Fluoren-9-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

GLP OECD 471: positive

Additional literature data for in vitro genotoxicity was found, however, the reliability was considered limited. In particular substance information and details on experimental conditions were missing.

Seifried et al. summarize the available in vitro data as shown below:

Ames Salmonella Assay (four strains only): negative with and without metabiolic activation

Mouse Lyphoma Assay: inconclusive (without S9), negative (with S9)

Reference:

Seifried HE, Seifried RM, Clarke JJ, Junghans TB, San RHC. A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella typhimurium and L5178Y Mouse Lymphoma Cell Mutation Assays. Chemical Research in Toxicology. 2006 May;19(5):627–44.

An evaluation of the available in vitro data is included as supporting data in Section 7.6.2.

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:

2017-10-24 to 2018-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

8 June 2000

Deviations:

no

Principles of method if other than guideline:

n.a.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB-, rfa- + R-factor (pKM101)

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB-, rfa- + R-factor (pKM101)

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB-, rfa-

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB-, rfa-

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

trp-, uvrA-

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from beta-Naphthoflavone/Phenobarbital-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EC, OECD and Japanese guidelines for this test system. The test material showed best solubility in DMSO and 5000 µg/plate was chosen as the appropriate maximum concentration.
1. Series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate
2. Series: 158, 281, 500, 889 and 1580 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: Daunomycin

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

Bacterial strains were tested in accordance with the plate incorporation method. 3 parallel plates were used for each concentration step of the test material. The incubation of plates was performed at 36-38°C for 2 days. Liver S9 mix from rats pre-treated with beta-Naphthoflavone/Phenobarbital was used as the metabolic activation system. Two experimental series were performed, containing 10% S9 ind the 1st and 20% S9 in the 2nd series.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the con-current negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response (OECD TG 471, 1997).

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was considered, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1580 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1580 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=889 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=889 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Precipitation: >=500 µg/plate and >= 1580 µg/plate at the beginning and the end of the experiment, respectively

Conclusions:

Under the conditions described, the test item induced relevant increases in revertant numbers (more than 3-fold of the negative control value) in TA1537 in both experimental series, indicating the potential of the test item to induce frameshift mutations in the absence of metabolic activation. Based on these results, it was concluded that the test item was mutagenic in this bacterial reverse mutation test under the described experimental conditions.

Executive summary:

Objective

The present study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix).

Study Design

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coil WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pre-treated with beta-Naphthoflavone/Phenobarbital was used. In this study, two independent experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series. The test item was dissolved in DMSO and tested at concentrations ranging from 5 to 5000 µg/plate.

Results

Precipitation of the test material on the agar plates occurred at concentrations >=500 µg/plate and >= 1580 µg/plate at the beginning and the end of the experiment, respectively. Toxicity to the bacteria, evident as a reduction of revertant numbers below 0.5 of the concurrent negative control, was observed in all tester strains at concentrations >=500 µg/plate (TA98), >=889 µg/plate (TA1537, WP2 uvrA) or >=1580 µg/plate (TA100, TA1535).

The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Under the conditions described, the test item induced relevant increases in revertant numbers (more than 3-fold of the negative control value) in TA1537 in both experimental series, indicating the potential of the test item to induce frameshift mutations in the absence of metabolic activation. Based on these results, it was concluded that the test item was mutagenic in this bacterial reverse mutation test under the described experimental conditions.

Conclusion

Based on these results for the described experimental conditions, it was concluded that the test item was mutagenic in this bacterial reverse mutation test .

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Nov 2017 - 18 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Commission Regulation No. 2017/735. Method B.49: Mutagenicity – In Vitro Mammalian Cell Micronucleus Test. OJ L 112/109.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human blood

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors:
Blood was collected from two healthy adult, non-smoking volunteers (aged 18 - 35 years).

Blood samples
Human blood was collected aseptically, pooled (in equal volumes from each donor) and diluted with HML media. Phytohaemagglutinin (PHA) was added to stimulate cell division.

Culture medium
HML Media: RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.

Lymphocyte cultures
Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each culture contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 34 to 39°C, and the cells were re-suspended (twice daily) by gentle inversion.

Cytokinesis block (if used):

Cytochalasin B: 6 μg/mL

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Sprague-Dawley rats
- method of preparation of S9 mix : rats were dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, S9 fraction was extracted and stored at -90 to -70°C.
- concentration or volume of S9 mix and S9 in the final culture medium: S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM).
- quality controls of S9: all the cofactors were filter sterilized before use.

Test concentrations with justification for top dose:

Preliminary toxicity test: 1.76, 3.52, 7.04, 14.08, 28.16, 56.31, 112.63, 225.25, 450.5 and 901 μg/mL

Main tests:
-S9 mix (3 hours): 5.85, 58.46, 116.92, 129.91, 144.34, 160.38, 178.2, 198 and 220 μg/mL
+S9 mix (3 hours): 5.85, 58.46, 116.92, 129.91, 144.34, 160.38, 178.2, 198 and 220 μg/mL
-S9 mix (20 hours): 1.21, 12.13, 24.26, 30.33, 37.91, 47.39, 52.65, 58.5 and 65 μg/mL

Justification for top dose: based on the results of the preliminary toxicity test, in which overt cytotoxicity was observed above the following concentrations
-S9 mix (3 hours): 112.63 μg/mL
+S9 mix (3 hours): 112.63 μg/mL.
-S9 mix (20 hours): 56.31 μg/mL

Vehicle / solvent:

- Solvent used: DMSO (0.5 % v/v)
- Justification for choice of solvent/vehicle: the solubility of the test item in a vehicle compatible with this test system was assessed and the test item was found to be soluble at 360.4 mg/mL in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Remarks:

Positive controls were solved in sterile purified (reverse osmosis) water

Details on test system and experimental conditions:

TEST ITEM PREPARATION AND ANALYSIS:
The effects of the test item on the osmolality and pH of the culture medium were measured by analysing samples of HML media treated with either the vehicle or test item at appropriate concentrations. Precipitate was assessed by eye at the end of the exposure period in treated HML media-only cultures as part of the preliminary toxicity and 3-hour main micronucleus tests.The test item was dissolved in DMSO shortly before dosing.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration:
Test item: duplicate
Positive controls: duplicate
Solvent controls: quadruplicate

METHOD OF TREATMENT/ EXPOSURE:
- Exposure duration/duration of treatment: 3-Hour treatment in the absence and presence of S9 Mix and 20-Hour treatment in the absence of S9 Mix
- Method:
3-hour exposure:
Lymphocyte cultures were incubated for approximately 48 hours following stimulation with PHA before the test item was added. Before treatment all cultures were centrifuged and re-suspended in the required volume of fresh medium. Test item preparations were added to cultures at 0.5% v/v. Cultures were incubated at 34 to 39°C for 3 hours. The cells were centrifuged and the medium was replaced with fresh medium. Cytochalasin B, at a final concentration of 6 μg/mL, was then added to all cultures. The cultures were incubated for a further 17 hours until the scheduled harvest time.
20-hour exposure:
Human lymphocyte cultures were set up as previously described. A 20-hour continuous treatment (1.5 to 2 normal cell cycles) at 34 to 39°C was used in the absence of S9 mix. Test item preparations were added to cultures at 0.5% v/v in the presence of Cytochalasin B (6 μg/mL).

CELL HARVESTING AND FIXATION:
The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 34 to 39°C, cultures were then incubated for 3 minutes at 34 to 39°C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix.
The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re-suspended. A further 4 mL of fresh fixative was then added and the cells stored at 2 to 8°C until slide preparation.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinese block: 6 µg/mL Cytochalasine B for 17 hours
- Methods of slide preparation: The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. The slides were then stained using an acridine orange solution at 0.0125 mg/mL in purified water. The remaining cell cultures were stored at 2 to 8°C until slide analysis was complete.
- Methods of evaluation: fluorescence microscopy
- Number of cells evaluated: 1000 binucleate cells per culture
- Determination of cytotoxicity: using the cytokinesis-block proliferation index (CBPI index).

Evaluation criteria:

ACCEPTABILITY CRITERIA (tests that did not fulfill the required criteria were rejected and therefore are not reported):
- The concurrent vehicle control must be considered acceptable for addition to the laboratories historical vehicle control database (lie within or close to the 95% confidence limits).
- Concurrent positive controls must induce responses that are compatible with the laboratories historical positive control database and produce statistically significant increases compared with the concurrent vehicle control.
- The criteria for selection of the top dose concentration are consistent with those outlined in the study plan.

EVALUATION CRITERIA:
The test item was considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
- The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% confidence limit).

A negative response was claimed if in any of the experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical vehicle control data (below the 95% confidence limit).

Statistics:

An arcsine square-root transformation was used to transform the data. Test item treated groups were then compared to control using Williams’ tests. Positive controls were compared to control using t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.

Statistical significance was declared at the 5% level for all tests.

Data were analyzed using SAS (SAS Institute 2002) and Quasar (Quasar 1.5 2017).

Key result

Species / strain:

lymphocytes: human blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

51.3% cytotoxicity at 129.91 μg/m (3-hour exposure); 56.8% cytotoxicity at 47.39 μg/mL (20-hour exposure)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human blood

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

52.7% cytotoxicity at 116.92 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control at either concentration
- Data on osmolality: for medium dosed with the test item at 1802 μg/mL fluctuations in the osmolality of the medium of more than 50 mOsmol/kg were observed. At 901 μg/mL no fluctuations in osmolality of the medium of more than 50 mOsmol/kg were observed.

- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

PRELIMINARY TOXICITY TEST:
- Precipitation was observed at the end of treatment at and above 225.25 μg/mL
- Overt cytotoxicity was observed above 112.63 μg/mL (3-hour treatment, with and without S9-mix) and above 56.31 μg/mL (20-hour treatment, without S9).

MAIN TEST:
- Precipitation:
-S9, 3-h: 220 μg/mL
+S9, 3-h: 220 μg/mL
-S9, 3-h: not observed at up to the highest tested concentration

- Cytotoxicity:
-S9, 3-h: 51.3% cytotoxicity at 129.91 μg/mL
+S9, 3-h: 52.7% cytotoxicity at 116.92 μg/mL
-S9, 20-h: 56.8% cytotoxicity at 47.39 μg/mL

- Concentrations selected for micronucleus analysis:
-S9, 3-h: 5.85, 116.92 and 129.91 μg/mL.
+S9, 3-h: 5.85, 58.46 and 116.92 μg/mL.
-S9, 20-h: 1.21, 30.33 and 47.39 μg/mL.

- MIcronucleus analysis: in the absence of S9-mix (3-h and 20-h treatment) the test item caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear dose-concentration relationship.
In the presence of S9-mix (3-h treatment) the test item caused statistically significant increases in the number of binucleate cells containing micronuclei at 58.46 and 116.92 μg/mL, these increases were associated with a statistically significant linear trend. However, because the mean micronucleus frequencies were within the laboratory historical 95% confidence limits the increases observed were considered to be of questionable biological relevance and the results negative overall.

HISTORICAL CONTROL DATA:
- Positive historical control data: for all treatments, the positive control compounds caused statistically significant increases in the number of binucleate cells containing micronuclei, compatible with the laboratory’s historical positive control data, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.
- Negative (solvent/vehicle) historical control data: All of the mean micronucleus frequencies for the solvent cultures were within the laboratory historical 95% confidence limits.

Conclusions:

An in vitro micronucleus assay performed according to OECD 487 and GLP principles showed that 9-Fluorenone is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A combined in vivo Micronucleus/ Comet assay was performed with 9-Fluorenone in rats according to OECD/EC guidelines and GLP principles. The study was performed as part of the hazard identification of the substance for its use in food contact applications for products regulated by the USFDA and the European Commission (with scientific support from EFSA).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Combined Micronucleus and Comet assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2018 - 19 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed as part of its hazard identification for use in food contact applications for products regulated by the USFDA and the European Commission (with scientific support from EFSA).

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Commission Regulation No. 2017/735. Method B.62: Mutagenicity – In Vivo Mammalian Alkaline Comet Assay. OJ L 112/180

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 05/06/2018

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Correction factor for formulation: None (tested as supplied).
The pH was recorded in the raw data.

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: ca 47-53 days old (preliminary and main toxicity test, males and females)
- Weight at study initiation: 180-194 g (males) and 170-177 g (females) (preliminary toxicity test); 172-212 g (males) and 154-191 g (females) (main test);
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: in cages seperated by sex; Animals were given access to small soft white untreated wood chew blocks, a red plastic shelter for environmental enrichment.
- Diet: Free access to pelleted Envigo Teklad 2014C diet
- Water: tap water, ad libitum
Food, chew blocks and tap water were routininely analysed for quality at source.
- Acclimation period:minimum of 5 days

ENVIRONMENTAL CONDITIONS (set target ranges)
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5% methylcellulose
- Batch no.: MKBN2740V and SLBR8963V (Sigma-Aldrich)
- Dose volume: 10 mL/kg (test concentrations: 50, 100 and 200 mg/mL)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Accurately weighed test item was ground in a mortar with a pestle. Accurately measured vehicle was added gradually during continuous rubbing. The resulting formulations were then homogenised for 5 minutes and magnetically stirred.

Stability and homogeneity of the test item in the vehicle were not determined in this test. Chemical analysis of dosing formulations for achieved concentration was not performed in this study.

Duration of treatment / exposure:

Three administrations within 48 hours

Frequency of treatment:

The test item was administered on three occasions, the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose, approximately 3 hours before sampling.

Post exposure period:

3 hours

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Comet: Three males and three females were treated with ethyl methanesulfonate at 200 mg/kg bw/day once approximately 3 hours prior to termination at a dose volume of 10 mL/kg.
Micronucleus: Positive controls from a different study were used in which 3 rats were dosed with 20 mg/kg bw/day Cyclophosphamide.

Tissues and cell types examined:

Comet assay: Liver and duodenum;
Micronucleus: Bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Comet Phase
Frosted-end glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer. Sections of the liver and duodenum were placed into ice cold mincing solution, all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using a tissue specific method. For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75μL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped.
Comet slides were prepared from all cell suspensions. Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored at 2 - 8ºC overnight prior to electrophoresis. Sections of the liver and duodenum were stored in 10% buffered formalin and stored.
Electrophoresis: after alkali unwinding at 18 V, approximately 300 mA (between 0.7 and 1.0 V/cm) for 30 minutes.
Microscopic Examination of Comet Phase: Fifty cells were scored per slide to give a total number of 150 cells per tissue per animal.

Micronucleus Phase
One femur was dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the heads of the femurs removed from each bone. The bone marrow of one femur from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal bovine calf serum by aspiration. The resulting cell suspensions were centrifuged at 1000 rpm for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal bovine calf serum to facilitate smearing in the conventional manner on glass microscope slides. The slides were fixed in methanol and allowed to air dry. They were then rinsed in purified water and stained using an acridine orange solution at 0.0125 mg/mL and stored at room temperature in the dark until required. Prior to scoring the slides were wet mounted with coverslips using purified water.

Microscopic Examination of Micronucleus Phase: Four thousand polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal. The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

Evaluation criteria:

Comet: a test item is considered to be clearly positive if: a) at least one of the test doses exhibits a statistically significant increase compared with the concurrent vehicle control, b) the increase is dose-related when evaluated with an appropriate trend test, and c) any of the results are outside the distribution of the historical vehicle control data. A test item is considered clearly negative if: a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control, b) there is no concentration-related increase when evaluated with an appropriate trend test and c) all results are inside the distribution of the historical vehicle control data. The test item is then considered unable to induce DNA strand breakage in the tissues studied in this test system.
Micronucleus: a test item is considered clearly negative if, in all experimental conditions examined: a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent vehicle control, b) There is no dose-related increase at any sampling time when evaluated by an appropriate trend test, c) All results are inside the distribution of the historical vehicle control data (95% confidence limits), and d) Bone marrow exposure to the test item(s) occurred. A test chemical is considered clearly positive if: a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent vehicle control, b) This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and c) Any of these results are outside the distribution of the historical vehicle control data (95% confidence limits).

Statistics:

Comet Phase
Separate analyses were performed for the control groups and the low, mid and high dose group, and for the control group and the high dose group. Sexes were analysed separately.
For the control groups and the low, mid and high dose group, Bartlett’s test for variance homogeneity (Bartlett 1937) was applied.
If this test was not significant at the 1% level, then parametric analysis was applied. The F1 approximate test was applied to Groups 1 to 4. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test. The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, MSE. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/MSE which can be compared with standard tables of the F distribution with 1 and error degrees of freedom. If the F1 test was not significant at the 1% level, two-tailed Williams' test for a monotonic trend was applied to compare the low, mid and high dose group to the vehicle control group; otherwise two-tailed Dunnett's test was performed instead.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

Comet assay, liver

Toxicity:

yes

Remarks:

Slight body weight loss in all treated animals (not seen in controls)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Sex:

male/female

Genotoxicity:

positive

Remarks:

Comet assay, duodenum

Toxicity:

yes

Remarks:

Slight body weight loss in all treated animals (not seen in controls)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: See remark

Remarks:

Observed increases in DNA strand breaks are directly associated with an increase in hedgehog cells. It is therefore considered unlikely that the DNA strand breaks observed in the duodenum are representative of a true genotoxic response.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

Micronucleus assay

Toxicity:

yes

Remarks:

Slight body weight loss in all treated animals (not seen in controls)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No mortalities were observed throughout the duration of the test.
At 1000 mg/kg bw/day pilo-erection was observed in one female animal. No clinical signs of toxicity were observed for the vehicle control, positive control, or any other animal administered 9-Fluorenone at 500, 1000 and 2000 mg/kg bw/day in both male and female animals.
Small incidences of bodyweight loss were observed in all groups throughout the main test. Group mean body weight loss of 3.1%, 2.8% and 1.7% were observed from day 1 to day 2 at treatment in male animals administered 9-Fluorenone at 500, 1000 and 2000 mg/kg bw/day respectively. Group mean body weight loss of 2.7%, 3.6% and 1.7% were observed in female animals administered 9-Fluorenone at 500, 1000 and 2000 mg/kg/day respectively. No body weight loss was noted in control animals.
Histopathology showed that there was no detectable increase in apoptotic/necrotic cells in the duodenum related to treatment with 9-Fluorenone.

Systemic exposure to 9-Fluorenone was confirmed in male and female animals administered with the test item at 500, 1000 and 2000 mg/kg bw/day. The group mean plasma concentration of 9-Fluorenone in male animals administered with the test item at 500, 1000 and 2000 mg/kg bw/day was 359.0, 631.4 and 430.6 ng/mL, respectively. The group mean plasma concentration of 9-Flourenone in female animals administered with the test item at 500, 1000 and 2000 mg/kg bw/day was 639.2, 650.8 and 702.6 ng/mL, respectively.

Comet assay (tabular results included below):
The vehicle control group mean and median % tail intensity (TI) values for the liver and duodenum of both male and female animals were within the 95% confidence limits of the current vehicle historical control range for the individual tissues. The positive control compound, EMS, produced a statistically significant increase (p<0.001) in the group median % TI when compared to the vehicle control values in both tissues analysed and all % TI values were comparable to the positive control range.
Liver: There were no statistically significant increases in the group median % TI in the liver of male Crl:CD(SD) rats administered 9-Fluorenone at 500 mg/kg bw/day, compared to vehicle control values. The group mean and median % TI values from all treatment groups were within the 95% confidence limits of the current vehicle historical control range. There was a statistically significant increase (p<0.05) in the group median % TI in the liver of male Crl:CD(SD) rats administered 9-Fluorenone at 1000 and 2000 mg/kg bw/day, compared to vehicle control values. The group mean and median % TI values were with the 95% confidence limits of the current vehicle historical control range (attached below) and therefore the statistical significance observed was not considered to be biologically relevant.
There were no statistically significant increases in the group median % TI in the liver of female Crl:CD(SD) rats administered 9-Fluorenone at 500 and 1000 mg/kg bw/day, compared to vehicle control values. The group mean and median % TI values from all treatment groups were within the 95% confidence limits of the current vehicle historical control range. There was a statistically significant increase (p<0.05) in the group median % TI in the liver of female Crl:CD(SD) rats administered 9-Fluorenone at 2000 mg/kg/day, compared to vehicle control values. The group mean and median % TI values were with the 95% confidence limits of the current vehicle historical control range and therefore the statistical significance observed was not considered to be biologically relevant.
Duodenum: There were no statistically significant increases in the group median % TI in the duodenum of male or female Crl:CD(SD) rats administered 9-Fluorenone at 500 mg/kg bw/day, compared to vehicle control values. The group mean, and median % TI values from all treatment groups were within the 95% confidence limits of the current vehicle historical control range. There was a statistically significant increases (p<0.001) in the group median % TI were observed in the duodenum of male and female Crl:CD(SD) rats administered 9-Fluorenone at 1000 and 2000 mg/kg bw/day. The group mean and median % TI values for dose groups
administered 9-Fluorenone at 1000 and 2000 mg/kg bw/day were outside of the 95% confidence limits of the current vehicle historical control range.
There was a small increase in the number of hedgehog cells observed in the liver of female Crl:CD(SD) rats administered 9-Fluorenone at 2000 mg/kg bw/day, compared to the concurrent vehicle control (group mean: vehicle control, 500, 1000 and 2000 mg/kg bw/day: 0.0, 0.0, 0.0 and 3.6 respectively). There was a dose related increase in the number of hedgehog cells observed in the duodenum of male Crl:CD(SD) rats administered 9-Fluorenone at 500, 1000 and 2000 mg/kg bw/day, compared to the concurrent vehicle control (group mean: vehicle control, 500, 1000 and 2000 mg/kg bw/day: 0.0, 4.2, 17.2 and 49.4 respectively).
There was a dose related increase in the number of hedgehog cells observed in the duodenum of female Crl:CD(SD) rats administered 9-Fluorenone at 500, 1000 and 2000 mg/kg bw/day, compared to the concurrent vehicle control (group mean: vehicle control, 500, 1000 and 2000 mg/kg/day: 0.0, 2.8, 17.0 and 38.8 respectively).

Micronucleus assay (tabular results included below):
The data for the concurrent vehicle control (group mean %MPCE) were within the ranges determined by the laboratory historical control data (95% confidence limits). There were no statistically significant increases in the group %MPCE observed in either male or female Crl:CD(SD) rats administered 9-Fluorenone at any dose level, compared to vehicle control values. All group mean values were within the current vehicle historical range (95% confidence limits).
There were no significant increases in MNCE observed in male or female Crl:CD(SD) rats administered 9-Fluorenone at any dose level. The data for the concurrent vehicle control (group mean %PCE) were within the ranges determined by the laboratory historical control data (95% confidence limits). There were no statistically significant decreases in the group %PCE observed in male or female Crl:CD(SD) rats administered 9-Fluorenone at any dose level, compared to vehicle control values. The group mean %PCE values were all within the current vehicle historical control range (95% confidence limits).

Preliminary toxicity test

No mortalities were observed. At 2000 mg/kg/day clinical signs of toxicity observed in both male and female animals included flattened and hunched posture, partially closed eyelids, pilo-erection, decreased

activity, unsteady and elevated gait and reduced body tone. Some incidences of body weight loss were observed throughout the preliminary toxicity testing. Overall body weight loss (Day 1 to Termination) was observed in both male and female animals (male range: 7 - 7.7%, female range: 5.1 – 7.4%). The individual and group mean %PCE values in bone marrow smears of male and female animals administered 9-fluorenone at 2000 mg/kg/day were within the current vehicle historical control range. The plasma concentration of 9-Flourenone in the single male sample analysed was 318 ng/mL. The group mean plasma concentration of 9-Flourenone in female animals was 1034.5 ng/mL (this significant difference between the sexes prompted the inclusion of both sexes in the main study).

Conclusions:

Based on the results of a combined in vivo Micronucleus/ Comet assay performed with 9-Fluorenone in rats according to OECD/EC guidelines and GLP principles, it is concluded that 9-Fluorenone did not show genotoxicity in the liver and bonemarrow of male and female rats. 9-Fluorenone has shown evidence of causing an increase in DNA strand breaks in the duodenum of exposed rats. However, as these increases in DNA strand breaks are observed together with an increase in hedgehog cells, it is considered unlikely that the DNA strand breaks observed in the duodenum are representative of a true genotoxic response.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The potential genotoxicity of 9-Fluorenone was tested in a combined in vivo Micronucleus/ Comet assay performed in male and female rats according to OECD/EC guidelines and GLP principles. Base don the results it is concluded that 9-Fluorenone did not show genotoxicity in the liver and bonemarrow of male and female rats. 9-Fluorenone has shown evidence of causing an increase in DNA strand breaks in the duodenum of exposed rats. However, as these increases in DNA strand breaks are observed together with an increase in hedgehog cells, it is considered unlikely that the DNA strand breaks observed in the duodenum are representative of a true genotoxic response.

All data on genetic toxicology were used in a weight-of-evidence approach to conclude on genotoxic properties. This evaluation is attached as supporting material in Section 7.6.2.

Justification for classification or non-classification

Based on the available data, 9-Fluorenone is not classified for genotoxicity according to Regulation (EC) No 1272/2008.