Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP)

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-06-08 to 2006-06-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2005-06-09
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark, desiccated, under inert gas.
Target gene:
TA100 & TA1535: hisG46
TA1537: hisC3076
TA 98: hisD3052
E. coli WP2 uvrA (pKM101): trpE ochre
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix contained : 10 % v/v S9 fraction, sodium-ortho-phosphate buffer (100 mM, pH 7.4), NADP (4 mM), glucose-6-phosphate (5 mM), MgCl2 (8 mM), KCl (33 mM) in water
Test concentrations with justification for top dose:
Plate incorporation test:
-without and with metabolic activation: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Preincubation test:
-without and with metabolic activation: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The Sponsor indicated that DIGIRENA SA TEMABF4 anhydrous was soluble in water. Water was, therefore, used as the vehicle for this study.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 4-nitroquinoline-1-oxide (without metabolic activation) & 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar: A) plate incorporation and B) preincubation

A)
DURATION
- Exposure duration: 72 hours at 37°C

NUMBER OF REPLICATIONS: 3 plates were used for each treatment.

EVALUATION: After the incubation period, revertant colonies were counted using an automated colony counter (Perceptive Instruments Sorcerer).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
After the incubation period of 72 hours, the appearance of the background bacterial lawn was examined.
Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of 4 non-toxic concentrations should be obtained.

OTHER EXAMINATIONS:
If precipitate were observed on the plates at the end of the incubation period, at least 4 non-precipitating concentrations should be obtained, unless otherwise justified by the Study Director.

OTHER: Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer.


B) As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test.
DURATION
- Preincubation period: 30 minutes at 37°C
- Exposure duration: 72 hours at 37°C

NUMBER OF REPLICATIONS: 3 plates were used for each treatment.

EVALUATION: After the incubation period, revertant colonies were counted using an automated colony counter (Perceptive Instruments Sorcerer).
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.

If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.
Statistics:
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DIGIRENA SA TEMABF4 anhydrous at any concentration up to 5000 µg/plate.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DIGIRENA SA TEMABF4 anhydrous at any concentration up to 5000 µg/plate.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation. The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data (mean revertant colony counts) from the period 1 May 2001 to 30 April 2006 are available.

INFORMATION ON CYTOTOXICITY:
Plate incorporation test: No evidence of toxicity was obtained following exposure to DIGIRENA SA TEMABF4 anhydrous. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
Preincubation test: No evidence of toxicity was obtained following exposure to DIGIRENA SA TEMABF4 anhydrous.
Remarks on result:
other: plate incorporation and preincubation tests
Conclusions:
The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.
Executive summary:

In this in vitro assessment of the mutagenic potential of DIGIRENA SA TEMABF4 anhydrous, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to DIGIRENA SA TEMABF4 anhydrous diluted in water. Water was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9-mix) from rats. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

Concentrations of DIGIRENA SA TEMABF4 anhydrous up to 5000 µg/plate were tested.

No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration of DIGIRENA SA TEMABF4 anhydrous in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (OECD 471).

Justification for classification or non-classification

Genetic toxicity in vitro

The substance should not be considered to have a mutagenic potential based on a bacterial reverse mutation assay (OECD 471). The substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.