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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to Ethylparaben at concentrations of 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I) and 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II). Ethylparaben was tested up to the limit concentration of 5000 µg/plate in all tester strains used. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
Link to relevant study records
Reference
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:
2011-12-14 to 2012-01-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study equivalent to guideline, tested with the source substance Ethylparaben. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Reference:
Composition 0
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:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
His
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction (activity of metabolic activation system was validated in Ames tests using 2-aminoanthracene and benzo[a]pyrene)
Test concentrations with justification for top dose:
Experiment I (plate incorporation):
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II (preincubation):
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
yes
Remarks:
A. dest., BSL Lot No. 111102, 111201, 111219
Solvent controls:
yes
Remarks:
DMSO, Applichem Lot No. 1P008076
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
-S9: 10 µg/plate sodium azide in Aqua dest. (TA100, TA1535); 4-nitro-o-phenylene-diamine in DMSO (10 µg/plate for TA98, 40 µg/plate for TA1537); 1 µL/plate methylmethanesulfonate in Aqua dest. (TA102); +S9: 2-aminoanthracene in DMSO for all strains
Details on test system and conditions:
METHOD OF APPLICATION:
experiment I: in agar (plate incorporation)
Experiment II: preincubation

DURATION
- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates each in two independent experiments were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.

In experiment I toxic effects of the test item were observed in tester strains TA 98 and TA 1537 at a concentration of 5000 µg/plate (with and without metabolic activation). In tester strain TA 1535 toxic effects of the test item were noted at concentrations of 2500 µg/plate and higher (with and without metabolic activation). In tester strains TA 100 and TA 102 toxic effects of the test item were observed at concentrations of 2500 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation).

In experiment II toxic effects of the test item were noted in tester strains TA 98 and TA 102 at concentrations of 2500 µg/plate and higher (with and without metabolic activation). In tester strains TA 100 and TA 1535 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with and without metabolic activation).

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Ethylparaben did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Ethylparaben is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to Ethylparaben at concentrations of 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I) and 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II).

Ethylparaben was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

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

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
Study equivalent to guideline, tested with the source substance Ethylparaben. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Justification for classification or non-classification

In conclusion, as deduced from the results of an Ames test performed with the source substance ethylparaben, the test item is not mutagenic in the bacterial reverse mutation assay in the presence and absence of metabolic activation up to the tested concentrations. 

Therefore, it does not have to be classified for mutagenicity since this substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate.