4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-01-24 to 2003-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopter 21 July 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane

EC Number:

421-750-9

EC Name:

4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane

Cas Number:

57280-22-5

Molecular formula:

C7H12O3

IUPAC Name:

4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hanlbm: WIST (SPF)

Details on species / strain selection:

The rat is an animal which has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the UDS test. In addition, the rat is an experimental animal in many physiological, pharmacological, and toxicological studies. Data from such experiments may also be useful for the design and the performance of the UDS test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf
- Age at study initiation: 6-10 weeks
- Weight at study initiation: Mean value 183.6 g (SD*± 15.1 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single, Makrolon Type II, with wire mesh top (Ehret, D-79312 Emmendingen) with granulated soft wood bedding (Altromin, D-32791 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet (Altromin, D-32791 Lage/Lippe) ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.9% NaCl solution. The vehicle was chosen to itsnon-toxicity for the animals. The animals received a single standard volume of 10 mL/kg body weight orally

Duration of treatment / exposure:

treatment once and isolation of hepatocytes after 2 and 16 h

Frequency of treatment:

once

Post exposure period:

2 and 16 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

0 mg/kg bw/day

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Positive control(s):

none; no data; 2-acetylaminofluorene; N, N`-dimethylhydrazinehydrochloride
- Route of administration: oral
- Doses / concentrations: 40 mg/kg bw and 100 mg/kg bw, respectively

Examinations

Tissues and cell types examined:

liver, hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with two animals per group and sex under identical conditions as in the UDS study concerning: starvation period, animal strain, vehicle, route, frequency, and volume of administration. The animals were treated orally (gavage) and examined for acute toxic symptoms at intervals of 1 h, 2-4 h, 6 h, 24 h, and 48 h after administration of the test item.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment} before receiving the test item, the positive or the vehicle control substance. Water was available ad libitum. Before the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the body weigiht of the animals. The animals received the test item once. Four animals (males) were treated per dose group. After anaesthetizing the rats with Na-thiopental (Trapanal, Byk Gulden, D-78467 Konstanz) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL, D-76344 Eggenstein} supplemented with collagenase (0.05 % (w/v), Boehringer Mannheim, D-68305 Mannheim) adjusted to pH 7.4 and maintained at 37° C

DETAILS OF SLIDE PREPARATION: The washed hepatocytes were centrifuged and transferred into Williams medium E. At least three cultures were established from each animal. After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO 2 humidified incubator at 37° C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR (5 µCi/mL, specific activity 20 Ci/mmol; New England Nuclear, D-63033 Dreieich) in 2.0 mL culture medium (WME, 1 % (v/v), FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried. The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 (Tecnomara, D-35463 Fernwald) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4° C. The photographic emulsion was then developed with KODAK Dektol Developer (Tecnomara, D-35463 Fernwald) at room temperature, fixed in TETENAL (Tetenal, D-22844 Norderstedt) and stained with hematoxylin/eosin.

METHOD OF ANALYSIS: Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grains of the most heavily lableled nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal; and 50 cells per slide were evaluated. Heavily radiolabeled cells undergoing replicative DNA synthesis were excluded from counting. Three animals per group were evaluated as described above.
Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts. A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points. A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains. Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A test item producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system.

Evaluation criteria:

see above

Statistics:

non-parametric Mann-Whitney test

Results and discussion

Test results

Key result

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: 2000 mg/kg bw
- Solubility: not reported
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur.
- Evidence of cytotoxicity in tissue analysed: No
- Rationale for exposure: Based on preliminary acute toxicity testing

Any other information on results incl. tables

Treatment	Period	Animal No.	Viability* [%]	Number of isolated cells [x E+06]
Vehicle control 0.9% NaCl solution	2 h	2	75	341
		3	80	216
		4	90	576
500 mg/kg bw	2 h	5	79	111
		6	75	229
		7	92	354
2000 mg/kg bw	2 h	9	72	310
		10	70	294
		11	81	126
40 mg/kg bw DMH	2 h	13	84	185
		15	76	247
		16	84	139
Vehicle control 0.9% NaCl solution	16 h	17	77	370
		18	83	593
		19	90	171
500 mg/kg bw	16 h	22	76	365
		23	91	578
		24	75	420
2000 mg/kg bw	16 h	25	71	540
		26	78	270
		27	86	357
100 mg/kg bw 2-AAF	16 h	29	72	266
		30	81	150
		31	80	164

 

Treatment	Period	Grains per nucleus		Grains per cytoplasmic area		Net grains per nucleus
		Mean *	± SD**	Mean *	± SD**	Mean *	± SD**
Vehicle control 0.9% NaCl solution	2 h	9.41	4.69	14.58	5.29	-5.17	5.13
1000 mg/kg bw	2 h	10.68	5.00	17.65	7.24	-6.97	5.80
2000 mg/kg bw	2 h	13.77	7.01	18.44	7.27	-4.67	6.69
40 mg/kg bw DMH	2 h	17.91	11.19	12.02	6.83	5.89	7.82
Vehicle control 0.9% NaCl solution	16 h	8.86	4.30	11.40	4.49	-2.54	4.69
1000 mg/kg bw	16 h	10.20	5.40	15.47	6.29	-5.27	5.05
2000 mg/kg bw	16 h	10.23	4.14	17.25	5.42	-7.02	5.57
100 mg/kg bw 2-AAF	16 h	33.98	13.96	16.90	7.41	17.08	11.80
* Mean of 3 animals ** Standard deviation

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Trioxabicyclooctan did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

Executive summary:

In a test conducted according to OECD test guideline 486 (1995) male Wistar rats (3 animals per group) were treated with Trioxabicyclooctan at doses of 0, 1000 and 2000 mg/kg bw once by oral gavage. Hepatocytes were isolated after 2h and after 16h after administration and incubated with 3HTdR for 4 h. Subsequently the hepatocytes were cultured over night with unlabelled thymidine. After the last incubation the DNA repair was measured as incorporation of 3HTdR into the DNA of the treated cells. The amount of incorporated radioactivity is determined by silver grain counting. 

The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test item at any of the treatment periods or dose groups.

No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals. as compared to the current vehicle controls.

Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 2 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test item were consistently negative. Appropriate reference mutagens (DMH, 40 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.) were used as positive controls. In vivo treatment with DMH or 2-AAF revealed distinct increases in the number of nuclear and net grain counts as indication of induced DNA-repair.