1,3,5-trioxane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-03 to 1992-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(adopted 1984)

GLP compliance:

yes

Remarks:

(Hoechst AG, Pharma Development Central Toxicology)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-trioxane

EC Number:

203-812-5

EC Name:

1,3,5-trioxane

Cas Number:

110-88-3

Molecular formula:

C3H6O3

IUPAC Name:

1,3,5-trioxane

Details on test material:

- Name of test material (as cited in study report): 1,3,5-trioxane
- Physical state: white crystals
- Analytical purity: 99.9%
- Lot/batch No.: 438 To II
- Stability under test conditions: stable during study period
- Storage condition of test material: dark at approx. 20°C

Method

Target gene:

hprt gene, which codes for hypoxanthine-guanine phosphoribosyl transferase

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Large stocks of the mycoplasma-free V79 cell line were stored in liquid nitrogen in the laboratory of Genetic Toxicology of Hoechst AG, allowing the repeated use of the same cell culture batch for many experiments. Consequently, the parameters of the experiments remained similar because of the identical characteristics of the cells.
- Thawed stock cultures were kept at 37 °C and 5% CO2 in plastic-flasks. Seeding was carried out with about 8 to 10 x 10E+5 cells per flask in 30 ml of MEM-Medium supplement with approx. 10% fetal calf serum, containing approx. 2 mM l-glutamine and approx. 0.01 % neomycinsulfate.
- The cells were subcultured twice a week.
- For the selection of mutants the medium was supplemented with approx. 11 ug/ml thioguanine.

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix prepared from S-9 liver fraction obtained from Aroclor 1254-treated Sprague-Dawley male rats.

Test concentrations with justification for top dose:

Both, in the absence and the presence of S9 mix, following concentrations were tested:
100, 250, 500 and 900 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua bidest.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated, with and without S9 mix, cells in MEM medium (containing Hanks salts and Hepes buffer).

Negative solvent / vehicle controls:

yes

Remarks:

treated with solvent (aqua bidest.), with and without S9 mix, cells in MEM medium (containing Hanks salts and Hepes buffer).

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Migrated to IUCLID6: 1 mg/ml, dissolved in cell culture medium

Controls 2

Untreated negative controls:

yes

Remarks:

untreated, with and without S9 mix, cells in MEM medium (containing Hanks salts and Hepes buffer).

Negative solvent / vehicle controls:

yes

Remarks:

treated with solvent (aqua bidest.), with and without S9 mix, cells in MEM medium (containing Hanks salts and Hepes buffer).

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

with S9 mix

Migrated to IUCLID6: dissolved in DMSO, 7.7 µg/ml in culture medium

Details on test system and experimental conditions:

- The dose levels for the main test were selected on the basis of the results of a preliminary cytotoxicity assay;
- A stock solution containing 9% of test substance was prepared, taking into account the limit of solubility of the test substance in culture medium (10 mM, determined analytically);
- At the day of testing, dilutions were made from the stock solution until reaching the scheduled concentrations;
- Two independent experiments, with and without S9 mix, were conducted.

PROCEDURE FOR TESTING:

1)- Two days old, exponentially growing cultures which were more than 90 % confluent were trypsinated and a single cell suspension was prepared. Subsequently the cells were replated for mutagenicity testing and for determination of plating efficiency.

2)- Day 1, about 6 x 10E+5 to 10E+6 cells in 175 cm2 flasks with 30 ml medium were used for mutagenicity testing. About 400 cells in 25 cm2 flasks with 5 ml medium were used for determination of plating efficiency.

3)- Day 2, the cultures were treated with the selected concentrations of test substance for 4 hours.

4)- Day 5, the cultures for mutagenicity testing were then subcultured (4 days).

5)- Day 8, the cultures for determination of plating efficiency were fixed and stained (10% methylene blue).

6)- Day 9, the cultures for mutagenicity testing were further subcultured, first, in presence of 6-thioguanine for mutant selection, and then again for determination of plating efficiency.

7)- Day 16, colonies obtained from the subcultured cultures of the mutagenicity assay were fixed and stained.

8)- Only colonies with more than 50 cells were counted.

Evaluation criteria:

- The test substance is classified as mutagenic if it reproducibly induces with one of the test substance concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in the experiment.
- The test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

Mann-Whitney-U-Test

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

in the preliminary cytotoxicity assay, trioxane was not cytotoxic to the V79 cells, with and without S9-mix, up to the limit of solubility (i.e 10 mM). In the mutagenicity assay, no cytotoxicity was observed up to 900 µg/ml (with/without S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed

ADDITIONAL INFORMATION:
The mutagenicity assay consisted of 2 independent experiments (Table 1).

- In the second experiment and in the absence of S9 mix, a slight but statistically significant enhancement of the mutation rate over the range of the negative controls was induced at a test concentration of 500 µg/ml. In fact, a mutation frequency of 18.2 mutant colonies per 10E+6 cells was reported, versus 7.7 for the solvent control.
For the negative control, mutant frequency was 15.9;
For the positive control, mutant frequency was 811.4;
For the remaining test concentration of trioxane, the mutant frequencies were 6.1, 12.1 and 10.3, for 100, 250 and 900 µg/ml, respectively.

- In the same experiment and in presence of S9 mix, a slight but statistically significant enhancement of the mutation rate over the range of the negative controls also was seen, however at a test concentration of 250 µg/ml. In fact, a mutation frequency of 24.5 was reported, versus 12.3 for the solvent control.
For the negative control, mutant frequency was 8.9;
For the positive control, mutant frequency was 209.5;
For the remaining test concentration of trioxane, the mutant frequencies were 11.4, 17.6 and 10.3, for 100, 500 and 900 µg/ml, respectively.

- Since the findings reported were not reproducible and showed no concentration-relationship, they were considered to have no biological relevance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1.

Test group	Dose (µg/ml)	S9 mix	Mutation frequency
			Main Expt.	Repeat Expt.
Negative control	0	-	32.5	15.9
Solvent control	0	-	32.4	7.7
EMS	1000	-	893.4	811.4
Test article	100	-	28.4	6.1
Test article	250	-	24.8	12.1
Test article	500	-	11.5	18.2*
Test article	900	-	11.2	10.3
Negative control	0	+	29.9	8.9
Solvent control	5	+	38.2	12.3
DMBA	7.7	+	239.3	209.5
Test article	100	+	10.4	11.4
Test article	250	+	28.3	24.5*
Test article	500	+	21.9	17.6
Test article	900	+	38.6	10.3

Mutation frequency (mutant colonies per 10⁶cells)

EMS: ethylmethanesulphonate

DMBA: 9,10-dimethylbenzanthracene

* p < 0.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative