Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-Jan-2016 to 09-Feb-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance: Brown coloured, brittle solid
- Storage condition of test material: At room temperature
- Chemical name: Zinc modified rosinate, hydrogenated zinc rosinate
- CAS No.: 68425-02-5

Method

Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 5.4, 17, 52, 164 and 512 µg/ml
Since the test item was poorly soluble in the exposure medium, the highest concentration was 512 µg/ml

Experiment 1
Without S9-mix: 1, 10, 15, 20, 25, 30, 35 and 40 μg/ml
With S9-mix: 5, 10, 20, 30, 40 and 60 μg/ml
The test item was tested up to cytotoxic dose levels.



Vehicle:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: No workable solution or suspension could be obtained in culture medium, dimethyl sulfoxide, ethanol, acetone and dimethyl formamide. The test item was dissolved in tetrahydrofuran.
Tetrahydrofuran has been accepted and approved by authorities and international guidelines

Controlsopen allclose all
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: cyclophosphamide 10 µg/mL
Remarks:
with S9
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours


- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a)The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b)The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c)The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment).
d)The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10-6).


DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: Test system L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation:Resin acids and Rosin acids, hydrogenated, zinc salts precipitated in the exposure medium at concentrations of 512 μg/ml and above.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, the relative suspension growth was 101% at the test item concentration of 17 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 52 μg/ml and above.
In the presence of S9-mix, the relative suspension growth was 35% at the test item concentration of 52 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 164 μg/ml and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test item concentration was 36% compared to the total growth of the solvent controls.
In the presence of S9-mix, the relative total growth of the highest test item concentration was 25% compared to the total growth of the solvent controls.

Applicant's summary and conclusion

Conclusions:
It is concluded that Resin acids and Rosin acids, hydrogenated, zinc salts is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Executive summary:

The mean spontaneous mutation frequency of the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced an expected and appropriate response. In addition, the spontaneous mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, Resin acids and Rosin acids, hydrogenated, zinc salts induced an up to
2.3-fold dose related increase in the mutation frequency. The increase was just above the 95% control limits of the distribution of the historical negative control databaseand also abovethe GEF + MF(controls)(237 per 10E6survivors).

 

In the presence of S9-mix, Resin acids and Rosin acids, hydrogenated, zinc salts induced an up to 4.3-fold dose related increase in the mutation frequency. The increase was above the95% control limits of the distribution of the historical negative control databaseand also abovethe GEF + MF(controls)(229 per 10E6survivors).In addition, astatistical significant dose related trend (p<0.001) was observed.