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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The mutagenic potential of test item was tested in cultured Chinese Hamster ovary (CHO) cells. Mutation at the hypowanthine guanine phosphoribosyl transferase (HGPRT) gene locus was measured. Mutagenicity testing of test item was performed initially in a range of Aroclor 1254 -induced rat liver homogenate (S9) concentrations (0 -10%) followed by a confirmatory experiment at 0 and 5%S9. In mutagenicity assays, test item was tested up to concentrations which induced significant cytotoxicity (>50% cell killing).
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Di(morpholin-4-yl) disulphide
EC Number:
203-103-0
EC Name:
Di(morpholin-4-yl) disulphide
Cas Number:
103-34-4
Molecular formula:
C8H16N2O2S2
IUPAC Name:
4-(morpholin-4-yldisulfanyl)morpholine

Method

Target gene:
HGPRT (hypoxanthine guanine phosphoribosyl transferase)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The subclone K1BH4 of CHO cells was used. The cells were routinely maintained in our laboratory as log growing monolayer cultures in Ham's F12 medium supplemented with heat-inactivated 10% newborn calf serum in an incubator-controlled environment of a 95% humidified atmosphere of 5% +/-1% CO2 and 95% air at 37.5°C +/-2.
Metabolic activation:
with and without
Metabolic activation system:
S9 = Rat liver homogenate induced by Aroclor 1254
Test concentrations with justification for top dose:
Without S9 : 3.4-10 µg/ml
With S9 (1, 2, 5%): 10-34 µg/ml
With S9 (10%): 3.4-10 µg/ml
Vehicle / solvent:
Methanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
methanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: benzo(a)pyrene with S9, ethyl methane sulfonate without S9
Details on test system and experimental conditions:
Cytotoxicity determination :
CHO cells were seeded in 25cm² plastic culture flasks at 0.5 x 10^6 cells per flask in growth medium 18-24 hours before treatment. On the day of treatment, the medium was changed to Ham's F12 medium without serum (5 ml) with or without activation. Volumes of 25µl of different concentrations of the test chemical were added. After an incubation of 3 hours at 37.5°C+/-2, the treatment medium was discarded. The wells were washed with 5 ml Hank's balanced salt solution and the cells were removed from the flasks by trypsinization and counted. Three aliquots of approximately 200 cells were plated per sample for determination of cloning efficiency. The plates were returned to incubation for 6-9 days. The colonies developed were fiwed with 70% methanol, stained with 10% Giemsa, and counted by hand. Cytotoxicity was expressed as relative survival.

Mutagenesis :
CHO cells were plated on the day before treatment. The next day, they were treated with test chemicals, positive controls and negative solvent control. The cells were when processed as described above for cytotoxicity determination, except that in addition to plating 200 cells for cloning efficiency, 10^6 cells per sample were plated in 10mL of subculture medium. The cells were subcutured every 2-3 days as attached cultures for 7-9 days for the expression of the mutant phenotype. Mutant selection was performed using selective medium consisting of hypoxanthine-free ham's F12 medium supplemented with mM 6TG and 5%% dialyzed newborn calf serum. Approximatesly 10^6 cells per sample were plated in 100 mm plates (5 plates, 2x10^5 cells per plate) in 8 ml of selective medium plate for mutant selection. Three aliquotsof approximately 200 cells per sample were plated in 2 ml of selective medium without 6TG for the determination of cloning efficiency. The plates were returned to the incubator for 7-9 days. The colonies developed were fixed, stained and counted. Results were expressed as mutant frequency.
Evaluation criteria:
not precised
Statistics:
Mutagenicity data were analyzed according to the statistical method of Snee and Irr (1981) designed specifically for the CHO/HGPRT mutation assay. Mutant frequency values were transformed according to the equation Y=(X+1)^0.15, where Y= transformed lutant frequency and X= observed mutant frequency. Student's t-test was then used to compare treatment data to solvent data. The Snee and Irr analysis also allowed the determination of dose-response relationship as linear, quadratic, or higher-order.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 3.4 µg/ml and greater
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µg/ml (1%), and at 34 µg/ml (2-10%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically significant increases in mutant frequency were observed in these experiments.
The positive controls yielded expected positive responses in mutagenicity indicating the adequacy of the experimental conditions.

RANGE-FINDING/SCREENING STUDIES:
An initial experiment was performed to test the cytotoxicity of the test material. A wide range of test material. A wide range of test concentrations (0.034, 0.065, 0.10, 0.34, 0.65, 1.0, 3.4, 10, 34 and 100 µg/ml) were used. Because of limited solubility of Sulfasan R in aqueous medium, 100 µg/ml was the higher dosage tested. The results of this experiment were used for a dose-selection purposes for the subsequent mutagenicity experiments.
The initial cytotoxicity determination of Sulfasan R in CHO cells at different S9 concentrations showed that the cytotoxicity of Sulfasan R decreased with increased S9 concentration. The concentrations for the cytotoxicity rangefinding experiment that induced significant cytotoxicitywere 10, 34, 34, 34 and 10 µg/ml in absence of S9, and in presence of 1%, 2%, 5% and 10% S9 respectively.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of S9 activation the cytotoxicity of the test sample decreased with increasing S9 concentration. Significant cytotoxicity was demonstrated for treatment levels of 10 µg/ml in the presence of 1% of S9 and at 34 µg/ml in the presence of 2, 5 and % S9.

Applicant's summary and conclusion

Conclusions:
No test chemical related mutagenicity was observed in the initial mutagenicity test or in two confirmation experiments conducted in the absence of S9 or in the presence of S9. The test item is therefore concluded not to be a mutagen in CHO cells.
Executive summary:

The mutagenic potential of 4,4'-dithiodimorpholine was tested in cultured Chinese Hamster ovary (CHO) cells. Mutation at the hypowanthine guanine phosphoribosyl transferase (HGPRT) gene locus was measured. Mutagenicity testing of Sulfasan R was performed initially in a range of Aroclor 1254 -induced rat liver homogenate (S9) concentrations (0 -10%) followed by a confirmatory experiment at 0 and 5%S9. In mutagenicity assays, Sulfasan R was tested up to concentrations which induced significant cytotoxicity (>50% cell killing). The ranges of concentrations for all experiments, including the cytotoxicity were 3.4 -10 µg/ml, 10 -34µg/ml, 10 -34 µg/ml, 10 -34 µg/ml and 3.4 µg/ml in the absence of S9 and in the presence of 1%, 2%, 5% and 10% of S9 respectively. No test chemical related mutagenicity was observed in the initial mutagenicity test or in two confirmation experiments conducted in the absence of S9 or in the presence of S9. 4,4'-dithiodimorpholine is therefore concluded not to be a mutagen in CHO cells.