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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-320-2
EC Name:
-
Cas Number:
24748-23-0
Molecular formula:
C12H24O6
IUPAC Name:
3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexaoxonane
Details on test material:
Batch no.: 1400-005
Stored from 14-20 March 1997 at ca. 25 degr. C, thereafter at ca. 4 degrc. C.

Method

Species / strain
Species / strain / cell type:
bacteria, other: Salmonella typhimurium strains TA98, 100, 1535, 1537 and Escherichia coli strain WP2uvrA-
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix.
Test concentrations with justification for top dose:
Concentration range in the main test (with and without metabolic activation): 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
Solvent: acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ENNG, 9-AA, 4-NQO
Remarks:
without S9-Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9-mix
Details on test system and experimental conditions:
Five concentrations of the test material were assayed in triplicate against
each tester strain, using the direct plate incorporation method in
accordance with the standard methods for mutagenicity tests using bacteria.

Known al iquots (0.1 ml) of one of the bacterial suspensions were
dispensed into sets of sterile test tubes followed by 2.0 ml of molten
trace histidine/tryptophan supplemented top agar at 45°C, 0.1 ml
of the appropriately diluted test material or vehicle control and
either 0.5 ml of the 59 liver microsome mix or phosphate buffer.
The contents of each test tube were mixed and equally distributed
onto the surface of Vogel-Bonner Minimal agar plates (one tube per
plate). This procedure was repeated, in triplicate, for each bacterial
strain and for each concentration of test material with and without
59-mix.

All of the plates were incubated at 37°C for approximately 48 hours
and the frequency of revertant colonies assessed using a Domino
colony counter. Due to the potentially volatile nature of the test
material, all of the plates were sealed in stainless steel containers
(one dose group per container) for the duration of the incubation
period.

The second experiment was performed using methodology as described for
experiment 1, using fresh bacterial cultures, test material and control
solutions in triplicate.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have
induced a dose-related and statistically(S) significant increase in mutation rate
(of at least twice the spontaneous reversion rate) in one or more strains of
bacteria in the presence and/or absence of the 59 microsomal enzymes in both
experiments at sub-toxic dose levels. In the event of the two experiments
giving conflicting or equivocal results, then a third experiment may be
performed to confirm the correct response. To be considered negative the
number of induced revertants compared to spontaneous revertants should be
less than twofold at each dose level employed, the intervals of which should
be between two and five fold and extend to the limits imposed by toxicity,
solUbility or up to the maximum recommended dose of 5000 tJg/plate. In this
case the limiting factor was the maximum recommended dose.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
Observations:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: main test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

This study was conducted to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers (1, 2, 3) and Garner et al (4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test material. This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. This method also conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471, Method B14 in EC Commission Directive 92/69/EEC and the USA, EPA (TSCA) guidelines. A copy of the Certificate of Compliance with GLP, issued by the UK Department of Health, is included as Appendix IV.

The substance was considered to be non-mutagenic under the conditions of this test.