Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Sept 2019 - 9 Oct 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Palladium(II) acetate
EC Number:
222-164-4
EC Name:
Palladium(II) acetate
Cas Number:
3375-31-3
Molecular formula:
C2H4O2.1/2Pd
IUPAC Name:
palladium(2+) diacetate
Details on test material:
- Name of test material (as cited in study report): palladium(II)-acetate trimer
- Substance type: brown powder
- Physical state: solid
- Analytical purity: 47.15% palladium
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Purity test date: no data
- Lot/batch No.: 6597
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable throughout the test period “according to the sponsor”
- Storage condition of test material: no data
- Other:

Specific details on test material used for the study:
Palladium(II) acetate (99.9% purity)
Pd content 48.22%

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S-9)
S-9 prepared from male Sprague Dawley rats induced with Aroclor 1254.
S-9 supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use.
Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 10% S-9 mix respectively.
Test concentrations with justification for top dose:
Treatments in this study were performed using suspensions of test item in vehicle up to a maximum concentration of 5000 μg/plate in Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997).
For Experiment 1 repeat treatments and Experiment 2 the maximum concentration tested was selected on the basis of toxicity seen in Experiment 1.
Toxicity assessed as diminution of background bacterial lawn and/or marke d reduciton in revertant numbers.
Experiment 1: 5, 16, 50, 160, 500, 1600,5000 µg/plate (+ and - S9)
Experiment 1 (repeat) and Experiment 2: 0.65536, 1.6384, 4.096, 10.24, 25.6, 64 and 160 µg/plate (+ and - S9), treatments +S9 further modified by inclusion of pre-incubation step.
Vehicle / solvent:
Preliminary solubility data indicated that Palladium acetate was soluble in dimethylformamide (DMF) at concentrations equivalent to at least 55.2 mg/mL.
Test article stock solutions were prepared by formulating Palladium acetate under
subdued lighting in DMF with the aid of vortex mixing, ultrasonication and warming
at 37°C (as required) to give the maximum required treatment concentration.
Subsequent dilutions were made using DMF. The test article solutions were protected
from light and used within approximately 4.5 hours of initial formulation.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL DMF
Positive controls:
yes
Remarks:
0.05 mL additions
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
0.1 mL volume additions of test article suspension were used for all treatments.
Plating details:
-0.1 mL of bacterial culture
-0.1 mL of test article suspension/vehicle control or 0.05 mL of positive control
-0.5 mL of 10% S-9 mix or buffer solution,
ollowed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated protected from light for 3 days in an incubator set to 37°C. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.
Due to extensive test article related toxicity following these initial Mutation
Experiment 1 treatments, Mutation Experiment 1 repeat treatments were performed in
all the tester strains in order to provide thorough and robust mutation data. These
treatments were performed using the methodology described above for Mutation
Experiment 1, but using lower test article concentrations.
It may be noted that initial Experiment 1 repeat treatments of strain TA102 in the
presence of S-9 were invalidated due to uncharacteristic vehicle control counts (data
not reported). These treatments were repeated (with diagnostic control treatments in
the absence of S-9; data not reported) in order to provide the mutation data for this
strain presented in this report.
As the results of Experiment 1 (and repeat) were negative, treatments in the presence
of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article,
vehicle control solution or positive control, bacteria and S-9 mix detailed above, plus
an additional 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4), were mixed
together and placed in an orbital incubator set to 37°C for 20 minutes, before the
addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded
as for the normal plate-incorporation procedure. In this way, it was hoped to increase
the range of mutagenic chemicals that could be detected in the assay.
The addition of 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4) to these
Experiment 2 treatments in the presence of S-9 was employed to reduce the solvent
concentration during the pre-incubation period. DMF, and some other organic
solvents, are known to be near to toxic levels when added at volumes of 0.1 mL in
this assay system when employing the pre-incubation methodology. By employing the
modification indicated, the DMF concentration in the pre-incubation mix was
decreased, and it was hoped that this would minimise or eliminate any toxic effects of
the solvent that may have otherwise occurred. In order to ‘correct’ for the additional
volume in the pre-incubation mix, these were plated out using 2 mL of 1.125% soft
agar, therefore the additions to each plate were comparable to that of the plateincorporation
treatments.
Rationale for test conditions:
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Statistics:
triplicate plates per concentration.
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of critical importance.

Results and discussion

Test results
Key result
Species / strain:
other: all tester strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Although in each experiment the change in pH units across the concentration range was >1, lower pH values were expected with increasing
Pd acetate concentration. These data were not considered to provide any concerns for the assay or test compound, and so treatments proceeded as planned.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Any other information on results incl. tables

Following test item treatments of all the test strains in the absence and presence of S-9, no notable or concentration-related increases in revertant numbers were observed, and none that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any test item mutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:
It was concluded that Palladium acetate did not induce mutation in five histidinerequiring
strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella
typhimurium when tested under the conditions of this study. These conditions
included treatments up to toxic concentrations in the absence and in the presence of a
rat liver metabolic activation system (S-9).
Executive summary:

Palladium acetate was assayed for mutation in five histidine-requiring strains (TA98,

TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the

absence and in the presence of metabolic activation by an Aroclor 1254-induced rat

liver post-mitochondrial fraction (S-9), in two separate experiments.

All Palladium acetate treatments in this study were performed using formulations

prepared in dimethylformamide (DMF).

Mutation Experiment 1 treatments of all the tester strains were performed in the

absence and in the presence of S-9, using final concentrations of Palladium acetate at

5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following these treatments, evidence of

toxicity was observed in all the tester strains and extended down to either 50 μg/plate

or 160 μg/plate in each case. Due to the extent of this toxicity, mutation data were

available from fewer than 5 analysable concentrations, and therefore all strain

treatments were repeated to provide a thorough and robust assessment of the

mutagenicity of the test article. Mutation Experiment 1 repeat treatments of all the

tester strains were therefore performed in the absence and in the presence of S-9 using

final concentrations of Palladium acetate at 0.65536, 1.6384, 4.096, 10.24, 25.6, 64

and 160 μg/plate, these concentrations being selected to test up to the lower limit of

toxicity. Following these treatments, evidence of toxicity was observed in all strains

in the absence and presence of S-9 and extended down to 25.6, 64 or 160 μg/plate in

each strain. Mutation data were available from at least 5 analysable concentrations in

each strain.

Mutation Experiment 2 treatments of all the tester strains were performed in the

absence and in the presence of S-9. The maximum test concentration of 160 μg/plate

was retained for all strains, as were the rest of the treatment concentrations.

Treatments in the presence of S-9 were modified by the inclusion of a pre-incubation

step. In this way, it was hoped to increase the range of mutagenic chemicals that could

be detected using this assay system. Following these treatments, evidence of toxicity

was again observed in all the strains in the absence and presence of S-9 and extended

down to either 25.6 μg/plate, 64 μg/plate or 160 μg/plate in each case. Mutation data

were available from at least 5 analysable concentrations from each strain.

The test article was completely soluble in the aqueous assay system at all

concentrations treated, in each of the experiments performed.

Vehicle and positive control treatments were included for all strains in all

experiments. The mean numbers of revertant colonies were comparable with

acceptable ranges for vehicle control treatments, and were elevated by positive control

treatments.

Following Palladium acetate treatments of all the test strains in the absence and

presence of S-9, no notable or concentration-related increases in revertant numbers

were observed, and none that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains

TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle

control. This study was considered therefore to have provided no evidence of any

Palladium acetate mutagenic activity in this assay system.