Iridium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity potential of iridium (tested as powder) is determined using an in vitro reverse mutation assay with bacteria according to protocol OECD 471 (GLP compliant). The assay provided no evidence of any iridium mutagenic activity.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Sept 2019 - 13 Dec 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to GLP

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

Specific details on test material used for the study:

purity >= 99.90% (ASTM powder)

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).
Toxicity assessed as diminution of background bacterial lawn and/or marked reduciton in revertant numbers.
Experiment 1: 5, 16, 50, 160, 500, 1600,5000 µg/plate (+ and - S9)
Experiment 2: 51.2, 128, 320, 800, 2000, 5000 µg/plate (+ and - S9), treatments +S9 further modified by inclusion of pre-incubation step.

Vehicle / solvent:

test item insoluble in THF, DMF, Acetone, ethanol, N-Methyl pyrrolidinone.
DMSO not to be used as vehicle due to potential test-item complexation.
Stable suspension in 0.5% methylcellulose at concentrations up to at least 50 mg/mL.
Test article stock suspensions were prepared by suspending test item under subdued lighting in 0.5% MC, with the aid of Silverson mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using 0.5% MC. The test article suspensions were protected from light, stirred continuously throughout dilutions and treatment and used within approximately 4.5 hours of initial formulation.

Negative solvent / vehicle controls:

yes

Remarks:

0.1 mL 0.5% MC

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.
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 control solution or positive control, bacteria and S-9 mix detailed above, were mixed together and placed in an orbital incubator set to 37°C for 20 minutes, before the addition of 2 mL of supplemented 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.
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as small colonies, bubbles or splits in the agar or contamination affected the accuracy of the automated counter.

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.

Key result

Species / strain:

other: all tester strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 treated as a suspension rather than a solution (and therefore the significance of these pH values is unclear), the change in pH units across the concentration range was <1, and these data were not considered to provide any concerns for the assay or test compound.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

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.

Conclusions:

It was concluded that Iridium 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 (treatments at concentrations up to 5000 μg/plate, the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Iridium 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 S-9, in two separate experiments.

All Iridium treatments in this study were performed using formulations as suspensions prepared in 0.5% methylcellulose (0.5% MC).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations up to 5000 μg/plate. Following these treatments, no evidence of toxicity was observed, as would normally be manifest as a thinning of the background bacterial lawn and/or a marked reduction in revertant numbers.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9 with the maximum test concentration of

5000 μg/plate retained for all strains. Narrowed concentration intervals were employed covering the range 51.2-5000 μg/plate, in order to examine more closely

those concentrations of Iridium approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In

addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. Following these treatments,

there was again no evidence of toxicity observed. As the test article was treated as a suspension, any observations regarding the presence or otherwise of particulate test article (precipitate) in the assay system were not considered relevant, and therefore are not reported.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies were comparable with

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

Following Iridium 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 Iridium mutagenic activity in this assay system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In vitro testing provided no evidence of any Iridium mutagenic activity, and the substance does not require a classification for this endpoint.