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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-09-25 to 1996-02-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

26 May 1983

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lymphocytes from peripheral blood

For lymphocytes:
- Sex, age and number of blood donors: not specified
- Whether whole blood or separated lymphocytes were used: yes, whole blood
- Whether blood from different donors were pooled or not: two different donors, male in experiment 1 and female in experiment 2
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA, reagent grade) 10 µg/mL

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9-Mix
- source of S9: The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, Annapolis, Maryland, USA.
- concentration or volume of S9 mix and S9 in the final culture medium: Glucose-phosphate (180 mg/mL), NADP (25 mg/mL), 150 mM KCl and rat liver S-9 (3.3) were mixed in the ratio 1:1:1:2. An aliquot of the resulting S-9 mix was added to each cell culture containing ,the test article to achieve the required final concentration in a total of 10 mL. The final concentration of liver homogenate in the test system was 2 %.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch was checked by the manufacturer for sterility, protein content (minimum 32 mg/mL), ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Experiment 1: 0, 28.51, 40.73, 58.19, 83.13, 118.8, 169.7, 242.4, 346.2, 494.6, 706.6, 1009, 1443 µg/mL
Experiment 2: 0, 342.2, 456.3, 608.3, 811.1, 1082, 1442 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : duplicate, controls quadruplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 3h and 20h
- Harvest time after the end of treatment (sampling/recovery times): 17h and 41h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Approximately 1 ½ hours prior to harvest, colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Cultures were then centrifuged at 200 x g for 10 minutes,. the supernatant was carefully removed and cells were resuspended in 4 mL hypotonic (0.075 M) KCl and incubated at 37°C for 15 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (200 x g, 10 minutes) and resuspension. This procedure was repeated several times (centrifuging at 1250 x g, 2-3 minutes) until the supernatants were clean. Lymphocytes were kept in fixative in the refrigerator before slides were prepared but slides were not made on the day of harvest to ensure cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative so as to give a milky suspension. Several drops of 45 % (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides. After the slides bad dried the cells were stained for 5 minutes in 4% (v/v) filtered
Giemsa stain in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Twenty-five cells from each of the selected NQO and CPA positive control cultures were analysed to ensure that the system was operating satisfactorily.
Slides from the selected treatments and from solvent controls were coded using randomly generated letters by a person not connected with the scoring of the slides. Labels bearing only the study reference number, the sex of the donor and the code
were used to cover treatment details on the slides. Each experiment was analysed by two analysts only. One hundred metaphases from each code were analysed for chromosome aberrations. Only cells with 44-46 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 46 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately. Classification of structural aberrations was based on the scheme described by ISCN. Observations were recorded on raw data sheets
with the microscope stage coordinates of any aberrant cell. After completion of microscopic analysis, data were decoded. The aberrant cells in each culture were categorised as follows:
1) cells with structural aberrations including gaps
2) cells with structural aberrations excluding gaps
3) polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in negative control cultures were used to determine whether the assay was acceptable or not. The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. The proportion of cells in category 2 for each test treatment condition, was compared with the proportion in concurrent negative controls using Fisher's exact test. Probability values of p ≥ 0.05 were accepted as significant.
The proportions of cells in categories 1, 2 and 3 were examined in relation to historical negative control (normal) ranges.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)

Rationale for test conditions:

As recommended by the test guideline

Evaluation criteria:

Evaluation criteria
The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and
2) . the proportion of cells with structural aberrations at such doses exceeded the normal range, and
3) the results were confirmed in the second experiment.

A positive result only at the delayed harvest or pulse -S-9 treatment in Experiment 2 was to be taken as evidence of clastogenicity provided criteria 1 and 2 were met. Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations, not exceeding the normal range or occurring only at very high or very toxic concentrations, were likely to be concluded as "equivocal". Full assessment of the biological importance of such increases is likely only to be possible with reference to data from other test systems. Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

ZK 39.294: summary of the numbers and types of structural aberrations observed
20+0 hours, -S9, Experiment 1
Treatment (µg/mL)	Rep	Cells*	g	Chr del	Chr exch	Ctd del	Ctd exch	Other	Abs +g	Abs -g
Solvent	A	100	2	0	1	1	0	0	4	2
	B	100	5	1	0	2	0	0	8	3
	A+B	200	7	1	1	3	0	0	12	5
706.6	A	100	8	2	0	7	1	0	18	10
	B	100	5	3	0	9	0	0	17	12
	A+B	200	13	5	0	16	1	0	35	22
1009	A	100	0	1	0	6	0	0	7	7
	B	100	7	1	0	9	0	0	17	10
	A+B	200	7	2	0	15	0	0	24	17
1442	A	100	6	7	0	14	1	0	28	22
	B	100	8	2	0	17	0	0	27	19
	A+B	200	14	9	0	31	1	0	55	41
NQO, 2.5	A	25	0	2	0	19	4	1	26	26
	B	25	1	1	1	9	4	0	16	15
	A+B	50	1	3	1	28	8	1	42	41
ZK 39.294: summary of the numbers and types of structural aberrations observed
3+17 hours, +S9, Experiment 1
Treatment (µg/mL)	Rep	Cells*	g	Chr del	Chr exch	Ctd del	Ctd exch	Other	Abs +g	Abs -g
Solvent	A	100	1	0	0	1	0	0	2	1
	B	100	2	2	0	1	0	0	5	3
	A+B	200	3	2	0	2	0	0	7	4
706.6	A	100	3	0	0	2	0	0	5	2
	B	100	3	2	0	1	0	0	6	3
	A+B	200	6	2	0	3	0	0	11	5
1009	A	100	6	0	0	1	0	0	7	1
	B	100	4	0	0	0	0	0	4	0
	A+B	200	10	0	0	1	0	0	11	1
1442	A	100	2	0	0	1	0	0	3	1
	B	100	4	0	0	2	0	0	6	2
	A+B	200	6	0	0	3	0	0	9	3
CP, 25	A	25	4	4	0	20	0	0	28	24
	B	25	8	5	0	15	2	3	33	25
	A+B	50	12	9	0	35	2	3	61	49
ZK 39.294: summary of the numbers and types of structural aberrations observed
.20+0 hours, -S9, Experiment 2
Treatment (µg/mL)	Rep	Cells*	g	Chr del	Chr exch	Ctd del	Ctd exch	Other	Abs +g	Abs -g
Solvent	A	100	0	1	0	0	0	0	1	1
	B	100	3	0	0	1	0	0	4	1
	A+B	200	3	1	0	1	0	0	5	2
811.1	A	100	2	0	0	3	0	0	5	3
	B	100	10	7	0	20	0	0	37	27
	A+B	200	12	7	0	23	0	0	42	30
1082	A	100	2	2	0	4	0	0	8	6
	B	100	24	4	0	52	0	0	80	56
	A+B	200	26	6	0	56	0	0	88	62
1442	A	100	7	5	0	14	0	1	27	20
	B	100	17	2	0	30	1	2	52	35
	A+B	200	24	7	0	44	1	3	79	55
NQO, 2.5	A	25	2	2	0	1	5	0	10	8
	B	25	5	1	0	5	2	1	14	9
	A+B	50	7	3	0	6	7	1	24	17
ZK 39.294: summary of the numbers and types of structural aberrations observed
3 + 17 hours, +S9, Experiment 2
Treatment (µg/mL)	Rep	Cells*	g	Chr del	Chr exch	Ctd del	Ctd exch	Other	Abs +g	Abs -g
Solvent	A	100	1	1	0	2	0	0	4	3
	B	100	0	0	1	1	0	0	2	2
	A+B	200	1	1	1	3	0	0	6	5
811.1	A	100	3	0	0	3	0	0	6	3
	B	100	1	1	0	3	1	0	6	5
	A+B	200	4	1	0	6	1	0	12	8
1082	A	100	0	4	0	2	0	0	6	6
	B	100	0	1	0	1	0	0	2	2
	A+B	200	0	5	0	3	0	0	8	8
1442	A	100	2	0	0	1	0	0	3	1
	B	100	1	1	0	1	0	0	3	2
	A+B	200	3	1	0	2	0	0	6	3
CPA, 25	A	25	3	7	0	3	1	0	14	11
	B	25	4	4	0	10	2	0	20	16
	A+B	50	7	11	0	13	3	0	34	27
ZK 39.294: summary of the numbers and types of structural aberrations observed
3+41 hours, +S9, Experiment 2
Treatment (µg/mL)	Rep	Cells*	g	Chr del	Chr exch	Ctd del	Ctd exch	Other	Abs +g	Abs -g
Solvent	A	100	2	0	0	1	0	0	3	1
	B	100	0	0	1	0	0	0	1	1
	A+B	200	2	0	1	1	0	0	4	2
1442	A	100	1	0	0	0	0	0	1	0
	B	100	0	2	0	1	0	0	3	3
	A+B	200	1	2	0	1	0	0	4	3
ZK 39.294: summary of the numbers and types of numerical aberrations observed
20+0 hours, -S9, Experiment 1 Donor sex: Male
Treatment (µg/mL)	Rep	Cells**	H	E	P	Tot abs	% with num abs
Solvent	A	100	0	0	0	0	0
	B	101	0	0	1	1	1.0
	A+B	201	0	0	1	1	0.5
706.6	A	100	0	0	0	0	0
	B	101	0	0	1	1	1.0
	A+B	201	0	0	1	1	0.5
1009	A	100	0	0	0	0	0
	B	101	0	1	0	1	1.0
	A+B	201	0	1	0	1	0.5
1442	A	100	0	0	0	0	0
	B	100	0	0	0	0	0
	A+B	200	0	0	0	0	0
NQO, 2.5	A	25	0	0	0	0	0
	B	25	0	0	0	0	0
	A+B	50	0	0	0	0	0
3+ 17 hours, +S9, Experiment 1 Donor sex: Male
Treatment (µg/mL)	Rep	Cells**	H	E	P	Tot abs	% with num abs
Solvent	A	100	0	0	0	0	0
	B	103	2	0	1	3	2.9
	A+B	203	2	0	1	3	1.5
706.6	A	101	1	0	0	1	1.0
	B	100	0	0	0	0	0
	A+B	201	1	0	0	1	0.5
1009	A	100	0	0	0	0	0
	B	100	0	0	0	0	0
	A+B	200	0	0	0	0	0
1442	A	101	0	0	1	1	1.0
	B	101	1	0	0	1	1.0
	A+B	202	1	0	1	2	1.0
CPA, 25	A	25	0	0	0	0	0
	B	25	0	0	0	0	0
	A+B	50	0	0	0	0	0
ZK 39.294: Summary of the numbers and types of numerical aberrations observed
20+0 hours, -S9, Experiment 2 Donor sex: Female
Treatment (µg/mL)	Rep	Cells**	H	E	P	Tot abs	% with num abs
Solvent	A	100	0	0	0	0	0
	B	102	2	0	0	2	2.0
	A+B	202	2	0	0	2	1.0
811.1	A	100	0	0	0	0	0
	B	100	0	0	0	0	0
	A+B	200	0	0	0	0	0
1082	A	100	0	0	0	0	0
	B	100	0	0	0	0	0
	A+B	200	0	0	0	0	0
1442	A	100	0	0	0	0	0
	B	100	0	0	0	0	0
	A+B	200	0	0	0	0	0
NQO, 2.5	A	25	0	0	0	0	0
	B	25	0	0	0	0	0
	A+B	50	0	0	0	0	0
ZK 39.294: Summary of the numbers and types of numerical aberrations observed
3+ 17 hours, +S9, Experiment 2 Donor sex: Female
Treatment (µg/mL)	Rep	Cells**	H	E	P	Tot abs	% with num abs
Solvent	A	100	0	0	0	0	0
	B	101	0	1	0	1	1.0
	A+B	201	0	1	0	1	0.5
811.1	A	101	1	0	0	1	1.0
	B	101	0	0	1	1	1.0
	A+B	202	1	0	1	2	1.0
1082	A	100	0	0	0	0	0
	B	100	0	0	0	0	0
	A+B	200	0	0	0	0	0
1442	A	100	0	0	0	0	0
	B	100	0	0	0	0	0
	A+B	200	0	0	0	0	0
CPA, 25	A	25	0	0	0	0	0
	B	25	0	0	0	0	0
	A+B	50	0	0	0	0	0
ZK 39.294: Summary of the numbers and types of numerical aberrations observed
Donor sex: Female 3+41 hours, +S9, Experiment 2
Treatment (µg/mL)	Rep	Cells**	H	E	P	Tot abs	% with num abs
Solvent	A	100	0	0	0	0	0
	B	100	0	0	0	0	0
	A+B	200	0	0	0	0	0
1442	A	100	0	0	0	0	0
	B	100	1	0	1	2	2.0
	A+B	200	1	0	1	2	1.0
* = Total cells examined for structural aberrations ** = Total cells examined for numerical aberrations

Applicant's summary and conclusion

Conclusions:

It is concluded that Trioxabicyclooctan induced chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 10 mM, and this effect was only seen in the absence of S-9 under the conditions of the test.

Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration) according to OECD test guideline 473 (1983), human peripheral lymphocytes were exposed to Trioxabicyclooctan, (100 % a.i.), in DMSO at concentrations of Experiment 1: 0, 28.51, 40.73, 58.19, 83.13, 118.8, 169.7, 242.4, 346.2, 494.6, 706.6, 1009, 1443 µg/mL in the first experiment for 3h and 20 h and at  0, 342.2, 456.3, 608.3, 811.1, 1082, 1442 µg/mL for the 3h treatment with 17h and 41 h post-treatment recovery with and without metabolic activation [S9-liver mix].

Trioxabicyclooctan was tested up to the limit concentration. Cultures treated with the test item in the absence of S9 mix showed statistically significant and biologically relevant increases of numbers of metaphases with aberrations, starting at 706.6 µg/mL without S9 mix. Positive controls induced the appropriate response. There was evidence of Chromosome aberrations induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.