Reaction mass of Resin acids and Rosin acids, hydrogenated, potassium salts and potassium [1R-(1α,4aβ,10aα)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylate

Genetic toxicity: in vitro

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:

9 April 2003 to 30 July 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Fully Guideline- and GLP-compliant, read-across from related substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

Reaction mass of Rosin, hydrogenated and [1R-(1a,4aß,10aa)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid

IUPAC Name:

Reaction mass of Rosin, hydrogenated and [1R-(1a,4aß,10aa)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid

Constituent 2

Reference substance name:

Resin 835 A

IUPAC Name:

Resin 835 A

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 / strain

Species / strain 1

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 2

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

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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 results

Test results 1

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

Test results 2

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.