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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Palladium di(4-oxopent-2-en-2-oate) was negative in good quality, guideline, in vitro studies for genotoxicity.

In a bacterial reverse mutation (Ames) test, conducted according to OECD Test Guideline 471 and to GLP, palladium di(4-oxopent-2-en-2-oate) failed to induce an increase in mutation frequency in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested at concentrations of up to 5000 μg/plate or up to the limit of cytotoxicity, in the absence and presence of rat liver S9 (Mc Garry, 2013).

In an in vitro mammalian cell gene mutation assay, conducted in accordance with OECD Test Guideline 476 and to GLP, palladium di(4-oxopent-2-en-2-oate) did not induce biologically relevant increases in mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to cytotoxic concentrations in the absence and presence of S9 (Lloyd, 2014).

In an in vitro micronucleus assay, conducted according to OECD Test Guideline 487 and to GLP, palladium di(4-oxopent-2-en-2-oate) failed to induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity, for 3 hours in the absence and presence of S9, and for 24 hours without S9 (Lloyd, 2013).

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:
28 June - 22 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Minor study deviation did not affect overall interpretation of study findings or compromise integrity of the study
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
Range-Finder (TA98; TA100; TA102 only):
0, 5, 15.81, 50, 158.1, 500, 1581, 5000 ug/plate (with and without S9)

Expt1
0, 0.1581 (without S9 only), 0.5, 1.581, 5, 15.81, 50, 158.1 ug/plate (with and without S9), 500 ug/plate (with S9 only)

Expt2
0, 1.563 (without S9 only), 3.125, 6.25, 12.5, 25, 50 ug/plate (with and without S9), 100 ug/plate (with S9 only)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl formamide.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation). In the presence of S9 in experiment 2, there is also a pre-incubation step.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Background lawns examined.
Evaluation criteria:
An increase in revertant numbers that give a significant response which was concentration-related.

A positive trend/effect described are reproducible.

The test article was considered positive in this assay if all the above criteria are met.
Statistics:
Dunnett's test was used to compare the counts at each concentration with the controls.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The concentrations tested in the Ames test were determined in a preliminary cytotoxicity range-finder experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants in negative (vehicle) and positive controls fell within the 99% confidence intervals of the current observed historial ranges.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In a bacterial reverse mutation (Ames) test, conducted according to OECD Test Guideline 471 and to GLP, palladium di(4-oxopent-2-en-2-oate) failed to induce an increase in mutation frequency in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested at concentrations of up to 5000 μg/plate or up to the limit of cytotoxicity, in the absence and presence of S9.

Executive summary:

Palladium di(4-oxopent-2-en-2-oate) was tested in a bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assayed 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 (S9), in two separate experiments. The highest concentrations of test article analysed were limited by toxicity and were determined following a preliminary toxicity range-finder experiment.

In experiment 1, cells were treated with the test article at a maximum concentration of 158.1 µg/plate in the absence of S9, and 500 µg/plate in the presence of S9. Following these treatments, evidence of toxicity was observed in all strains at 15.81 µg/plate and above in the absence of S9 and at 50 µg/plate and above in the presence of S9.

In experiment 2, the maximum concentration was reduced to 50 µg/plate for all treatments in the absence of S9, and 100 µg/plate for treatments in the presence of S-9 based on toxicity observed in Experiment 1. All treatments in the presence of S9 were further modified by the inclusion of a pre-incubation step.

Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and fit the acceptance criteria. Following palladium di(4-oxopent-2-en-2-oate) treatments of all the test strains in the absence and presence of S9, no statistically significant increases in revertant numbers were observed when the data were analysed at the 1% level using Dunnett’s test.

It is concluded that palladium di(4-oxopent-2-en-2-oate) did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of S. typhimurium when tested at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines) or up to the limit of toxicity, in the absence and in the presence of S9. 

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:
19 Spetember 2013-24 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Minor protocol deviation not expected to impact interpretation of results or study integrity: pH values for some stock test solutions were not taken
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Range-finder experiment (without S9):
0.1563, 0.3125, 0.6250, 1.250, 2.500, 5.000 and 10.00 ug/ml

Range-finder experiment (with S9):
4.688, 9.375, 18.75, 37.50, 75.00 and 150.0 ug/ml

(The data presented for the Range-Finder Experiment in the absence of S-9 were generated from a second experiment. In the first Range-Finder, complete toxicity was observed at all concentrations analysed in the absence of S 9, therefore a further experiment was performed using lower concentrations. Data from the first Range-Finder in the absence of S-9 are not further reported.)

Experiment 1 (without S9):
0.1, 0.2, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.75 and 1 ug/ml

Experiment 1 (with S9):
0.5, 1, 2, 4, 6, 8, 9, 10, 12.5, 15 and 20 ug/ml

Experiment 2 (without S9):
0.1, 0.2, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.7, 0.8 and 1 ug/ml

Experiments 2 and 3 (with S9):
0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7 and10 ug/ml



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: Test substance was described as "soluble" in DMF at up to 14.97 mg/ml (note: at 15 mg/ml in DMF, presumably in the test article formulation, it was described as a "cloudy yellow suspension"; the solubility limit in culture medium was around 50 ug/ml.)
Untreated negative controls:
yes
Remarks:
Culture medium alone
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
NQO without S9; BaP with S9
Details on test system and experimental conditions:
For all treatments 0.2 mL vehicle, test article, culture medium or positive control solution was added to cells, with S 9 mix or 150 mM KCl buffer; the final treatment volume was 20 mL.

Treatments were performed in duplicate (except single cultures only used for positive control treatments).

Exposure duration: 3 hours.
Expression period: 7 days

Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p less than 0.05)
2. There was a significant concentration relationship as indicated by the linear trend analysis (p less than 0.05)
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance.
Statistics:
All calculations (i.e. cloning efficiency, relative survival, mutant frequency) were performed by computer using validated software. Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. (These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
Experiment 1 and experiment 2
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Experiment 1 and experiment 3
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Experiment 2 - Mutant frequencies increased; significant linear trend also. Not considered biologically relevant (see additional information, below).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A weak, statistically significant linear trend (p≤0.05) was observed in Experiment 2 without S9. However, there were no significant increases in mutant frequencies at any concentration analysed in this experiment, the observation was considered not biologically relevant.

The absolute mutant frequency (MF) values in the presence of S-9 in Experiment 2 were generally similar in magnitude to those observed in the absence of S-9 in the same experiment. However, the mean vehicle control MF values differed between the two treatment conditions, hence the statistical significance observed in the presence of S 9 was accentuated by the lower vehicle control MF value. Also, compared to historical control, "significant" increases in MF were small in magnitude. The increases seen in the presence of S-9 in Experiment 2 were investigated further in Experiment 3, where the same test article concentrations were tested, and neither significant increases in MF nor a significant linear trend were found despite duplicate tests/ Therefore the results of experiment 2 (with S9) were considered not biologically relevant.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Experiment 1

Concentration ug/ml

-S-9

Concentration ug/ml

+S-9

% RS

MF$

% RS

MF$

0

100

2.28

0

100

1.63

UTC

110

1.65

UTC

108

4.24

0.1

96

1.97 NS

0.5

78

2.65 NS

0.2

90

2.73 NS

1.0

97

2.96 NS

0.3

82

2.17 NS

2.0

79

2.11 NS

0.35

58

2.44 NS

4.0

15

2.33 NS

0.4

46

1.86 NS

 

 

 

0.5

25

2.93 NS

 

 

 

0.55

20

2.96 NS

 

 

 

NQO 0.15

21

54.41

B[a]P 2

54

15.81

NQO 0.2

10

64.77

B[a]P 3

65

43.79

Linear Trend test –S-9: NS

Linear Trend test +S-9: NS

UTC       Untreated control

$             6-TG resistant mutants/106viable cells 7 days after treatment

% RS     Percent relative survival adjusted by post treatment cell counts

NS         Not significant

a             Comparison of each treatment with control: Dunnett’s test (one-sided), significant at 5% level

 

Experiment 2

Concentration ug/ml

-S-9

Concentration ug/ml

+S-9

% RS

MF$

% RS

MF$

0

100

4.62

0

100

2.44

UTC

100

5.23

UTC

114

4.40

0.1

85

3.13 NS

0.5

108

5.59*

0.2

77

2.91 NS

1

103

5.27*

0.3

70

4.38 NS

2

75

5.34*

0.35

52

5.04 NS

2.5

41

5.39*

0.4

31

5.76 NS

3

27

8.26*

0.45

24

6.39 NS

3.5

6

4.93 NS

0.5

9

6.23 NS

 

 

 

NQO 0.15

57

35.34

B[a]P 2

77

26.38

NQO 0.2

51

54.40

B[a]P 3

45

2.55

Linear Trend test –S-9: * P<0.05

Linear Trend test +S-9: ** P<0.01

UTC                      Untreated control

$                            6-TG resistant mutants/106viable cells 7 days after treatment

% RS                    Percent relative survival adjusted by post treatment cell counts

NS                        Not significant

*                           Comparison of each treatment with control: Dunnett’s test (one-sided), significant at 5% level

*,**,***            Test for linear trend: X2(one-sided), significant at 5%, 1% and 0.1% level, respectively   

 

Experiment 3

Concentration ug/ml

+S-9

% RS

MF $

0

100

3.32

UTC

99

2.47

0.5

91

3.58 NS

1

96

3.20 NS

2

80

2.30 NS

2.5

59

4.96 NS

3

44

3.65 NS

3.5

19

5.18 NS

B[a]P 2

57

16.99

B[a]P 3

49

27.97

Linear Trend test +S-9: NS

UTC                      Untreated control

$                            6-TG resistant mutants/106viable cells 7 days after treatment

% RS                    Percent relative survival adjusted by post treatment cell counts

NS                        Not significant

Conclusions:
Interpretation of results (migrated information):
negative

In an in vitro mammalian cell gene mutation assay, conducted in accordance with OECD Test Guideline 476 and to GLP, palladium di(4-oxopent-2-en-2-oate) did not induce biologically relevant increases in mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to cytotoxic concentrations in the absence and presence of S9.
Executive summary:

In an in vitro GLP study, conducted in accordance with OECD Test Guideline 476 (in vitro mammalian cell gene mutation assay), palladium di(4-oxopent-2-en-2-oate) (in DMF) was tested for its ability to induce gene mutations at the hprt locus in mouse lymphoma L5178Y cells. A 3 hr treatment incubation period was used for all experiments.

In Experiment 1, no significant increases in mutant frequency (MF) were observed up to cytotoxic concentrations (0.55 or 4 μg/mL in the absence or presence of S9, respectively).

A weak, statistically significant linear trend (p≤0.05) was observed in Experiment 2 without S9. However, there were no significant increases in mutant frequencies at any concentration analysed in this experiment (at up to cytotoxic concentrations of 0.5 μg/mL). As such, the observation was considered not biologically relevant. The absolute MF values in the presence of S9 in Experiment 2, when tested at up cytotoxic concentrations (3.5 μg/mL) were generally similar in magnitude to those observed in the absence of S9 in the same experiment. However, the mean vehicle control MF values differed between the two treatment conditions, hence the statistical significance observed in the presence of S9 was accentuated by the lower vehicle control MF value. Also, compared to historical control, "significant" increases in MF were small in magnitude. The increases seen in the presence of S9 in Experiment 2 were investigated further in Experiment 3, where the same test article concentrations were tested, and neither significant increases in MF nor a significant linear trend were found despite duplicate tests. Therefore the results of Experiment 2 (with S9) were considered not biologically relevant.

Overall, palladium di(4-oxopent-2-en-2-oate) did not induce biologically relevant increases in gene mutations at the hprt locus of L5178Y mouse lymphoma cells when tested up to cytotoxic concentrations in three independent experiments in the absence and presence of S9

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June to October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, to GLP.
Qualifier:
according to guideline
Guideline:
other: OECD guideline 487. In Vitro Mammalian Cell Micronucleus Test
Deviations:
yes
Remarks:
Study deviations did not affect overall interpretation of study findings or compromise the integrity of the study.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: Human peripheral blood
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
0, 0.4, 0.9, 1.2 (without S9 for 3 hour exposure)
0, 2.5, 3.5, 4.5, 5.25 (with S9 for 3 hour exposure)
0, 0.4, 1.05, 1.35 (24 hour exposure)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl formamide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Vinblastine
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 or 24 hours
- Expression time (cells in growth medium): 21 hours or 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours
- Cyto-B added at time of treatment

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 500

DETERMINATION OF CYTOTOXICITY
- Method: Cells examined for proportions of mono-, bi- and multinucleate cells to determine a replication index (RI).
Evaluation criteria:
A statistically significant increase in the frequency of binucleate cells with micronuclei (MNBN) at one or more concentrations.

An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates.

A concentration-related increase in the proportion of MNBN cells.

All of the above criteria must be met for the test article to be considered positive in this assay.
Statistics:
The proportions of binucleate cells with micronuclei for each treatment condition were compared with the proportion in vehicle controls by using the Fisher's exact test. Probability values of p
Species / strain:
lymphocytes: Human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested up to limit of cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The concentrations tested in the micronucleus assay were determined in a preliminary cytotoxicity range-finder experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: The frequency of MNBN cells in vehicle controls fell within the 95th percentile of the current observed historial vehicle control (normal) ranges.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In an in vitro micronucleus assay, conducted according to OECD Test Guideline 487 and to GLP, palladium di(4-oxopent-2-en-2-oate) failed to induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity, for 3 hours in the absence and presence of S9, and for 24 hours without S9.
Executive summary:

Palladium di(4-oxopent-2-en-2-oate) was tested in an in vitro micronucleus assay conducted according to OECD Test Guideline 487 and to GLP. Human lymphocyte cultures prepared from the pooled blood of two healthy male donors were used. The highest concentrations of test article analysed were limited by toxicity and were determined following a preliminary cytotoxicity range-finder experiment. Cells were treated with the test article (in dimethyl formamide (DMF)) at 0, 0.4, 0.9 or 1.2 µg/ml for 3 hours (with 21 hours recovery time) in the absence of metabolic activation (S9), and at 0, 2.5, 3.5, 4.5 and 5.25 µg/ml in the presence of S9 from Aroclor 1254-induced rats. Additionally, cells were exposed to the test substance at 0, 0.4, 1.05 and 1.35 µg/ml for 24 hours in the absence of S9. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and matched the acceptance criteria.

Treatment of cells with palladium di(4-oxopent-2-en-2-oate) for 3 hours and for 24 hours in the absence of S9 resulted in frequencies of micronucleated binucleate MNBN cells that were generally similar to (marginally but not significantly higher than) those observed in concurrent vehicle controls at the highest concentrations analysed under both treatment conditions. There were no clear concentration-related increases in MNBN cell frequency and the evaluation criteria for a positive result were not fulfilled under either treatment condition. Therefore these isolated observations were considered not biologically relevant.

Treatment of cells for 3 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.05), compared to those observed in concurrent vehicle controls, at the lowest and highest concentrations analysed (2.50 and 5.25 μg/mL). However, the MNBN cell frequency of all treated cultures under this treatment condition cultures fell within the normal range and there was no evidence of a concentration-related response. Therefore, the small and sporadic statistically significant increases were considered to be of no biological relevance.

Overall, it is concluded that palladium di(4-oxopent-2-en-2-oate) did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3 hours in the absence and presence of S9, and for 24 hours in the absence of S9

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

No studies conducted in humans were identified (although effects in human lymphocytes were assessed in vitro).

Palladium di(4-oxopent-2-en-2-oate) was tested in a bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assayed 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 (S9), in two separate experiments. The highest concentrations of test article analysed were limited by toxicity and were determined following a preliminary toxicity range-finder experiment. In experiment 1, cells were treated with the test article at a maximum concentration of 158.1 µg/plate in the absence of S9, and 500 µg/plate in the presence of S9. Following these treatments, evidence of toxicity was observed in all strains at 15.81 µg/plate and above in the absence of S9 and at 50 µg/plate and above in the presence of S9. In experiment 2, the maximum concentration was reduced to 50 µg/plate for all treatments in the absence of S9, and 100 µg/plate for treatments in the presence of S-9 based on toxicity observed in Experiment 1. All treatments in the presence of S9 were further modified by the inclusion of a pre-incubation step. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and fit the acceptance criteria. Following palladium di(4-oxopent-2-en-2-oate) treatment of all the test strains in the absence and presence of S9, no statistically significant increases in revertant numbers were observed when the data were analysed at the 1% level using Dunnett’s test. It is concluded that palladium di(4-oxopent-2-en-2-oate) did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of S. typhimurium when tested at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines) or up to the limit of toxicity, in the absence and in the presence of S9 (Mc Garry, 2013). 

In an in vitro GLP study, conducted in accordance with OECD Test Guideline 476 (in vitro mammalian cell gene mutation assay), palladium di(4-oxopent-2-en-2-oate) (in DMF) was tested for its ability to induce gene mutations at the hprt locus in mouse lymphoma L5178Y cells. A 3-hour treatment incubation period was used for all experiments. In Experiment 1, no significant increases in mutant frequency (MF) were observed up to cytotoxic concentrations (0.55 or 4 μg/mL in the absence or presence of S9, respectively). A weak, statistically significant linear trend (p ≤ 0.05) was observed in Experiment 2 without S9. However, there were no significant increases in MF at any concentration analysed in this experiment (at up to cytotoxic concentrations of 0.5 μg/mL). As such, the observation was considered not biologically relevant. The absolute MF values in the presence of S9 in Experiment 2, when tested at up cytotoxic concentrations (3.5 μg/mL) were generally similar in magnitude to those observed in the absence of S9 in the same experiment. However, the mean vehicle control MF values differed between the two treatment conditions, hence the statistical significance observed in the presence of S9 was accentuated by the lower vehicle control MF value. Also, compared to historical control, "significant" increases in MF were small in magnitude. The increases seen in the presence of S9 in Experiment 2 were investigated further in Experiment 3, where the same test article concentrations were tested, and neither significant increases in MF nor a significant linear trend were found despite duplicate tests. Therefore the results of Experiment 2 (with S9) were considered not biologically relevant. Overall, palladium di(4-oxopent-2-en-2-oate) did not induce biologically relevant increases in gene mutations at the hprt locus of L5178Y mouse lymphoma cells when tested up to cytotoxic concentrations in three independent experiments in the absence and presence of S9 (Lloyd, 2014).

Palladium di(4-oxopent-2-en-2-oate) was tested in an in vitro micronucleus assay conducted according to OECD Test Guideline 487 and to GLP. Human lymphocyte cultures prepared from the pooled blood of two healthy male donors were used. The highest concentrations of test article analysed were limited by toxicity and were determined following a preliminary cytotoxicity range-finder experiment. Cells were treated with the test article (in dimethyl formamide (DMF)) at 0, 0.4, 0.9 or 1.2 µg/ml for 3 hours (with 21 hours recovery time) in the absence of metabolic activation, and at 0, 2.5, 3.5, 4.5 and 5.25 µg/ml in the presence of S9from Aroclor 1254-induced rats. Additionally, cells were exposed to the test substance at 0, 0.4, 1.05 and 1.35 µg/ml for 24 hours in the absence of S9. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and matched the acceptance criteria. Treatment of cells with palladium di(4-oxopent-2-en-2-oate) for 3 hours and for 24 hours in the absence of S9 resulted in frequencies of micronucleated binucleate MNBN cells that were generally similar to (marginally but not significantly higher than) those observed in concurrent vehicle controls at the highest concentrations analysed under both treatment conditions. There were no clear concentration-related increases in MNBN cell frequency and the evaluation criteria for a positive result were not fulfilled under either treatment condition. Therefore these isolated observations were considered not biologically relevant. Treatment of cells for 3 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.05), compared to those observed in concurrent vehicle controls, at the lowest and highest concentrations analysed (2.50 and 5.25 μg/mL). However, the MNBN cell frequency of all treated cultures under this treatment condition cultures fell within the normal range and there was no evidence of a concentration-related response. Therefore, the small and sporadic statistically-significant increases were considered to be of no biological relevance. Overall, it is concluded that palladium di(4-oxopent-2-en-2-oate) did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3 hours in the absence and presence of S9, and for 24 hours in the absence of S9 (Lloyd, 2013).

 

No in vivo data were identified.


Justification for selection of genetic toxicity endpoint
GLP study, conducted according to OECD guidelines.

Justification for classification or non-classification

No evidence of genotoxic activity has been seen in reliable in vitro assays in bacterial or mammalian somatic cells, including GLP guideline studies assessing mutagenic and clastogenic activity. No studies specifically assessing the mutagenic activity in germ cells were identified. However, no effects on reproductive parameters were seen in the combined repeated dose and reproductive/developmental toxicity screening assay on palladium hydroxide. As such, classification of palladium di(4-oxopent-2-en-2-oate) for germ cell mutagenicity is not warranted, according to EU CLP criteria (EC 1272/2008).