Fluoren-9-one

Genetic toxicity: in vivo

Administrative data

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).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

Fluoren-9-one

EC Number:

207-630-7

EC Name:

Fluoren-9-one

Cas Number:

486-25-9

Molecular formula:

C13H8O

IUPAC Name:

9H-fluoren-9-one

Test material form:

solid: crystalline

Remarks:

yellow

Details on test material:

Purity: >99%

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.

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Mid dose group

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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.

Test results 3

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).

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.