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
Study period:
from 2010-03-31 to 2010-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2009-01-13
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name : LCE 10031
Lot : T92235
Container : plastic flask (n = 1)
Quantity : 41.094 g (content + container)
Aspect : pearls
Storage : Room temperature (20 ± 5° C)
Purity : 100 %
Date of reception : 29th March 2010
LEMI Code : DTT290310

Method

Target gene:
Salmonella typhimurium: gene histidine
Escherichia coli: tryptophane gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
other: uvr A-, pKM101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50 / 150 / 500 / 1500 / 500 ug/plate
Vehicle / solvent:
ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Dimethyl-benzanthracene, CAS n° : [57-97-6]
Remarks:
for Escherichia coli WP2 (uvr A-) (pKM101) strain, with S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: cis-platin (II), Diammine Dichloride, CAS n° : [15663-27-1]
Remarks:
for Escherichia coli WP2 (uvr A-) (pKM101) strain, without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for S typhimirium TA 98, without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for S typhimurium strain 1535 and strain 100, without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for strain S typhimurium TA 3537, without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
for S. typhimurium strains with S9-mix
Details on test system and experimental conditions:
Preliminary cytotoxicity testing (strain TA 100)
In order to choose the range of concentration to be tested, the bacteriostatic activity of the test
substance is evaluated. The test substance is diluted in the appropriate solvent.
Five concentrations are studied. The recommended maximum test concentration (5 000 Rg/plate), or
the highest concentration compatible with the test substance solubility, and four half-log dilutions are
used.
100 RL of the bacterial suspension (1-5 x 109 bacteria/mL) and the different concentrations of the test
substance are successively added to 2 mL of overlay agar at 45° C, containing 10 % (v/v) of a solution of
L-Histidine-D-Biotine (2.5 mM). After homogeneization, the content of the tube is poured onto a Petri
plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration are incubated
for 24 or 48 hours at 37° C, and the number of colonies counted. A negative control containing the
solvent alone is run in parallel.
For test substances that are cytotoxic already below 5 000 Rg/plates, the highest concentration to be
retained is that exhibiting a bacteriostatic activity of 75 % or less. The precipitate, if present, should
not interfere with the scoring. Four other followed doses, distributed by half log ratio are used.

Main test- Assay procedure
- Assay without metabolic activation
Salmonella Typhimurium strains: for every strain, 0.1 mL of the bacterial suspension containing
1-5 x109 bacteria/mL and 0.1 mL of every test substance concentration are successively added to two
mL of overlay agar maintained surfusion in 45° C containing 10 % (v/v) of a L-Histidin-D-Biotin
solution (0.5 mM).
Escherichia coli strain: in a test tube 0.1 mL of the bacterial suspension containing
1-5 x 109 bacteria/mL and 0.1 mL of every test substance concentration are successively added to 2 mL
of overlay agar maintained surfusion in 45° C containing 5 % (v/v) of nutrient broth n° 2 to which are
added 5 RL of a L-Tryptophan solution at 2 mg/mL.
Plates are incubated at 37° C over a 48-72 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 (see table 2).

- 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 RL of S9-mix fraction
is quickly mixed, or the pre-incubation assay where the test substance is preincubated with the test strain, and
500 RL of S9-mix fraction usually for 20 min, or more 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). For the first assay it is the method by direct incorporation that is used.

Assay repetition
If the first assay is positive, the second one is performed in the same manner.
If the first assay is negative, the pre-incubation test is performed for the second assay

Presentation of data
After a 48-72 hour incubation period at 37° C, revertant colonies per plate are counted (n = 3).
Data are presented as the number of revertant colonies (mean ± standard deviation) per plate.
The following ratio is calculated :
R =Number of revertant colonies in the presence of the test substance/Number of revertant colonies in the absence of the test substance
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(uvrA-) (pKM 101) strains with
and/or without metabolic activation.
The validity criteria are as follows :
- bacteriostatic activity of the highest concentration shall be equal to or less than 75 %,
- the spontaneous reversion rate of the absolute negative control shall comply with the
laboratory’s historical control data,
- the mean number of revertant colonies obtained for each strain and the corresponding positive
control, with and/or without metabolic activation shall comply with laboratory’s historical
control data.
The result of the test is considered positive if a concentration – related increase is obtained in
one, or several of the 5 strains, 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(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Species / strain:
other: WP2(uvrA-) (pKM 101)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
One can observe in presence of 5 000 Rg/plate, 20 % cytotoxic levels respectively compatible with the
maximal acceptable (75 %). There is no evidence of any bacteriostatic activity in the presence of the
test substance (1500,500, 150 and 50 Rg/plate).

There is no significant difference between the number of spontaneous reversions, the number of
reversions obtained in the positive controls (without and with 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, 1 500, 500, 150 and 50 Rg/plate) without and with metabolic activation for bacterial
strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli
WP2(uvrA-) (pKM 101).
Results were confirmed in a second independent experiment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance does not induce reverse mutation on four Salmonella typhimurium strains and one Escherichia coli WP2 (uvrA-) (pKM 101) strain according to the OECD guidelines n°471 and to the method B13/B14 of the directive 2000/32/EC
Executive summary:

The purpose of this procedure is to quantitatively assess the mutagenic effect of a test substance in four Salmonella typhimurium strains (Ames and al) and one Escherichia coli WP2(uvrA-) (pKM 101) strain (Matsuchima and al), in compliance with the OECD guidelines 471 for the testing of chemicals. The assay is performed using five concentrations of the test substance, in both the presence and absence of an appropriate metabolic activation system (rat liver microsome fraction).

Five strains are exposed to the test substance, in the presence or in the absence of an exogenous metabolic activation system, onto minimal medium. After a suitable period of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates. The test substance is studied in the presence, or absence, of metabolic activation in order to distinguish between promutagens and direct mutagens.

strain tested: Salmonella typhimurium TA 98 TA 1537 TA 1535 TA 100

Escherichia coli WP2 (uvr A-) (pKM101)

concentration tested 50 / 150 / 500 / 1500 / 500 ug/plate (20% of cytotoxicity for the highest concentration)

There is no significant difference between the number of spontaneous reversions, the number of

reversions obtained in the positive controls (without and with 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, 1 500, 500, 150 and 50 Rg/plate) without and with metabolic activation for bacterial

strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli

WP2(uvrA-) (pKM 101).

Results were confirmed in a second independent experiment.

The test substance does not induce reverse mutation on four Salmonella typhimurium strains and one Escherichia coli WP2 (uvrA-) (pKM 101) strain according to the OECD guidelines n°471 and to the method B13/B14 of the directive 2000/32/EC