Palladium dichloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Palladium dichloride was not mutagenic in good-quality bacterial reverse mutation (Ames) assays when tested up to the limit of toxicity in the presence and absence of metabolic activation (Ballantyne 2018; Mortelmans et al., 1986).

In a GLP-compliant guideline in vitro mammalian cell gene mutation assay, Palladium dichloride did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested up to toxic and/or precipitating concentrations in the absence of a rat liver metabolic activation system (S-9) and up to toxic concentrations in the presence of S-9.

In a limited study, palladium dichloride did not significantly increase the number of micronuclei in human lymphocytes, in the absence of S9 (Gebel et al., 1997). Whereas, in another limited study, palladium chloride did cause a weak but statistically significant micronuclei induction in a human lymphocyte micronucleus assay in the absence of S9 (Migliore et al., 2002).

 

No evidence of DNA damage was seen with palladium dichloride in a limited bacterial SOS chromotest using the E. coli strain PQ37, in the absence of S9 (Gebel et al., 1997). Similarly, according to a published paper, in a limited study, palladium dichloride did not induce DNA damage in human leukocytes when tested at concentrations of up to 300 μM in the absence of metabolic activation (Migliore et al., 2002).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not given

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although not a standard (guideline) study, it appears well conducted and scientifically acceptable.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A modified alkaline single cell gel electrophoresis (SCGE) assay in human leukocytes, performed according to Singh NP et al. (1988). Exp. Cell Res. 175, 184-191 with some modifications (Klaude M et al (1996). Mutat. Res. 363, 89-96).

GLP compliance:

not specified

Type of assay:

comet assay

Species / strain / cell type:

primary culture, other: human leukocytes

Details on mammalian cell type (if applicable):

Whole blood obtained from a young, healthy, non-smoking male donor

Metabolic activation:

without

Test concentrations with justification for top dose:

100, 200 and 300 μM (It is unclear whether the units of measurement are mM or μM as the concentrations given in the text and in graphs for other chemicals tested in the same study report the concentrations in μM; however a table detailing the results gives the test concentrations in mM.)

Vehicle / solvent:

Bi-distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: hydrogen peroxide at concentrations of 50, 100, 150 and 200 μM (or possibly mM)

Details on test system and experimental conditions:

Human leukocytes were mixed with trypan blue and, after 15 min, counted and checked for viability. They were resuspended in RPMI 1640 and treated with the test substance for 2 hr at 37°C and, immediately afterwards, assayed for DNA damage under yellow light to prevent additional DNA damage. 3x10+5 cells (10 μl cell suspension) was added to a slide surface together with agarose. Slides were immersed in ice-cold freshly prepared lysing solution to lyse the cells and allow DNA unfolding. After at least 1 hr at 4°C in the dark, slides were placed on a horizontal electrophoresis unit and the unit filled with buffer to cover the slides. The slides were allowed to set for 20 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was conducted for 20 min at 25 V and slides were gently washed in neutralization buffer to remove alkali and stained with ethidium bromide. Endonuclease III (endo III) and formamidopyrimidine glycosylase (fpg) were used to detect oxidised pyrimidines and damaged purines (including 8-oxoguanine) respectively.The enzymes were diluted in enzyme buffer, placed on gels and covered with coverslips. Slides were put into a moist box and incubated at 37°C for 45 (endo III) or 30 min (fpg).

Evaluation criteria:

Images of 100 randomly selected cells (50 cells from each of 2 replicate slides) were analysed from each sample under a fluorescence microscope using a calibration scale considering 2 variables: nucleus diameter and comet tail length, which includes the nucleus diameter plus tail length. Two independent cultures were performed per experimental point, for a total of 100 cells, and the mean was calculated.

Statistics:

The effects of dose, culture and experiment were evaluated by multifactor analysis of variance (MANOVA). The multiple range test was performed (p<0.05) in order to detect differences in DNA migration among doses.

Species / strain:

primary culture, other: human leukocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Relative viability >80% to prevent potential artefacts due to indirect toxicity-induced damage

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

There were no statistically significant effects on DNA migration or on oxidised pyrimidines and purines, indicating no evidence of DNA damage.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

According to a published paper, palladium dichloride did not induce DNA damage in human leukocytes when tested at concentrations of up to 300 μM in the absence of metabolic activation.

Executive summary:

According to a published paper, the ability of palladium dichloride to induce DNA damage in human leukocytes in a modified alkaline single cell gel electrophoresis (SCGE) assay was assessed at concentrations of up to 300 μM, in the absence of a mammalian (S9) metabolic activation system.

There were no statistically significant effects on DNA migration or on oxidised pyrimidines and purines, indicating no DNA damaging effect of treatment.

In conclusion, palladium dichloride did not induce DNA damage in human leukocytes in vitro at concentrations of up to 300 μM in the absence of S9.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: chromosome damage (micronuclei)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Limited study. Although not a standard (guideline) study, it appears well conducted and scientifically acceptable. However, only two concentrations were analysable (as severe cytotoxicity seen at the highest concentration); whereas current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated. In the absence of S9 only.

Qualifier:

according to guideline

Guideline:

other: method as described by Fenech M (1993) Mut Res 285, 35-44

Principles of method if other than guideline:

Mammalian cytokinesis-block micronucleus assay similar to that described by OECD TG487. Principal differences were that only two concetrations were analysable (as severe cytotoxicity seen at the highest concentration); whereas current OECD guidelines recommend that at least 3 analysable concentrations should be evaluated. Also, test was carried out only in the absence of metabolic activation.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human peripheral mononuclear blood cells (lymphocytes)

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentrations of 0, 100, 300 or 600 µM

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Briefly, blood from healthy, non-smoking donors (aged 25-35-years) was obtained, and the lymphocytes isolated, stained and counted. The lymphocytes were then cultured in medium at a concentration of 500,000/ml, and cell mitosis was stimulated. The test substance was disolved in DMSO and added 24 hr later to the culture in a volume of 20-30 µl. Seventy hours after cell mitosis was stimulated, the cells were harvested, fixed and prepared for microscopy. Micronuclei were scored in 1000 binucleate cells with two nuclei of equal size, and the nuclear division index (NDI) was calculated. Duplicate or triplicate experiments were carried out on different donors.

Evaluation criteria:

Test substance was considered genotoxic if a statistically significant (p<0.05) increase in the mean number of micronuclei in binucleate cells were observed.

Statistics:

Number of micronuclei analysed with the X2 test

Species / strain:

lymphocytes: Human peripheral mononuclear blood cells (lymphocytes)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Severe cytotoxicity reported at 600 µM

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The mean numbers of micronuclei in binucleate cells were 6.0, 4.5 and 3.3 at concentrations of 0, 100 and 300 µM, respectively, so treatment with palladium dichloride produced no statistically significant change from negative control. At 600 µM, severe cytotoxicity was seen.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In a limited study, palladium dichloride did not significantly increase the number of micronuclei in human lymphocytes, in the absence of S9.

Executive summary:

In a limited study, the ability of palladium dichloride (in DMSO) to induce micronuclei in human peripheral mononuclear blood cells (lymphocytes) was assessed, in the absence of S9 activation only.

The mean numbers of micronuclei in binucleate cells were 6.0, 4.5 and 3.3 at concentrations of 0, 100 and 300 µM, respectively, so treatment produced no statistically significant change from the negative control. At 600 µM, severe cytotoxicity was seen and no assessment of chromosome damage was possible.

In conclusion, the test substance did not induce chromosome damage in a limited cytokinesis-block micronucleus test with human lymphoctes that tested only two analysable concentrations, in the absence of S9. [Current OECD guidelines recommend that at least 3 analysable test concentrations should be evaluated, with and without S9.]

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not given

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: From published paper, appears to be a well conducted study and scientifically acceptable. Lymphocytes from only one man assessed.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A micronucleus assay in human lymphocytes, coupled with fluorescence in situ hybridization.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Whole blood obtained from a young, healthy, non-smoking male donor

Metabolic activation:

without

Test concentrations with justification for top dose:

50, 100, 200, 300, 400 and 600 μM (presumably the test concentration is measured in μM, as given in the text, in a graph depicting the results and a table giving the results of the FISH analysis; however, a table of the results quotes the test concentrations in mM).

Vehicle / solvent:

Bi-distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: Griseofulvin also employed for the FISH analysis

Details on test system and experimental conditions:

Lymphocytes were stimulated with phytohaemagglutinin (PHA) and cultured, in duplicate, for 72 hr at 37°C. Treatment with the test substance was performed 24 hr after PHA stimulation. To block the cytokinesis of interphase cells, cytochalasin B was added at 44 hr. Binucleated lymphocytes were harvested after 72 hr culture (48 hr treatment time), treated with potassium chloride to lyse erythrocytes, prefixed in methanol, washed twice with fixative and dropped onto a clean glass slide and, once air-dried, stained in Giemsa solution or hybridized within 1 week of preparation. For FISH analysis, a digoxigenin-labelled α-satellite DNA probe specific for the centromeres of all human chromosomes was used. Prewarmed slides were denatured and dehydrated and, after heating at 70°C for 5 min, probes were placed on the slide and incubated overnight. Post-hybridization washes were performed, followed by preincubation in an immunological buffer to minimize the background. Anti-digoxigenin, TRITC-conjugated anti-mouse and TRITC-conjugated anti-rabbit anti-bodies were alternately incubated for 30 min, slides were washed and dehydrated after each incubation and counterstained with DAPI to detect the digoxigenin-labelled probe.

Evaluation criteria:

Giemsa stained slides were scored blind for micronucleus analysis under an optical microscope. The results were expressed as the average number of micronucleated cells ± SD from 2 observations of 1000 cells on 2 different slides from 2 culture tubes. Micronucleus frequency was expressed as the number of micronucleated binucleate cells per 1000 cells. For FISH analysis, preparations were analysed on a fluorescence microscope equipped with a triple bandpass filter for simultaneous visualization of TRITC, FITC and DAPI fluorescence. A sufficient number of lymphocytes were scored in order to record 50 micronuclei for each experimental point. Micronuclei were analysed for the presence of the fluorescent signal by considering a TRITC-labelled micronucleus as centromere-positive micronucleus (C+MN) and a non-labelled micronucleus as cetromere-negative micronculeus (C-MN).

Statistics:

Data were analysed by Fisher's exact test to determine the significant difference between the treated and the control cultures for micronucleated binucleate cells and to analyse the results obtained with the FISH technique (% C+MN or % C-MN) in treated compared with control cultures.

Species / strain:

lymphocytes: Human

Metabolic activation:

without

Genotoxicity:

other: weak but statistically positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic at 600 μM

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Weak but statistically significant increases in the number of micronucleated binucleate cells were reported following exposure to 100, 200 and 300 μM (p<0.05). In the FISH analysis, although a slight prevalence of C-MN was observed, there was no statistically significant difference in the frequency of C+MN and C-MN.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
other: weakly positive, without activation

In a limited study, palladium chloride caused weak but statistically significant micronuclei induction in a human lymphocyte micronucleus assay in the absence of S9.

Executive summary:

According to a published paper, in a limited study, the ability of palladium dichloride to induce micronuclei in lymphocytes from a healthy, non-smoking man was assessed in the absence of a metabolic (S9) activation system. Weak but statistically significant increases in the frequency of micronucleated binucleate cells were noted, indicating a genotoxic effect.

 

In conclusion, palladium dichloride caused weak but statistically significant micronuclei induction (chromosome damage) in human lymphocytes in vitro, in the absence of S9.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited study. Although not a standard (guideline) study, it appears well conducted and scientifically acceptable.

Qualifier:

according to guideline

Guideline:

other: non standard method as described by Quillardet P and Hofnung M (1985) Mut Res 147, 65-78

Deviations:

yes

Remarks:

As described in Mersch-Sundermann V et al. (1991) Mut Res 252, 51-60; Mersch-Sundermann V et al.(1992) Mut res 278, 1-9; Mersch-Sundermann V et al. (1993) Anticancer Res 13, 2037-2044

Principles of method if other than guideline:

SOS chromotest for the identification of the sfiA gene inducing DNA damage in the strain E. coli PQ37

GLP compliance:

not specified

Type of assay:

SOS/umu assay

Target gene:

sfiA gene

Species / strain / cell type:

E. coli, other: PQ37

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentrations range of 7-458 µM

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

Briefly, test substance was dissolved in DMSO (20 ul) and given to 600 ul portions of the bacterial nutrient broth dilution (5 000 000 CFU/ml). The highest concentration of the substance was taken as "reagent blank" (unless substance was of intensive colour, in which case a blank of each dilution was taken to avoid false negative results). The mixtures were incubated at 37 degrees centigrade for 2.5 hr and afterwards the beta-galactosidase activity (SOS induction) and the alkaline phosphatase activity (toxicity assay) were determined. 4-NQO was used as positive control.

Evaluation criteria:

In order to classify a substance as genotoxic, a continuous increase in beta-galactosidase activity with increasing substance concentration was needed. Maximum Induction Factor (IFmax) in the non-cytotoxic dose range was also utilised for evaluation as follows:
Not genotoxic: IFmax less than 1.5
Moderately genotoxic: IFmax between 1.5 and 2.0
Genotoxic: IFmax greater than 2.0

Species / strain:

E. coli, other: PQ37

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The maximum induction factor (IFmax) was 1.31.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

In a limited study, palladium dichloride (at up to the limit of solubility) did not induce DNA damage in a bacterial SOS chromotest using the E. coli strain PQ37, in the absence of S9.

Executive summary:

In a limited study, the ability of palladium dichloride (at 7-458 µM) to induce DNA damage in the bacteria E. coli (strain PQ37), in an SOS chromotest, was assessed (in the absence of a mammalian metabolic activation system). Cytotoxicity was not seen but was tested up to the limit of solubility.

A maximum induction factor (IFmax, in the absence of cytotoxicity) of 1.31 was reported, indicating that the test substance had no genotoxic effect. In conclusion, the test substance did not show any ability to induce DNA damage in a bacterial SOS chromotest using E. coli PQ37, without S9.

Reference 5

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:

19 April 2018 - 30 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 99.6% PdCl2 (59.78% Pd content)

Target gene:

Histidine

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:

S-9 derived from Aroclor 1254-treated male Sprague-Dawley rats

Test concentrations with justification for top dose:

Mutation Experiment 1 (TA100,1535,1537,102; with and without S9)
2.5, 8, 25, 80, 250, 800, 2500 ug/plate

Mutation Experiment 1 (TA98; with and without S9)
0.08, 0.25, 0.8, 2.5, 8, 25, 80, 250 ug/plate

Mutation Experiment 2 (all strains; with and without S9)
0.33, 0.82, 2.05, 5.12, 12.8, 32, 80, 200 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test article stock solutions were prepared by suspending Palladium dichloride under subdued lighting in purified water with the aid of vortex mixing, ultrasonication and warming at 37°C (where required).

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Palladium dichloride was soluble in water for irrigation (purified water) at concentrations equivalent to approximately 5 mg/mL.

Untreated negative controls:

yes

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

Remarks:

2NF for TA98 (-S9); NaN3 for TA100 and TA1535 (-S9); AAC for TA1537 (-S9); MMC for TA102 (-S9); BaP for TA98 (+S9); AAN for TA100, TA1535, TA1537 and TA102 (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In agar (plate incorporation); preincubation (for experiment 2 in the presence of S9).
- 0.5 mL volume additions of test article solution were used for all Experiment 1 treatments, except those of strain TA98 which used 0.1 mL additions, as did the Experiment 2 treatments for all strains.

Triplicate plates for test substance, vehicle and positive controls.

Prepared test suspensions were protected from light and used within approximately5 hours of initial formulation.

DURATION
As the results of Experiment 1 were negative, treatments in the presence of S-9 in
Experiment 2 included a pre-incubation step. Quantities of test article, vehicle,
untreated or positive control, bacteria and S-9 mix detailed above, were mixed
together and incubated for 20 minutes at 37±1°C, with shaking, 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.

DETERMINATION OF CYTOTOXICITY
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity included a marked reduction in revertants compared to the concurrent vehicle controls.

Evaluation criteria:

Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges.

The assay was considered to be valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of > (or equal to) 1.5-fold (in strain TA102), > (or equal to) 2-fold (in strains TA98 and TA100) or > (or equal to) 3-fold (in strains TA1535 and TA1537) the concurrent vehicle control, confirming discrimination between different strains, and an active S 9 preparation.

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 and TA100) or ≥3-fold (in strains TA1535 and 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 either of the above criteria were met.

Statistics:

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.

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

Remarks:

Exp1: evidence of toxicity was observed at 80 µg/plate and above in all strains in the absence of S-9 and in strain TA98 in the presence of S 9, and at 250 µg/plate and above in all other strains in the presence of S-9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The pH range of the treatment concentrations for each experimental occasion was notably greater than 1 pH unit. The Sponsor however advised that the observations of decreasing pH with increasing test article concentration are to be expected, as the test article (Palladium dichloride) acidifies the medium upon dissolution. Treatments were therefore continued for each experiment, as this reduction in pH was not considered to have indicated any adverse effect on the test article, and the assay system is buffered at pH 7.4, and in any case the test bacteria will tolerate quite acidic conditions.

It was noted that due to the extent of the test article-related toxicity, fewer than 5 analysable concentrations remained for all Experiment 1 strain treatments, with the exception of those of strain TA98. However, as at least 5 analysable concentrations remained for all strain treatments in Experiment 2, the study as a whole is considered to have provided a thorough and robust assessment of the mutagenicity of Palladium dichloride in this assay system.

A small increase (2.2-fold the concurrent vehicle control level) in revertant numbers was seen in strain TA1535 in the absence of S-9, but this increase was largely attributable to a single elevated replicate plate count, and was not reproducible in Experiment 2, and was therefore not sufficient to be considered to have been a true compound-related effect. This study was considered therefore to have provided no evidence of any Palladium dichloride mutagenic activity in this assay system.

Conclusions:

In a guideline Ames test, it was concluded that Palladium dichloride did not induce mutation in five histidine-requiring 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 dichloride 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 dichloride treatments in this study were performed using formulations prepared in water for irrigation (purified water).

Mutation Experiment 1 treatments for strains TA100, TA1535, TA1537 and TA102 were performed in the absence and in the presence of S-9, using final concentrations of Palladium dichloride up to 2500 µg/plate (maximum concentration limited by solubility in the primary vehicle, purified water). For the Mutation Experiment 1 treatments of strain TA98, final concentrations up to 250 µg/plate were used. Following these Mutation Experiment 1 treatments, toxicity or evidence of toxicity was observed at 80 µg/plate and above in all strains in the absence of S-9 and in strain TA98 in the presence of S‑9, and at 250 µg/plate and above in all other strains in the presence of S-9.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum treatment concentrations were reduced to estimates of the lower limit of toxicity, this being 80 µg/plate for all strains except TA100 and TA1535 in the absence of S-9, and 200 µg/plate for all strains in the presence of S-9 and for strains TA100 and TA1535 in the absence of S-9. Narrowed concentration intervals were employed covering the ranges 0.33-80 µg/plate or 0.82-200 µg/plate, apart from treatments of strain TA98 in the presence of S-9 where concentrations covered the range 0.33-200 µg/plate. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. Following these treatments, toxicity or evidence of toxicity was again observed at 80 µg/plate (and above where applicable) in all strains in the absence of S-9, and at 200 µg/plate in all strains in the presence of S-9.

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 both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Palladium dichloride treatments of all the tester strains in the absence and presence of S-9, no notable and 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 dichloride mutagenic activity in this assay system.

Reference 6

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:

5 April 2018 - 29 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Specific details on test material used for the study:

purity: 99.6% (based on Pd content 59.78%)

Target gene:

Thymidine kinase (tk) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Growth media (RPMI 1640; containing L-glutamine and HEPES) prepared with horse serum, penicillin, streptomycin, amphotericin B, sodium pyruvate acid and pluronic; tissue culture flasks were gassed with 5±1% v/v CO2 in air.
- Properly maintained: yes. The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells were stored as frozen stocks in liquid nitrogen.
- Periodically checked for Mycoplasma contamination: yes. Each batch of frozen cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: yes. Each batch of frozen cells was purged of mutants.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9) from male Sprague-Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Cytotoxicity range-finder experiment: 15.63, 31.25, 62.50, 125, 250, 500 μg/mL (3-hr treatment; both with and without S9)

Mutation experiment: 20, 40, 80, 100, 120, 160, 200, 240, 280, 300, 500 μg/mL (3-hr treatment; without S9) and 10, 20, 40, 60, 80, 120, 160, 200, 240, 300 μg/mL (3-hr treatment; with S9)

In the mutation experiment, the dose levels were based on the observed effects on relative suspension growth (RSG) with increasing test concentration in the range-finder experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Palladium dichloride was soluble in purified water at a maximum concentration of 5 mg/mL. The solubility limit in culture medium was at least 500 µg/mL, as indicated by the absence of precipitate at this concentration following 3 hours’ incubation at approximately 37°C.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle (purified water) diluted 10-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: For the mutation experiments, at least 10^7 cells in a volume of 16 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2 mL vehicle or test article, or 0.2 mL positive control solution (plus 1.8 mL purified water) was added. S 9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S 9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 50 mL RPMI 10 medium.
Cells were transferred to tissue culture flasks for growth throughout the expression period. The solubility of the test article in culture was assessed, by eye, at the beginning and end of treatment.
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described above for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL. Following treatment, cells were centrifuged (200 g), washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium. All cultures were incubated at 37±1°C for 1 day, recounted and where possible, diluted to 2 x 10^5 cells/mL. Cultures were incubated for a further day and counted. At the end of the expression period, toxicity was assessed by measuring suspension growth (and hence relative suspension growth (RSG)), compared to the concurrent vehicle control values. Cultures were not plated for viability assessment.


DURATION
- Preincubation period: Not applicable
- Exposure duration: 3 hours (with and without S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: For viability: 192 wells averaging 1.6 cells/well; for TFT resistance (384 wells at 2000 cells/well).

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test substance was measured by calculating the relative total growth (RTG) according to the following formula: RTG = RSG x ([Individual viability value]/[Mean control viability value]), where RSG = ([Individual SG value]/[Mean control SG value]) x 100. Viability (cloning efficiency [CE]) was calculated as follows: CE = ([-ln P(0)]/[Number of cells per well]) x 100, where P(0) = (Number of wells with no colony)/(Total number of wells)

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the vehicle control mutant frequency plus GEF
2. The linear trend test was statistically significant.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Linear regression was performed on ranked mutant frequency against ranked concentration to test for a linear trend. The linear trend was performed on the overall MF per concentration level, not individual replicates (i.e. one MF value per concentration). The test for linear trend is one-tailed, therefore negative trend was not considered relevant. The positive control was excluded from the test for linear trend.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

3-hr: highest concentration analysed was 240 µg/mL (+/- S-9), MF of concentrations plated were all

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest concentrations analysed -S9 (300 and 500 µg/mL) were too toxic; 240 µg/mL retained as max concentration (20%RTG). Highest concentration retained + S9 was 240 µg/mL (12% RTG).

Vehicle controls validity:

valid

Remarks:

MF in vehicle control cultures fell within acceptable ranges.

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Clear increases in mutation were induced by the positive control chemicals.

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed (250 or 125 µg/mL in the absence and presence of S 9. respectively) as compared to the concurrent vehicle controls.
- Evaporation from medium: No data
- Water solubility: Preliminary solubility data indicated that Palladium dichloride was soluble in purified water at a maximum concentration of 5 mg/mL.
- Precipitation: solubility limit in culture medium was at least 500 µg/mL, as indicated by the absence of precipitate at this concentration following 3 hours’ incubation at approximately 37°C.
- Definition of acceptable cells for analysis: Two days after treatment the highest two concentrations tested in the absence of S-9 (300 and 500 µg/mL) were considered too toxic for selection to determine viability and TFT resistance. In addition, the lowest two concentrations tested in the presence of S-9 were not selected as there were sufficient non-toxic concentrations. The highest concentration analysed to determine viability and TFT resistance was 240 µg/mL in the absence and presence of S 9, which gave 20% and 12% RTG, respectively.

RANGE-FINDING/SCREENING STUDIES:
the cytotoxicity Range-Finder Experiment, six concentrations were tested, in the absence and presence of S-9, ranging from 15.63 to 500 µg/mL (limited by solubility in the primary vehicle). Following the 3 hour treatment incubation period, post treatment precipitate was observed at the highest two concentrations tested in the absence of S-9 (250 and 500 µg/mL) and at the highest three concentrations tested in the presence of S-9 (125 to 500 µg/mL). The lowest concentration, at which precipitate was observed at the end of the treatment incubation period, in the absence and presence of S-9, was retained and higher concentrations were discarded. The highest concentrations to provide >10% RSG were 250 µg/mL in the absence of S-9 and 125 µg/mL in the presence of S-9, which gave 15% and 44% RSG, respectively.

MUTAGENICITY (INCLUDING COMPARISON WITH HISTORICAL CONTROL DATA):
In the Mutation Experiment, the MF of the concentrations plated were all less than the sum of the mean control MF plus the GEF and there were no statistically significant linear trends, indicating a negative result.

Mutation data in absence of S-9
Concentration%RTG MF
µg/mL
0 100 57.02
20 87 76.39
40 79 77.51
80 76 60.57
100 75 59.99
120 83 55.95
160 54 85.71
200 43 75.60
240 20 102.30
MMS 15 54 358.54
MMS 20 47 475.52
linear trend test on MF: p= 0.1431

Mutation data in presence of S-9
Concentration%RTG MF
µg/mL
0 100 76.78
40 50 103.12
60 42 116.26
80 38 103.44
120 33 124.38
160 42 109.94
200 25 96.58
240 PP 12 149.46
B[a]P 2 47 411.70
B[a]P 3 33 664.84
linear trend test on MF: p= 0.0.0914

For the negative and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated.
For the vehicle controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 35% to 38%.
Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals MMS and B[a]P.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The highest concentration analysed to determine viability and TFT resistance was 240 µg/mL in the absence and presence of S 9, which gave 20% and 12% RTG, respectively.

Conclusions:

In a GLP-compliant guideline in vitro mammalian cell gene mutation assay, Palladium dichloride did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested up to toxic and/or precipitating concentrations in the absence of a rat liver metabolic activation system (S-9) and up to toxic concentrations in the presence of S-9.

Executive summary:

Palladium dichloride was assayed for the ability to induce mutation at the tk locus in mouse lymphoma cells. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by S-9. The test article was formulated in purified water. A 3 hour treatment incubation period was used.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested, in the absence and presence of S-9 (15.63 - 500 µg/mL; limited by solubility in the primary vehicle). The highest concentrations analysed were limited by the appearance of post treatment precipitate to 250 µg/mL in the absence of S-9 and 125 µg/mL in the presence of S-9, which gave 15% and 44% relative suspension growth (RSG), respectively.

In the Mutation Experiment, eleven concentrations were tested in the absence of S‑9 (20 - 500 µg/mL), and ten concentrations were tested in the presence of S‑9 (10 - 300 µg/mL). Two days after treatment the highest concentration analysed to determine viability and TFT resistance was 240 µg/mL in the absence and presence of S-9, which gave 20% and 12% relative total growth (RTG), respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals. Therefore the study was accepted as valid.

The MF of the concentrations plated were all less than the sum of the mean control MF plus the global evaluation factor (GEF) and there were no statistically significant linear trends, indicating a negative result.

It is concluded that Palladium dichloride did not induce mutation at the tk locus of L5178Y mouse lymphoma cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo data were identified.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No studies conducted in humans were identified (although in vitro studies using human lymphocytes are described below).

 

In two good quality studies (according to or similar to procedure described in OECD Test Guideline 471) palladium dichloride was tested up to the limit of toxicity in a bacterial reverse mutation assay, in the presence and absence of a S9 mammalian metabolic activation system. There was no evidence of a mutagenic effect, and no dose response in any of the strains tested, either in the presence or absence of S9. In conclusion, palladium dichloride was not mutagenic to four strains of S. typhimurium, tested up to the limit of toxicity, in the presence and absence of S9 (Ballantyne 2018; Mortelmans et al., 1986).

 

In a GLP-compliant guideline in vitro mammalian cell gene mutation assay, Palladium dichloride did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested up to toxic and/or precipitating concentrations in the absence of a rat liver metabolic activation system (S-9) and up to toxic concentrations in the presence of S-9 (Hargreaves 2018).

In a limited study, the ability of palladium dichloride (in DMSO) to induce micronuclei in human peripheral mononuclear blood cells (lymphocytes) was assessed, in the absence of S9 activation only. The mean numbers of micronuclei in binucleate cells were 6.0, 4.5 and 3.3 at concentrations of 0, 100 and 300 µM, respectively, so treatment produced no statistically significant change from the negative control. At 600 µM, severe cytotoxicity was seen and no assessment of chromosome damage was possible. In conclusion, the test substance did not induce chromosome damage in a limited cytokinesis-block micronucleus test with human lymphoctes that tested only two analysable concentrations, in the absence of S9. [Current OECD guidelines recommend that at least 3 analysable test concentrations should be evaluated, with and without S9.]

 

According to a published paper, in a limited study, the ability of palladium dichloride to induce micronuclei in lymphocytes from a healthy, non-smoking man was assessed in the absence of S9. Weak but statistically significant increases in the frequency of micronucleated binucleate cells were noted, indicating a genotoxic effect. In conclusion, palladium dichloride caused weak but statistically significant micronuclei induction (chromosome damage) in human lymphocytes in vitro, in the absence of S9 (Migliore et al., 2002).

 

The ability of palladium dichloride (at 7-458 µM) to induce DNA damage in the bacterium E. coli (strain PQ37) was assessed in an SOS chromotest (in the absence of a mammalian metabolic activation system). Cytotoxicity was not seen even at the limit of solubility. A maximum induction factor (IFmax, in the absence of cytotoxicity) of 1.31 was reported, indicating that the test substance had no genotoxic effect. In conclusion, the test substance did not show any ability to induce DNA damage in a bacterial SOS chromotest using E. coli PQ37, without S9 (Gebel et al., 1997).

 

Finally, according to a published paper, the ability of palladium dichloride to induce DNA damage in human leukocytes in a modified alkaline single cell gel electrophoresis (SCGE) assay was assessed at concentrations of up to 300 μM, in the absence of any mammalian metabolic activation system. There were no statistically significant effects on DNA migration or on oxidised pyrimidines and purines, indicating no DNA damaging effect of treatment. In conclusion, palladium dichloride did not induce DNA damage in human leukocytes in vitro at concentrations of up to 300 μM in the absence of S9 (Migliore et al., 2002).

 

Several Expert Groups have assessed the toxicity profile of palladium, and various palladium compounds, including the assessment of CMR properties. The overwhelming weight-of-evidence (including good-quality in vitro and in vivo studies) indicates that palladium and palladium compounds are of no concern for mutagenic activity (bibra, 2007; DFG, 2006; EFSA, 2012, 2014; WHO, 2002). In addition, proprietary genotoxicity studies (including in vitro bacterial and mammalian studies assessing mutagenicity and clastogenicity, and an in vivo micronucleus study) of palladium compounds were overwhelmingly negative.

No in vivo data were identified.

Justification for selection of genetic toxicity endpoint
Good quality study, similar to OECD guidelines.

Justification for classification or non-classification

Based on the existing data set, palladium dichloride does not meet the criteria for classification as a germ cell mutagen (category 1A or 1B) under EU CLP criteria (EC 1272/2008).