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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of studies with structural analogues 16alpha-hydroxyprednisolone and prednisolone, Prediac-Z is not mutagenic in Ames test, both with and without metabolic activation.

Link to relevant study records

Referenceopen allclose all

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:
20 March 2013 to 3 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500 and 5000 µg/plate of hydroxyprednisolone he tests on S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: hydroxyprednisolone was insoluble in water but soluble in DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
It was concluded that 16alpha-hydroxyprednisolone showed no evidence of mutagenic activity in this bacterial system at doses up to 5000 micrograms/plate under the test conditions employed.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study is classified as reliable with restrictions because, although it is not-GLP compliant, it is an acceptable and a well-documented study report, according or similar to a guideline, that meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Were used only two of the five bacterial strains recommended from the guideline.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Were used only two of the five bacterial strains recommended from the guideline.
Principles of method if other than guideline:
The test chemical in 0.1 ml dimethyl sulfoxide or H20 was placed in a tube and mixed with 0.5 ml S-9 mix (150 microliter of the S-9 fraction of rat liver pretreated with polychlorinated biphenyl, 2 micromol NADPH, 2 micromol NADH, 2.5 micromol glucose 6-phosphate, 0.25 unit glucose-6-phosphate dehydrogenase, 4 micromol MgCI2, 16.5 micromol KCI, and 50 micromol sodium phosphate buffer, pH 7.4 and 0.1 ml of culture of the bacterial tester strain (1 to 2 x 10E8 cells). Without metabolic activation 50 micromol sodium phosphate buffer, pH 7.4, in 0.5 ml were used instead of S-9 mix. The mixture was preincubated at 37° for 20 min and then mixed with 2 ml top agar (0.7% agar and 0.6% NaCI) at 45° and spread on a minimal glucose agar plate containing 0.1 micromol each L-histidine and biotin. Plates were incubated at 37° for 2 days, and then his+ revertant colonies were counted.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: This strain carryies R-factor plasmid pKM101.
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: This strain carryies R-factor plasmid pKM101.
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Prednisolone was tested at 8.3, 17, and 33 micrograms/plate. Drug contained some vehicle, and the amount of the drug was expressed as micrograms of active principle.
Untreated negative controls:
yes
Remarks:
Average of values in 20 experiments.
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle controls.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Also daunomycin hydrochloride, and adriamycin hydrochloride were reported as positive controls.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Prednisolone was not lethal at doses of up to 33 micrograms/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Prednisolone was not lethal at doses of up to 33 micrograms/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

               his+revertants/plate  
     TA100    TA98     
    micrograms/plate   + S-9 mix  - S-9 mix    + S-9 mix  - S-9 mix  his+revertants/nmola
 Prednisoloneb  8.3  135  125    37  34   0     
   17  145  145    39  33
   33  123  144    35  23
 Controlc    147  132    30  16  

aThe number ofhis+revertants per nmol was calculated from the data obtained under the most active conditions in

the linear dose-responsible range and a yield of 100 colonies more than the background yield.

bDrug contained some vehicle, and the amount of the drug was expressed as micrograms of active principle.

cAverage of values in 20 experiments.

Conclusions:
Prednisolone was not mutagenic to in S. typhimurium TA98 and TA100 strains with or without S-9 mix, and it was not cytotoxic at doses of up to 33 micrograms/plate.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
20 March 2013 to 3 April 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from a structural analogue
Justification for type of information:
The read-across rationale is attached in section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
In a reliable guideline study concluded with a structural analogue 16alpha-hydroxyprednisolone no evidence of mutagenic activity iat doses up to and including 5000 micrograms/plate were seen in S. typhimurium strains TA98, TA100, TA153 and TA1535 and E. coli strain WP2 uvrA, both with and without metabolic activation. This result can be read across to Prediac-Z.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from a structural analogue
Justification for type of information:
The read-across rationale is attached in section 13.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Were used only two of the five bacterial strains recommended from the guideline.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Were used only two of the five bacterial strains recommended from the guideline.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Prednisolone was not lethal at doses of up to 33 micrograms/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Prednisolone was not lethal at doses of up to 33 micrograms/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

               his+revertants/plate  
     TA100    TA98     
    micrograms/plate   + S-9 mix  - S-9 mix    + S-9 mix  - S-9 mix  his+revertants/nmola
 Prednisoloneb  8.3  135  125    37  34   0     
   17  145  145    39  33
   33  123  144    35  23
 Controlc    147  132    30  16  

aThe number ofhis+revertants per nmol was calculated from the data obtained under the most active conditions in

the linear dose-responsible range and a yield of 100 colonies more than the background yield.

bDrug contained some vehicle, and the amount of the drug was expressed as micrograms of active principle.

cAverage of values in 20 experiments.

Conclusions:
In a comparable to guideline study with a structural analogue of Prediac-Z, prednisolone, the test substance was not mutagenic to S. typhimurium strains TA98 and TA100 with or without S-9 mix, and was not cytotoxic at doses of up to 33 micrograms/plate. This result can be read across to Prediac-Z.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In a reliable GLP-compliant guideline study a stuctural analogue 16alpha-hydroxyprednisolone was found to be not mutagenic in Ames test in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA, when tested up to limit concentrations, both with and without metabolic activation.

In a study with a structural analogue prednisolone, in which only two S. typhimurium strains were tested (TA98 and TA100), prednisolone was not mutagenic when tested up to precipitating concentrations, both with and without metabolic activation.

Based on these results Prediac-Z is considered not mutagenic in the Ames test.

Justification for classification or non-classification

Based on the negative results of the Ames tests conducted with structural analogues 16alpha-hydroxyprednisolone and prednisolone, classification of Prediac-Z for genotoxicity is not warranted under Regulation (EC) 1272/2008.