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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:
9 April 2003 to 30 July 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully Guideline- and GLP-compliant

Data source

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

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:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Bacterial Reverse Mutation Test
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Resin 835 A (Lot:0161852)
- Molecular formula (if other than submission substance): main component C20H28O2
- Molecular weight (if other than submission substance): main component 300 g/mol
- Physical state: melt
- Composition of test material, percentage (w/w) of components: 56 % dehydroabietic acid, 20 % dihydroabietic acid, 9.8 % neutral components, 9 % non ident. rosin acids, 4 % Pimaric acids
- Purity test date: 11 March 2003
- Lot/batch No.: 0161852
- Expiration date of the lot/batch: 17 June 2003
- Stability: guaranteed for 4 hours in ethanol
- Storage condition of test material: at approx. 20 °C in a fume cupboard
- Concentration of stock solution: 50 mg/mL

Method

Target gene:
TA98: his D3052 rfa uvrB +R
TA100: hisG46 rfa uvrB +R
TA1535: hisG46 rfa uvrB
TA1537: hisC3076 rfa uvrB
TA102: hisC3076 rfa +R
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from Spraque Dawley rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
Without metabolic activation: 1.6 to 5000 µg/plate
With metabolic activation (10 % S9-mix): 50 to 5000 µg/plate
With metabolic activation (30 % S9-mix): 5 to 1600 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: soluble and stable in ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: dissolved in deion.w ater; conc. 1 µg/plate for TA1535, 2 µg/plate for TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: dissolved in DMSO; conc. 50 µg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: dissolved in DMSO; conc. 2.5 µg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: dissolved in deion. water; conc. 0.3 µg/plate for TA102
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, dissolved in DMSO; concn. 1.5 to 7.5 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS:
- 3 withut metabolic activation
- 2 with metabolic activation

DETERMINATION OF TOXICITY
- Method: relative total growth
Evaluation criteria:
The assay is considered valid if the followin 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.
Statistics:
No statistical analyses performed.

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
Remarks:
see Appendix for details
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see Appendix for details
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see Appendix for details
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see Appendix for details
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
ambiguous with metabolic activation

The results lead to the conclusion that RESIN 835 A is not mutagenic in these bacterial test systems either in the absence or in the presence of an exogenous metabolising system.
Executive summary:

Resin 835 A (Lot: 0161852) was tested for mutagenicity with the strains TA 100, Ta 1535, Ta 1537, TA 98 and TA 102 of Salmonella typhimurium. Two independent mutagenicity studies were conducted (one plate incorporation test with 10 % rat liver homogenate - and one with 30 % rat liver homogenate),each in the absence and in the presence of a metabolising system derived from a rat liver homogenate.

Additionally a repeat of the test with 10 % S9 -mix was performed with the strains TA 100, TA 1537 and TA 102 in the absence of S9 -mix because of heavy toxicity.

As ethanol had to be chosen as a vehicle, preincubation was not possible due to vehicle toxicity in high concentrations. Therefore, a modified plate incorporation test with 30 % S9 -mix was performed as second test. Other common vehicles (deionised water, DMSO) were not appropriate due to insolubility of the test compound.

For all studies, Resin 835 A was dissolved in ethanol, and each bacterial strain was exposed to 5 dose levels in the assays with 10 % S9 -mix. In the repeat assay with 10 % rat liver homogenate and in the assay with 30 % rat liver homogenate 6 dose levels were used.

The concentrations for the first plate incorporation assay were 50, 160, 500, 1600 and 5000 µg/plate.

Because of toxicity in the first assay dose levels from 1.6 to 5000 µg/plate were chosen for the repeat assay with 10 % rat liver homogenate.

Dose ranges for the assay with 30 % rat liver homogenate were variable across bacterial strains to account for varying toxicity. The lowest concentration was 1.6 µg/plate and the highest 5000 µg/plate.
Visible precipitation of Resin 835 A was observed at 1600 µg/plate and above.

Control plates without mutagen showed that the number of spontaneous revertant colonies were within the laboratory's historical control range. All the positive controls showed the expected increase in the number of revertant colonies.
The number of revertant colonies of the solvent controls in individual strains was slightly out of the historical control data range with ethanol. However the existing control data pool with ethanol is limited as this solvent is used rarely. All data are within the normal range of historical DMSO controls.
Additionally the number of revertant colonies of the negative controls with the strain TA 1537 (first test only) in the presence of S9-mix was slightly out of the historical control data range, the number of revertant colonies of the solvent controls with the strain TA 1537 (repeat test only) in the absence of S9-mix was slightly out of the historical control data range of DMSO and also the number of revertant colonies of the positive controls with the strain TA 102 (repeat and second test) in the absence of S9-mix was slightly out of the historical control data range, but in all cases the criteria for the negative/positive response were fulfilled.

Toxicity: In the plate incorporation test with 10 % rat liver S9-mix toxicity was observed with metabolic activation in a dose range of 160 to 5000 µg/plate and above.
In the absence of metabolic activation the test compound proved to be toxic to individual bacterial strains in a dose range of 50 to 1600 µg/plate and above.

In three strains (TA 100, TA 1535, TA 102) without S9-mix all doses used caused toxicity in form of reduced bacterial lawn. As toxicity might cover possible mutagenic effects, the test had to be repeated with these strains using a lower dose range. Slight differences in toxicity between the first and repeat test are probably caused by biological variability.
In the plate incorporation test with 30 % rat liver S9-mix toxicity was observed with metabolic activation in a dose range of 160 to 1600 µg/plate.
In the absence of metabolic activation Resin 835 A proved to be toxic to most of the bacterial strains at concentrations of 160 µg/plate and above, expect the strain TA 98, where toxicity was observed at the dose level of 1600 µg/plate and above.