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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003-04-15 to 2003-06-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21. July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Histidin auxotroph
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (fraction of Spraque Dawley rat liver incuded with Aroclor 1254). Obtained by Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, USA.
Test concentrations with justification for top dose:
First plate incorporation test serving as range finding test: with metabolic activation (10 % S9 liver homogenate): 50, 160, 500, 1600, 5000 µg/plate
without metabolic activation: 50, 160, 500, 1600, 5000 µg/plate
Second plate incorporation test: with metabolic activation (10 % S9 rat liver homogenate): 1.6, 5, 16, 50, 160, 500 µg/plate (TA 1537), and 5, 16, 50, 160, 500, 1600 µg/plate (TA 100, TA 1535, TA 98, WP2uvrA),
without metabolic activation: 0.5, 1.6, 5, 16, 50, 160 µg/plate (TA 100, TA 1537), 1.6, 5, 16, 50, 160, 500 µg/plate (TA 1535, TA 98) and 16, 50, 160, 500, 1600, 5000 µg/plate (WP2uvrA)
Third plate incorporation test: with metabolic activation (30 % S9 rat liver homogenate): 1.6, 5, 16, 50, 160, 500 µg/plate (TA 1537), and 5, 16, 50, 160, 500, 1600 µg/plate (TA 100, TA 1535, TA 98, WP2uvrA)
without metabolic activation: 0.5, 1.6, 5, 16, 50, 160 µg/plate (TA 100, TA 1537), and 1.6, 5, 16, 50, 160, 500 µg/plate (TA 1535, TA 98), and 5, 16, 50, 160, 500, 1600 µg/plate (WP2uvrA)
Repeat of third plate incorporation test: with metabolic activation (30 % S9 rat liver homogenat): 50, 160, 500, 1600, 5000 µg/plate (TA 100, WP2uvrA)
without metabolic activation: 50, 160, 500, 1600, 5000 µg/plate (WP2uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Other common vehicles (deionizede water, DMSO) were not appropriate due to insolubility of the test item. Stability and homogeneity (to be reached by stirring) of the test item in the vehicle is guaranteed by the sponsor (Report Ulrike Reploeg, dated 31-Mar-2003) for 4 hours.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: dissolved in DMSO, for TA 1537 (50.0 µg/plate)
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: dissolved in deionized water, for TA 100 (2.0 µg/plate), TA 1535 (1.0 µg/plate)
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: dissolved in DMSO, for TA 98 (2.5 µg/plate)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: dissolved in DMSO, for WP2uvrA (2.0 µg/plate, plate inc.)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (wiht metabolic activation) dissolved in DMSO: for TA 98, TA 100, TA 1535, TA 1537 (1.5 µg/plate); for WP2uvrA (20.0 µg/plate, plate inc.)
Remarks:
with metabolic activation (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) with 10 and 30 % S9-mix in the third assay.

DURATION
- Preincubation period: not done
- Exposure duration: 48 h

NUMBER OF PLATES EVALUATED: three per dose

NUMBER OF REPLICATIONS: Three independent experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: toxicity to bacteria was assessed by thinning of the bacterial lawns and/or reduction in the number of colonies.



Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range
Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn. If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
Statistics:
No

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
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A 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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item was dissolved in ethanol and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160, 50, 16, 5, 1.6 and 0.5 µg/plate were used in the different experiments
- Precipitation: visible precipitation of Praepagen TQ on the plates was observed at 1600 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
In the first plate incorporation test with 10 % S9-mix bacteria were treated with doses of 50, 160, 500, 1600 and 5000 µg/plate. Howeever the test item was toxic to all bacteria strains so that evaluation according to the guidelines (5 doses) was not possible. This test served therefore only as range finding test.

COMPARISON WITH HISTORICAL CONTROL DATA:
Values were within the laboratory´s historical control range. Only the number of revertant colonies of the solvent controls in strains TA 1537 and WP2 were slightly out of the historical control data range with ethanol in some experiments. However, the existing control data pool with ethanol is limited as this solvent is used rarely. The historical control data pool for DMSO indicating that all values are in normal range and do not influence the validity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses in the second plate incorporation test with 10 % S9-mix based on the toxicity to bacteria observed in the first test. In this test toxicity to bacteria in form of incomplete bacterial lawn was observed without metabolic activation at dose levels of 160 µg/plate and above with the strains TA 100, TA 1535, TA 1537 and TA 98 and at dose levels of 500 µg/plate and above with the strain WP2uvrA. In the presence of metabolic activation the test compound proved to be toxic to bacterial strains TA 100, TA 1537 and TA 98 at concentrations of 500 µg/plate and above and at a concentration of 1600 µg/plate to bacterial strains TA 1535 and WP2uvrA.
The top dose level for the third plate incorporation test (30 % S9-mix) was selected on the basis of toxicity to bacteria observed in previous tests. In the third plate incorporation test (with 30% rat liver homogenate) toxicity was observed without metabolic activation at dose levels of 160 µg/plate andabove with the tester strains TA 100, TA 1535, TA 1537 and TA 98. In the presence of metabolic activation the test compound proved to be toxic to the tester strain TA 1537 at a concentration of 500 µg/plate and at a concentration of 1600 µg/plate to bacterial strains TA 1535 and TA 98.

30 % S9-mix reduced toxicity to E. coli WP2 and TA 100 in the third test with the result that the maximum dose showed no toxicity to bacteria as required by the guideline. Therefore, the test with 30 % 89 had to be repeated with this strains (repeat plate incorporation test) using doses up to 5000 µg/plate, In the repeat test with 30 % rat liver homogenate toxicity to bacteria in form of incomplete bacteria lawn was observed with strain TA 100 (+ S9-mix) at a dose level of 1600 µg/plate and with E. coli WP2 at a dose level of 1600 µg/plate and above (-S9-mix).
Slight differences in toxicity to bacteria in the experiments with 30 % S9-mix (WP2 -S9-mix) are probably caused by biological variability.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Sterility control:

The plates for sterility control of test item and S9-mix showed no growth.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic
activation in this study.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, 1997, strains TA1535, TA 1537, TA 98 and TA 100 of S. typhimurium and E. coli WP2 were exposed to the  “partially unsaturated TEA-Esterquat”. Three independent experiments were performed up to the limit concentration of 5000 µg/plate with and without mammalian metabolic activation (rat liver S9-mix 10 and 30 %).

Significant bacteriotoxic effects of varying severeness were observed, depending on the test strain and the presence of metabolic activation. Generally, bacteriotoxicity was less pronounced in the presence of metabolic activation, especially at concentrations of 30 % S9-mix. Precipitation was observed at 1600 µg/plate and above. There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.

There was no evidence of induced mutant colonies over background.