25 well-spread metaphases, were analyzed for chromoatid-type (gaps, breaks, minutes, rings, dicentrics) of aberrations, and other anomalies (endoreduplication, polyploid cells, heavily damaged cells).
Evaluation criteria:
The criteria for a positive response was a clear increase in the percentage of cells with strucutural aberrations over the concurrent control frequencies (in the whole group).

A test substance was considered to be negative in the in vivo chromosome aberration test if it produced no increase in the number of structural chromosomal aberrations at any of the test points.

Both statistical significance and biological relevance were conisidered together in the evaluation.

Gaps (achromatic lesions) were recorded separately and not included in the final assessment of clastogenic activity.
Statistics:
Data were analysed statistically by the Fisher's exact probability test (two-sided) to determine signifcant differences between treated and control groups.
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations at the concentration of 2000 mg/kg bw at any of the time points analysed, when compared with the vehicle control values.

At the post-treatment time of 6 hours, the mean mitotic index of the female rats treated with the test substance was reduced to 1.62% when compared to the mean mitotic index of the female control rats (2.94%)

At the post treatment time of 48 hours, the mean mitotic index of the female rats treated with the test substance was reduced to 2.04% when compared to the mean mitotic index of the female control rats (2.80%)

The positive control substance, Mitomycin C, showed the expected statistically significant increase in the number of cells with structural chromosome aberrations.
Conclusions:
From the reported findings it is concluded that 9,9 -bis-(methoxymethyl)fluorene is considered not clastogenic because it did not induce a statistically significant increase in the number of cells with structural chromosome aberrations in bone marrow cells of male and female rats under the conditions employed in the study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Data available to assess the genetic toxicity includes bacterial reverse mutation assays, in vitro clastogenic assay, in vivo micronucleus, and in vivo clastogenic assay. Positive findings in the in vitro clastogenicity experiment with CHO cells was not observed in vivo with Wistar rats. There were no positive findings in the in vivo micronucleus assay or the bacterial reverse mutation assays. The weight of evidence indicates that this substance (Donor C20) is not a germ cell mutagen.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data set, it is concluded that the substance does not met the criteria to be classified as germ cell mutagen (negative results observed in all in vivo studies) and therefore classification under the CLP regulation (1272/2008 EC, as amended) is not warranted.