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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-12-29 to 2004-01-29
1 (reliable without restriction)

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
not specified
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): PROTEOL APL
- Lot/batch No.: 335JG


Target gene:
his D, his C, his G, tryp E
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
The genotype of bacterial strains is checked:
Histidine and tryptophan requirements
Loss of cell wall LPS
Ampicillin resistance
Δuvr B mutation i.e. U.V.B sensitivity for Salmonella typhimurium and Δuvr A mutation i.e. U.V.A sensitivity for Escherichia coli
Spontaneous revertant rate
Sensitivity to reference mutagens
Metabolic activation:
with and without
Metabolic activation system:
Test concentrations with justification for top dose:
15µg ; 45µg ; 150 µg ; 450 µg ; 1500 µg/plate
Vehicle / solvent:
physiological serum (NaCl 0,9 % (w/v)
Untreated negative controls:
Negative solvent / vehicle controls:
physiological serum (NaCl 0,9 % (w/v) + solvents of positive control substances
True negative controls:
Positive controls:
Positive control substance:
sodium azide
other: B-Propiolactone ; 2-Anthramine ; cis-Platinum (II)
Details on test system and experimental conditions:
Assay without metabolic activation:
For each of the strains, 100 μL of the bacterial suspension (1-5 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
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 S9-mix fraction is quickly mixed,
• or the pre-incubation assay where the test substance is preincubated with the test strain, and 500 μL of S9-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 preincubation by using a shaker. This method is known to increase the detection sensitivity of a number of promutagens like alkaloïds, aliphatic N-Nitroso compounds

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.
Evaluation criteria:
After a 48 hour incubation period at 37° C, revertant colonies per plate are counted.
Data are presented as the number of revertant colonies (mean ± standard deviation) per plate.
The following ratio is calculated :

Number of revertant colonies in the presence of the product
R = _______________________________________________________________________
Number of revertant colonies in the absence of the product

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
1500 µg/plate was the highest concentration shall be equal to or less than 75 % of cytotoxicity,
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):

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 (1 500 μg - 450 μg 150 μg - 45 μg - 15 μg) without and with metabolic activation.
Results were confirmed in a second independent experiment.

In summary:
At these doses the test substance, PROTEOL APL (LEMI code : DTT221203) supplied by SEPPIC does not induce any mutagenic change in Salmonella
typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), without or with metabolic activation, according to the
guideline OECD n° 471 and to the method B13/B14 of the directive CE/2000/32.
Executive summary:

Test substance :


Batch n° 335JG

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

Test result : 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 guideline n° 471 and to the method B13/B14 of the directive CE/2000/32.