Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OCDE n° 471 Dir. 92/69/CEE : B13/B14
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Product : LCA01006
- Product supplier : SEPPIC
- Received sterile on 19th June, 2001
- LEMI Reference : DIB190601-2

Method

Target gene:
For Salmnonella typhimurium: histidine
For Escherichia coli: tryptophane
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: strain TA 98, TA 100 and E coli wp2 contains PKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (10 % liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate
Vehicle / solvent:
distilled water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: cis-diammineplatinium (II) dichloride
Remarks:
for E. coli strain
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 100 strain, without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for TA 98 strain, without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA 1537 strain, without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
for all S. typimurium strains, with S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: B-propiolactone
Remarks:
E. coli strain without S9
Details on test system and experimental conditions:
Assay without metabolic activation
For each of the strains, 100 μL of the bacterial suspension (1-2 x 109 bacteria/mL) and 100 μL of the test
substance are mixed with 2.0 mL of overlay agar and poured over the surface of a minimal agar plate (90
mm in diameter) (n = 3). The overlay agar is allowed to solidify before incubation.
Plates are incubated at 37°C over a 48 hour period. The number of revertant colonies per plate is counted.
Moreover the following controls are carried out :
- negative controls
. absolute negative control (spontaneous reversion rate),
. positive control solvent.
- test substance solvent if necessary.
- positive control

Assay with metabolic activation
Two tests can be performed using
· either a standard plate incorporation method where the protocol is similar to that described
above, except that just before pouring the mixture onto the plates, 500 μL of S-9 mix fraction is
quickly mixed,
· or the pre-incubation assay where the test substance is preincubated with the test strain, and
500 μL of S-9 mix fraction for 1 hour at 37°C prior to mixing with the overlay agar and pouring
onto the surface of the minimal agar plate. Tubes should be aerated during pre-incubation by
using a shaker. This method is known to increase the detection sensitivity of a number of
promutagens like alkaloïds, aliphatic N-Nitroso compounds (OECD n° 471* * * ).

Assay are conduced in triplicate
Evaluation criteria:
The result of the test is considered as negative if the revertant number is below three fold the
number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of
spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2 strains with and without metabolic
activation.

The result of the test is considered positive if a concentration –related in crease is obtained in one,
or several of the 5strains, with and/or without metabolic activation; a mutagenic effect is taken into
account for a given concentration of the test substance if the number of revertant colonies is at least two
fold that of spontaneous revertant colonies number for TA 98, TA 100 and Escherichia coli (WP2), and
three fold for TA 1535 and TA 1537.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(up to 5000 µg/plate)
Vehicle controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

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

There is no significant difference between the number of spontaneous reversions, the number of
reversions obtained in the positive controls (with and without metabolic activation), and the mean of
corresponding experimental historic values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test
substance (5 000 μg - 1 500 μg - 500 μg - 150 μg - 50 μg) with and without metabolic activation.
Results were confirmed in a second independent experiment.
Executive summary:

Analysis : Reverse mutation assay.

Test substance : LCA01006 LEMI code : DIB190601-2

Test procedure : Reverse mutation assay on “Salmonella typhimurium his-“ and “Escherichia coli” WP2 (pKM 101) strains according to the OECD guideline n° 471 (LEMI operating procedure MB08/45).

Test result : The product did not induce reverse mutation, Salmonella typhimurium and Escherichia coli WP2 (pKM 101) strains according to the OECD guideline n° 471 and according to the test method B13/B14 of the E.C. Directive n° 2000/32.