3-phenyl-7-[4-(tetrahydrofurfuryloxy)phenyl]-1,5-dioxa-s-indacen-2,6-dione

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: 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:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

no

Principles of method if other than guideline:

Butterworth , B.E. et al. (1987). A protocol and guide for the in vivo rat hepatocyte DNA-repair assay. Mutation Research 189: 123-133.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

3-phenyl-7-[4-(tetrahydrofurfuryloxy)phenyl]-1,5-dioxa-s-indacen-2,6-dione

EC Number:

413-330-9

EC Name:

3-phenyl-7-[4-(tetrahydrofurfuryloxy)phenyl]-1,5-dioxa-s-indacen-2,6-dione

Cas Number:

134724-55-3

Molecular formula:

Hill formula: C27H20O6 CAS formula: C27H20O6

IUPAC Name:

6-{4-[(oxolan-2-yl)methoxy]phenyl}-12-phenyl-4,10-dioxatricyclo[7.3.0.0³,⁷]dodeca-1(12),2,6,8-tetraene-5,11-dione

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 199± 7 g; range: 187 - 214 g
- Assigned to test groups randomly: yes
- Fasting period before study: for animals of the 12-16 hours sampling time feed was withheld 4.5 h prior to dosing; animals of the 2-4 hours sampling time had access to a limited amount of food during the night before dosing (approx. 7 g/rat). Feeding regimen was chosen to facilitate the process of perfusion.
- Housing: 5 animals per cage in polycarbonate cages (type MIV height 18 cm) containing sterilised saw dust as bedding material (Litalabo; S.P.P.S., Argenteuil, France); paper bedding was provided as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiaeten GmbH, Soest, Germany), ad libitum. Feeding regimen during dosing mentioned before.
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 6 days before treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-22.2
- Humidity (%): 42-58
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 100, 200 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
SUMIKARON BRILLIANT RED S-BWF was suspended in corn oil (Roth, Karlsruhe, Germany). The concentrations were blended and heated to 37°C (only 12-16 hour sampling time) to obtain a homogeneous suspension. SUMIKARON BRILLIANT S-BWF concentrations were dosed within 1 hour after preparation.

Duration of treatment / exposure:

single exposure by gavage

Frequency of treatment:

once

Post exposure period:

2-4 or 12-16 hours (sampling times)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

3 per sampling time per dose (2 per vehicle and positive control group)

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene (2-AAF); N-dimethylnitrosamine (DMN)
- Justification for choice of positive control(s): commonly accepted
- Route of administration: route, frequency and volume adminstered consistent with those of the test substance
- Doses / concentrations: DMN 10 mg/kg bw in milli-Q water for the 2-4 h sampling time, 2-AAF 50 mg/kg bw in corn oil for the 12-16 h sampling time

Examinations

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose selection was based an a dose range finding study. The conditions were similar to those applied in the main test. One dose group, comprising of 3 males received a single dose of 2000 mg SUMIKARON BRILLIANT RED S-BWF/kg bw. The study duration was 2 days. During this period mortality and physical condition were recorded. A pilot perfusion was performed to examine possible hepatotoxic effects of SUMIKARON BRILLIANT S-BWF. The test substance showed no toxicity up to 2000 mg/kg bw, the highest dose required in the guidelines. Therefore, 2000 mg/kg was selected as highest dose and 1000 mg/kg was selected as the lower dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The negative control and all dose groups were each sampled at 2-4 and 12-16 hours after dosing. The positive control group which received DMN was sampled 2-4 hours after dosing, while the 2-AAF group was sampled after 12-16 hours

DETAILS OF SLIDE PREPARATION:
After liver perfusion and collagenase digestion for hepatocyte isolation dead cells were removed by centrifugation and 10e5 cells were seeded on 1 cm² coverslips in wells of a 24-well dish. Four cover slips were prepared per animal. All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80-100 % (actual range 88-95 %), containing 5.0±0.5 % CO2 in air in the dark at 37.0±1.0°C (actual range 34.9-37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations were considered not to affct the study integrity.

The cultures were labelled 1-2 hours after seeding of the cells by addition of 0.5 ml of culture medium supplemented with tritiated thymidine solution (370 KBq/ml (10µCi/ml); specific activity 66-1110 GBq/mol (18-30 x 10³ Ci/mol), Amersham Biosciences Benelux). The cultures were incubated for 4 hours at 37°C, 5.0±0.5 % CO2. After incubation, 1.0 ml of culture medium containing 0.25 x 10e-3 M unlabelled thymidine was added to each well to diminish unincorporated radioactivity.Cultures were incubated overnight (14-18 h). Thereafter the cultures were washed with HBSS and fixed with methanol-acetic acid (3:1 v/v). After fixation the coverslips were mounted on microscopic slides, dipped in Ilford K5D emulsion at 40-45°C and dried for 2 h at room temperature in the dark. After drying, the slides were placed in light tight boxes in the presence of silica gel. The photographic emulsion was exposed for 7 days at 4°C. The emulsion was developed for 4 min in Kodak D19 developer at 15°C, rinsed in Milli-RO water and fixed for 5 min in Kodak fixative. The slides were insed with running tap water and the cells were stained with haematoxylin/eosin. At least 3 scorable coverslips per animal were subjected to the autoradiographic procedure.

METHOD OF ANALYSIS:
The slides were checked for the presence of suficient cells of normal morphology to permit a meaningful assessment of UDS. Preparations were examined microscopically for signs of overt cytotoxicity (e.g. pyknosis). Grain counts were determined over the nuclei (nuclear grains, N) and the nucleus-equivalent areas over the cytoplasm (cytoplasmic grains, C) manually on the cover slips. Fifty cells were counted on each coverslip, 2 coverslips per animal (in total 100 cells per animal) and 3 animals per treatment group (except for the negative and the positive controls where 2 animals were examined).
The following criteria were used to determine if a cell was countable:
1) Cells with abnormal morphology, such as those with pyknotic or lysed nuclei, were not counted.
2) Isolated nuclei not surrounded by cytoplasm were not counted.
3) Cells with unusual staining artifacts or in th epresence of debris were not counted.
4) Heavily labelled cells in S-phase were not counted.
The normal cells observed while moving the microscope stage were counted.

Averages and standard deviations were calculated. Grain counts over nuclear areas (N) were compared to grain counts over the most heavily labelled adjacent cytoplasm area (C) of the same size as the corresponding nuclear area (Lonati-Galliani et al., 1983). The net nuclear grain count (NNG) was calculated for each cell by subtracting cytoplasmic grain counts from nuclear grain counts. The background counts of a single area from the same size as the corresponding nuclear area was recorded per coverslip.

Evaluation criteria:

A DNA-repair assay is considered acceptable if it meets the following criteria:
a) The viability of the hepatocytes from the negative control animals should be at least 50 %.
b) The positive control substances should produce increases in the number of net nuclear grain counts, yielding greater than or equal to 5 net nuclear grain counts per group average, and greater than or equal to 20 % of the cells responding.
c) The net nuclear grain count found in the negative controls should reasonably be within the laboratory historical control data range.
d) The selected dose range should include a toxic dose as demonstrated by the dose range finding study. In case of a non-toxic test substance the dose range should extend to 2000 mg/kg body weight or to the limit of solubility.

A test substance is considered positive in the DNA-repair assay if it yields greater than or equal to 5 net nuclear grain (NNG) counts per group average, and greater than or equal to 20 % of the cells responding (i.e. with NNG values of 5 or more). These values are based on the laboratory historical data.
A test substance is considered negative in the DNA-repair assay if it does not induce a net nuclear grain (NNG) count of at least 5 and 20 % or more of the cells responding.
In addition to the above mentioned criteria, biological relevance of the data was also considered, i.e. parameters such as an inter-animal variation, dose response relationship and cytotoxicity were taken into account. The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Statistics:

Averages and standard deviations were calculated.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: single dose of 2000 mg/kg bw
- Solubility: not indicated
- Clinical signs of toxicity in test animals: No treatment-related clinical signs or mortality were observed.
- Evidence of cytotoxicity in tissue analyzed: No macroscopic effects in the liver were observed, and the viability of the liver cells was 80 and 76 %, indicating that no hepatotoxicity would adversely affect the performance of the assay.
- Rationale for exposure: Highest dose required in the guidelines
- Observation period: 1 day


RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: Dose levels included the highest dose required in the guideline, and the oral route was chosen because of its relevance for human exposure.

None of the groups treated with the test substance nor the negative and positive control groups showed treatment-related clinical signs or mortality.

At the 2-4 hour sampling time, the viability of the hepatocytes used for the evaluation of DNA repair inducing ability was at least 77 %, indicating no direct liver toxicity. A viability of 75 and 78 % was found for the vehicle control cultures. At the 12-16 hours sampling time, the viability of the hepatocytes was at least 79 %, indicating no direct liver toxicity. A viability of 77 and 80 % was found for the vehicle control cultures.

In all slides used for grain counts sufficient cells of normal morphology were present. Preparations showed no or slight overt cytotoxicity (e.g. pyknosis ≤ 50 %). The NNG per coverslip and per animal, as well as the group average revealed no positive response in this assay at any of the dose levels. The percentage of cells in repair (repair taken as NNG ≥ 5), both per individual animal and for the group average, revealed no increase at any dose.

The NNG in the solvent-treated control cultures was within the historical control data range. Oral dosing of a male rat with DMN resulted in a NNG of 32.3 with 100 % of the cells in repair (NNG ≥ 5). Oral dosing of a male rat with 2-AAF resulted in a NNG of 32.8 with 100 % of the cells in repair.

In the scored coverslips the mean background of a single area of the same size as the corresponding nuclear area, located outside the nucleus in the cytoplasm, was always less than 11 grains, indicating that the autoradiographic procedure functioned adequately.

Any other information on results incl. tables

Results DNA repair assay:

		2-4 hours sampling time		12-16 hours sampling time
Chemical	Dose (mg/kg bw)	NNG group average	% cells in repair group average	NNG group average	% cells in repair group average
Negative control	0	0.7	0	0.8	0
DMN	10	32.3	100	-	-
2-AAF	50	-	-	32.8	100
SUMIKARON BRILLIANT RED S-BWF	1000	0.5	0	0.6	0
	2000	0.5	0	0.7	0

NNG = Net nuclear grain count. Calculated for each cell by subtracting the cytoplasmic count from the nuclear count.

Group average = average of 3 animals (controls groups: 2 animals).

% of cells in repair: repair is defined as NNG ≥ 5.

CONCLUSION:

Oral treatment of male Wistar rats with test substance dosaes of up to 2000 mg/kg bw induced no DNA repair in hepatocytes isolated 2 -4 or 12 -16 hours after dosing, respectively. Therefore, the test substance was not genotoxic in the DNA repair assay using hepatocytes obtained from male rats following in vivo exposure to the test substance under the conditions described.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative