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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant, near guideline study, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): test article API 83-06
- Substance type: Heavy catalytic reformed naphtha (petroleum)
-Description: A complex combination of hydrocarbons produced from the distillation of products from a catalytic reforming process. It consists of predominantly aromatic hydrocarbons having carbon numbers predominantly in the range of C7 through C12 (approx. 91% aromatics, 9% paraffins: no benzene reported present). It has a boiling range of approximately 90°C to 230°C (194-446°F).
- Physical state: non-viscous liquid
- Storage condition of test material: room temperature, protected from light

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- mouse lymphoma cells Clone 3.7.2C (Burroughs Wellcome, North Carolina< USA)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 0.01, 0.05, 0.1, 0.5, 1.0, 5.0 10.0, 50.0 and 100 µL/mL for the first test (with and without S9 activation) and 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 µL/mL for the second test (with S9 activation only).
Mutagenicity tests: Eight dose levels decreasing approximately 10-fold from highest to lowest: 0.1 µL/mL to 0.013 µL/mL for non-activated cultures and 0.5 µL/mL to 0.067 µL/mL for S9 activated cultures.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9

Migrated to IUCLID6: 1 and 0.5 µL/mL
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9

Migrated to IUCLID6: 5 and 7.5 µg/mL
Details on test system and experimental conditions:
PRELIMINARY TOXICITY TEST: With and without S9 activation. Tube cultures were initiated by seeding one centrifuge tube/dose level and 2 tubes/solvent control with 6 mL of a cell suspension containing 1 x 10E6 cells/mL. API 83-06 was solubilised and diluted in ethanol to intended concentrations and added to the tubes. Medium or S9 activation mixture was added. The tubes were exposed for 4 hours, after which time the API 83-06 was removed by centrifugation and the cells washed. Toxicity was determined by comparing the cell population growth at each concentration with the solvent controls. Cell population density was determined 24 and 48 hours after initial exposure.

MUTAGENICITY ACTIVITY TEST (Study number MAI #T2420.701)

METHOD OF APPLICATION: in culture

DURATION
- Exposure duration: 4 hours at 37°C in the dark.
- Expression time (cells in growth medium): 2 days (with a cell population adjustment at 24 and 48 hours).
CLONING: At the end of the expression period, 12 non-activated and 6 S9 activated cultures were cloned, in cloning medium containing 0.34% Noble agar, based on their degree of toxicity. The non-activated cultures that were cloned were treated with 0.075, 0.056, 0.042, 0.032, 0.024 or 0.018 µL/mL. The S9 activated cultures that were cloned were treated with 0.12, 0.089 or 0.067 µL/mL. TFT at a final concentration of 3 µg/mL was used as the restrictive agent.

NUMBER OF REPLICATIONS: Two

MUTAGENICITY ACTIVITY TEST (Study number MAI #T2420.701012)
The API 83-06 was re-tested in the presence of S9. The initial toxicity test was also repeated and based on these data API 83-06 was tested over a range of concentrations from 0.3 - 0.03 µL/mL.
- Exposure duration: 4 hours at 37°C in the dark.
- Expression time (cells in growth medium): 2 days (with a cell population adjustment at 24 and 48 hours).

CLONING: At the end of the expression period, 10 cultures were selected for cloning based on their degree of toxicity. The cultures that were cloned were treated with 0.22, 0.18, 0.15, 0.11 or 0.07 µL/mL.
Evaluation criteria:
Positive: if there is a positive dose response and one or more of the doses in the 10% or greater total growth range exhibit a mutant frequency which is 2-fold greater than background level. All data including that from cultures with less than 10% total growth used to establish the dose response relationship.
Equivocal: if there is no dose response but any one or more of the 3 highest doses with 10% or more total growth exhibit a 2-fold increase in mutant frequency over background, or if there is a dose response but no culture exhibits a 2-fold increase in mutant frequency over background.
Negative: if there is no dose response in cultures with 10% or greater total growth and none of these test cultures exhibit a 2-fold or greater increase in mutant frequency over background.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Three non-activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls (mutant frequencies 31.0, 3.6 and 2.0 times greater than controls and respective total growth was 1, 2 and 10%). As API 83-06 has a very steep toxic response curve in this system, minute differences in dose result in large differences in total growth. The total growth exhibited by each culture may be more representative of dose delivered than the concentration of API 83-06 indicated. A comparison of induced mutant frequency with total growth indicates a dose-dependent response and the data are judged equivocal.

 

Two S9 activated cultures that were cloned exhibited mutant frequencies which were more than twice the mean mutant frequency of the solvent controls. Total growth of these cultures was 4 and 7%. A dose-dependent response was noted.

 

API 83-06 was re-tested with S9 activation. The cultures that were cloned were treated with a range of concentrations which produced 10-91% total growth. One culture exhibited a mutant frequency which was 2.1 times solvent control mean frequency. However, the duplicate culture for this dose (0.22 µL/mL) did not exhibit a significant increase in mutant frequency. A dose-dependent response was noted in the treated cultures. The mutagenic response of API 83-06 in the presence of S9 activation is classed as “equivocal” since a reproducible positive response was not observed.

 

Under the conditions of these tests, Heavy catalytic reformed naphtha (petroleum), API 83-06, produced an equivocal response in the presence and absence of exogenous metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous with metabolic activation
ambiguous without metabolic activation

Under the conditions of these tests, Heavy catalytic reformed naphtha (petroleum), API 83-06, produced an equivocal response in the presence and absence of exogenous metabolic activation.
Executive summary:
API 83-06 (CAS 64741-68-0) was tested in the L5178 Y TK +/- mouse lymphoma mutagenesis assay in the presence and absence of Aroclor induced rat liver S9. Three non-activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls (mutant frequencies 31.0, 3.6 and 2.0 times greater than controls and respective total growth was 1, 2 and 10%). As API 83-06 has a very steep toxic response curve in this system, minute differences in dose result in large differences in total growth. The total growth exhibited by each culture may be more representative of dose delivered than the concentration of API 83-06 indicated. A comparison of induced mutant frequency with total growth indicates a dose-dependent response and the data are judged equivocal.   Two S9 activated cultures that were cloned exhibited mutant frequencies which were more than twice the mean mutant frequency of the solvent controls. Total growth of these cultures was 4 and 7%. A dose-dependent response was noted.   API 83-06 was re-tested with S9 activation. The cultures that were cloned were treated with a range of concentrations which produced 10-91% total growth. One culture exhibited a mutant frequency which was 2.1 times solvent control mean frequency. However, the duplicate culture for this dose (0.22 µL/mL) did not exhibit a significant increase in mutant frequency. A dose-dependent response was noted in the treated cultures. The mutagenic response of API 83-06 in the presence of S9 activation is classed as “equivocal” since a reproducible positive response was not observed. Under the conditions of these tests, Heavy catalytic reformed naphtha (petroleum), API 83-06, (CAS 6471-68-0) produced an equivocal response in the presence and absence of exogenous metabolic activation.