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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:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid methods, therefore the study is considered relevant, adequate and reliable for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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
Qualifier:
according to
Guideline:
other: ICH Harmonised Tripartite Guideline S2(R1 ): Guidance on GenotoxicityTesting and Data lnterpretation for Pharmaceuticals lntended for Human Use, Current Step 4 version dated November 9, 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Sodium bis (C11-14-isoalkyl, C13-rich) sulfosuccinate; Butanedioic acid, sulfo-, 1,4-ditridecyl ester, sodium salt
- Physical state: White solid
- Analytical purity: >99%
- Impurities (identity and concentrations): Not provided
- Composition of test material, percentage of components: Not provided
- Purity test date: September 10, 2012 (CoA)
- Lot/batch No.: S20283-66A
- Expiration date of the lot/batch: December 31, 2013
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature
-Other: Manufacturer/supplier: Cytec Industries Inc., Five Garret Mountain Plaza, Woodland Park, NJ 07424, USA

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media:
*Oxoid 2 nutrient broth
* Minimal Glucose Agar medium E
* 10 mL sterile solution of 0.5 mM L-histidine HCl/0.5 mM biotin were added to 100 mL of molten agar (containing 0.6% agar and 0.5% NaCl)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg/plate
Main tests: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiol. saline (aqua ad iniectabilia)
- Justification for choice of solvent/vehicle: The test item was completely dissolved in aqua ad iniectabilia. Aqua ad iniectabilia was used as vehicle control.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide in aqua ad iniectabilia
Remarks:
TA 1535, TA 100 ( 10 µg/plate): without S9-mix
Positive controls:
yes
Positive control substance:
other: 2-Nitro-fluorene in DMSO
Remarks:
TA 98 (10 µg/plate): without S9-mix
Positive controls:
yes
Positive control substance:
other: 9-Amino-acridine in ethanol, abs.
Remarks:
TA 1537 ( 100 µg/plate): without S9-mix
Positive controls:
yes
Positive control substance:
other: Mitomycin C in DMSO
Remarks:
TA 102 ( 10 µg/plate): without s9-mix
Positive controls:
yes
Positive control substance:
other: Benzo(a)pyrene in DMSO
Remarks:
TA 98, TA 102, TA 1537 ( 10 µg/plate): with S9-mix
Positive controls:
yes
Positive control substance:
other: 2-amino-anthracene in DMSO
Remarks:
TA 100, TA 1535 (2-4 µg/plate): with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation): 1st independent experiment
preincubation: 2nd independent experiment

DURATION
- Preincubation period: 20 min (2nd independent experiment)
- Exposure duration: 48 h to 72 h (1st independent experiment and 2nd independent experiment)
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.
Evaluation criteria:
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Historical background data of revertant colony numbers of the vehicle control and positive controls without and with metabolic activation for the experiments of the year 2011 (most recent background data, not audited by the QAU-department) are given in Appendix 3.
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.
Statistics:
In our laboratory, a test item is considered to show a positive response if
-the number of revertants is significantly increased (p≤0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA98, TA100 and TA102 and 3-fold of the vehicle control for TA1535 and TA1537 in both independent experiments;
Or
-a concentration-related increase of the revertants is observed. The Spearman's rank correlation coefficient may be applied.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
The recommended maximum test concentration for soluble non-cytotoxic test items is 5 mg/plate or 5 µL/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative with and without metabolic activation

Under the present test conditions the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was completely dissolved in aqua ad iniectabilia. Aqua ad iniectabilia was used as vehicle control.

Preliminary test

The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations of 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in all test strains.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, under the present test conditions the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.