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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
Study period:
From November 26, 1984 to March 11, 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1984
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dioxolane
EC Number:
211-463-5
EC Name:
1,3-dioxolane
Cas Number:
646-06-0
Molecular formula:
C3H6O2
IUPAC Name:
1,3-dioxolane
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity, including information on contaminants, isomers, etc.: 99.9%

Method

Target gene:
TK
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y TK+/- - 3.7.2C derived from Fischer L5178Y line of Dr. Donald Clive

For cell lines:
- Absence of Mycoplasma contamination: periodically checked by culturing methods
- Methods for maintenance in cell culture: exposure to methotrexate to reduce spontaneous TK-/- mutants and then returned to normal growth medium for 3 to 8 days before use


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Maintenance medium: Fischer's mouse leukemia medium supplemented with pluronic solution, L-glutamine, sodium pyruvate, antibiotics, and horse serum (10% by volume). Cloning medium: preceding growth medium with the addition of agar (0.35%). Selection medium: cloning medium containing 3 µg/ml of TFT
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver homogenate
- method of preparation of S9 mix: it is commercially prepared and consists of the 9000 x g supernatant prepared from Aroclor 1254-induced adult male rat livers
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): each batch of S9 is checked for sterility and assayed for AHH activity and protein content
Test concentrations with justification for top dose:
Test 1 (without metabolic activation): 1.5, 2.5, 3, 3.5, 4 and 5 µL/mL
Test 2 (with metabolic activation): 0.125, 1, 2, 2.5, 3, 4 and 5 µL/mL
Test 3 (with and without metabolic activation): 0.75, 1.5, 2.5, 3, 4, and 5 µL/mL
The test limit in this asay is 5 µL/mL because of the possibility of spurious results due to osmotic or salt effects that can occur at higher concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile deionized water
Controls
Negative solvent / vehicle controls:
yes
Remarks:
3 solvent controls are included in each assay. In the solvent controls, an equivalent volume of the solvent is included in the assay mixture. For test substances assayed with activation, the solvent controls include the activation mixture
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Remarks:
Non activation studies: Ethylmethane sulfonate (0.25 to 0.4 µL/mL)
Metabolic activation studies: 3-Methylcholanthrene (2.5 to 4.0 µg/mL)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): single for each treatment and triplcate for solvent control in each experiment
- Number of independent experiments: 3 but experiment 2 was performed only in the presence of metabolic activation

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3x10E6 cells/tube
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: cells added to normal growth medium for 3 to 8 days before use
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 10-14 days


- Selective agent: 5-trifluorothymidine 3µg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: A total sample size of 3x10E6 cells is suspended in selection medium to select for mutants. This sample is distributed into 3 dishes. The cloning effeciciency is determined by serially diluting the sample and seeding each of 3 dishes with approximately 100 cells in cloning medium. After 10 to 14 days in the incubator, the colonies are counted with an electronic colony counter.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: total viable colony count/number of cells seeded
Evaluation criteria:
Assay acceptance criteria:
1. Average absolute cloning efficiency of the negative controls: between 60% and 130%.
2. Minimum acceptable value for the suspension growth of the average negative control for 2 days: 8.0
3. Normal range of background frequencies for assays performed with different cell stocks: 10x10E-6 to 100x10E-6
4. A positive control is included with each assay. Minimum acceptable mutant frequency induced by 0.3µL/mL EMS: 200 x 10E-6; by 2.5 µg/mL MCA: 200x10E-6
5. For test articles with little or no mutagenic activity, an assay should include applied concentrations that reduce the relative growth to 10 to 20% of the average solvent control or reach the maximum applied concentrations given in the evaluation criteria.
6. An experimental mutant frequency will be considered acceptable for evaluation only if the relatvie cloning efficiency is 10 % or greater and the total number of viable clones exceeds about 30
7. An acceptable mutant frequency can be calculated from a minimum of 2 dishes per set if the colony numbers in the 2 dishes differ by no more than about 3-fold
8. The mutant frequencies for 5 treated cultures are normally determined in each assay. A minimum of 3 analyzed cultures is considered necessary.

Assay evaluation criteria:
Minimum criterion to demonstrate mutagenesis: mutant frequency that is at least 150% of the concurrent background frequency plus 10x10E-6
Positive substance:
- dose-related or toxicity-related increase in mutant frequency should be observed (for at least 3 doses)
- increase of about 2 times the minimum criterion or greater is observed for a single dose near the highest testable toxicity,
Substance evaluated as negative in a single assay only if the minimum increase in mutant frequency is not observable for 1) a range of applied concentrations that extends to toxicity causing 10 to 20% relative growth or 2) a range of applied concentrations extending to max 5 mg/mL (5µL/mL) (for nontoxic materials)

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: summary of mouse lymphoma (L5178Y) results - Trial 1





































































































































































Test condition



Daily Cell counts (cells/mL 10E5 units)



Suspension growth



Total mutant colonies



Total viable colonies



Cloning efficiency



Relative growth (%)



Mutant frequency (10E-6 units)



 



1



2



 



Average solvent control



 



 



 



Average solvent control



 



 



Nonactivation



Solvent control



12.8



12.0



17.1



16.1



37.0



490.0



81.7



90.1



100.0



15.1



Solvent control



13.9



10.0



15.4



40.0



557.0



92.8



100.0



14.4



Solvent control



11.0



13.0



15.9



38.0



574.0



95.7



100.0



13.2



EMS0.25 µL/mL



9.1



12.4



12.5



606.0



479.0



79.8



69.0



253.0



EMS 0.40 µL/mL



8.4



11.0



10.3



692.0



406.0



67.7



47.9



340.9



Test compound



 



Relative to solvent control (%)



 



Relative to solvent control (%)



 



1500.0000 nL/mL



14.0



13.7



132.4



35.0



404.0



74.7



98.9



17.3



2500.0000 nL/mL



10.0



13.5



93.2



51.0



482.0



89.2



83.1



21.2



3000.0000 nL/mL



9.6



12.4



82.2



49.0



602.0



111.4



91.6



16.3



3500.0000 nL/mL



9.0



11.7



72.7



43.0



654.0



121.0



88.0



13.1



4000.0000 nL/mL



7.6



11.8



61.9



65.0



537.0



99.3



61.5



24.2



5000.0000 nL/mL



7.0



11.0



53.1



38.0



628.0



116.2



61.7



12.1



Suspension growth = (day 1 count/3) * (day 2 count)/(3 or day 1 count if not split back)


Mutant frequency = (total mutant colonies/total viable colonies) x2x10E-4. Decimal is moved to express the frequency in units of 10E-6


Cloning efficiency = total viable colony count/number of cells seeded


Relative growth = (relative suspension growth * relative cloning efficiency)/100


 


Table 2: summary of mouse lymphoma (L5178Y) results - Trial 2
















































































































































































Test condition



Daily Cell counts (cells/mL 10E5 units)



Suspension growth



Total mutant colonies



Total viable colonies



Cloning efficiency



Relative growth (%)



Mutant frequency (10E-6 units)



 



1



2



 



Average solvent control



 



 



 



Average solvent control



 



 



S9 activation



Solvent control



11.2



13.7



17.0



15.2



146.0



689.0



114.8



112.4



100.0



42.4



Solvent control



10.0



14.4



16.0



101.0



577.0



96.2



100.0



35.0



Solvent control



10.6



10.6



12.5



180.0



758.0



126.3



100.0



47.5



MCA 2.5 µg/mL



7.5



10.4



8.7



426.0



319.0



53.2



27.0



267.1



MCA 4.0 µg/mL



5.3



6.7



3.9



448.0



266.0



44.3



10.2



336.8



Test compound



 



Relative to solvent control (%)



 



Relative to solvent control (%)



 



125.0000 nL/mL



9.7



10.0



70.9



142.0



594.0



88.1



62.5



47.8



1000.0000 nL/mL



8.0



10.2



59.6



159.0



729.0



108.1



64.4



43.6



2000.0000 nL/mL



8.1



10.0



59.2



189.0



609.0



90.3



53.5



62.1



2500.0000 nL/mL



7.1



10.2



52.9



181.0



616.0



91.3



48.3



58.8



3000.0000 nL/mL



7.6



10.1



56.1



196.0



601.0



89.1



50.0



65.2



4000.0000 nL/mL



5.6



10.0



40.9



191.0



584.0



86.6



35.4



65.4



5000.0000 nL/mL



6.0



9.2



40.4



189.0



576.0



85.4



34.5



65.6



Suspension growth = (day 1 count/3) * (day 2 count)/(3 or day 1 count if not split back)


Mutant frequency = (total mutant colonies/total viable colonies) x2x10E-4. Decimal is moved to express the frequency in units of 10E-6


Cloning efficiency = total viable colony count/number of cells seeded


Relative growth = (relative suspension growth * relative cloning efficiency)/100


Table 3: summary of mouse lymphoma (L5178Y) results - Trial 3













































































































































































































































































































Test condition



Daily Cell counts (cells/mL 10E5 units)



Suspension growth



Total mutant colonies



Total viable colonies



Cloning efficiency



Relative growth (%)



Mutant frequency (10E-6 units)



 



1



2



 



Average solvent control



 



 



 



Average solvent control



 



 



Nonactivation



Solvent control



14.9



11.2



16.5



18.3



77.0



807.0



134.5



125.7



100.0



19.1



Solvent control



12.6



12.9



18.1



73.0



749.0



124.8



100.0



19.5



Solvent control



12.8



12.8



18.2



72.0



706.0



117.7



100.0



20.4



EMS0.25 µL/mL



10.9



10.4



12.6



857.0



594.0



99.0



54.2



288.5



EMS 0.40 µL/mL



10.0



10.6



11.8



953.0



234.0



39.0



20.0



797.4



Test compound



 



Relative to solvent control (%)



 



Relative to solvent control (%)



 



750.0000 nL/mL



13.1



12.1



96.2



75.0



667.0



88.4



85.0



22.5



1500.0000  nL/mL



11.0



12.0



80.1



47.0



736.0



97.6



78.2



12.8



2500.0000  nL/mL



11.0



11.6



77.5



61.0



684.0



90.7



70.3



17.8



3000.0000  nL/mL



10.1



11.2



68.7



81.0



777.0



103.0



70.8



20.8



4000.0000  nL/mL



10.7



11.5



74.7



84.0



845.0



112.2



83.8



19.8



5000.0000  nL/mL



10.4



10.3



65.0



77.0



708.0



93.9



61.0



21.7



S9 activation



Solvent control



11.6



12.1



15.6



13.7



92.0



747.0



124.5



112.1



100.0



24.6



Solvent control



10.6



10.6



12.5



81.0



682.0



113.7



100.0



23.7



Solvent control



10.0



11.8



13.1



111.0



588.0



98.0



100.0



37.7



MCA 2.5 µg/mL



5.5



8.7



5.3



729.0



430.0



71.7



24.8



339.1



MCA 4.0 µg/mL



4.8



6.7



3.6



412.0



229.0



38.2



8.9



359.8



Test compound



 



Relative to solvent control (%)



 



Relative to solvent control (%)



 



750.0000 nL/mL



9.0



12.0



87.6



129.0



702.0



104.4



91.5



36.7



1500.0000 nL/mL



7.4



11.5



69.0



180.0



819.0



121.8



84.0



43.9



2500.0000 nL/mL



7.6



10.9



67.2



115.0



779.0



115.8



77.8



29.5



3000.0000 nL/mL



7.6



10.9



67.2



118.0



825.0



122.7



85.5



28.6



4000.0000 nL/mL



8.4



11.1



75.6



129.0



846.0



125.8



95.1



30.5



5000.0000 nL/mL



7.8



10.9



69.0



106.0



799.0



118.8



82.0



26.5




Suspension growth = (day 1 count/3) * (day 2 count)/(3 or day 1 count if not split back)


Mutant frequency = (total mutant colonies/total viable colonies) x2x10E-4. Decimal is moved to express the frequency in units of 10E-6


Cloning efficiency = total viable colony count/number of cells seeded


Relative growth = (relative suspension growth * relative cloning efficiency)/100


 

Applicant's summary and conclusion

Conclusions:
The test material, 1,3-Dioxolane, did not induce significant increases in the mutant frequency at the TK locus in L5178Y TK+/- cells. Treatments from 750 nL/mL to 5000 nL/mL were assayed for mutant induction and low to moderate toxicities were induced. Higher concentrations were not assayed because 5000 nL/mL is the testing limit in this assay. The test material was therefore considered inactive with and without metabolic activation in the Mouse Lymphoma Assay up to 5000 nL/mL
Executive summary:

A mouse lymphoma forward mutation assay was performed in order to evaluate the mutagenic potential of 1,3-Dioxolane. The objective of this assay was to determine the ability of 1,3-Dioxolane to induce forward mutations at the thymidine kinase (TK) locus as assayed by colony growth of L5178Y TK+/- mouse lymphoma cells in the presence of 5-trifluorothymidine (TFT).


During this study, 1,3-Dioxolane was dissolved in sterile deionized water and used in cytotoxicity and mutation assays.


In the preliminary cytotoxicity assay, the test material induced, at most, small decreases in the 24 hour growth even at 5000 nL/mL. Concentrations of 1,3-Dioxolane up to 5000 nL/mL were therefore used in the mutation assay.


Regarding the mutation assay, three trials were initiated. However, only data generated in the absence of metabolic activation are available for trial 1 and trial 2 was performed only in the presence of metabolic activation.


Under nonactivation conditions, six treatments from 1500 to 5000 nL/mL were chosen for trial 1 and from 750 to 5000nL/mL for trial 3. None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion. 1,3-Dioxolane was therefore considered nonmutagenic without metabolic activation in these assays.


In the presence of metabolic activation, seven treatments from 125 to 5000 nL/mL were analyzed for mutant induction. None of these treatments induced a mutant frequency that exceeded the minimum criterion. 1,3-Dioxolane was therefore considered nonmutagenic with metabolic activation in this assay. Another activation assay was initiated to confirm the absence of mutagenic activation. In this assay, six treatments from 750 to 5000 nL/mL were chosen. None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion. 1,3-Dioxolane was therefore considered nonmutagenic in the presence of metabolic activation up to the testing limit of 5000 nL/mL.


In the assays used in this evaluation, the average cloning efficiencies for the solvent controls varied from 90.1% and 125.7% without activation to 112.4% and 112.1% with activation which demonstrated good cloning conditions for the assays. The negative control mutant frequencies were all in the expected range and the positive control compounds yielded mutant frequencies that were greatly in excess of the background.


1,3-Dioxolane was considered inactive with and without metabolic activation in the Mouse Lymphoma Assay up to 5000 nL/mL.