Diammonium hexachloroiridate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Sept 2019 - 19 Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium hexachloroiridate

EC Number:

241-007-0

EC Name:

Diammonium hexachloroiridate

Cas Number:

16940-92-4

Molecular formula:

Cl6Ir.2H4N

IUPAC Name:

diammonium hexachloroiridate

Specific details on test material used for the study:

purity: 43.22% w/w iridium

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9)
S-9 prepared from male Sprague Dawley rats induced with Aroclor 1254.
S-9 supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use.
Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 10% S-9 mix respectively.

Test concentrations with justification for top dose:

Treatments in this study were performed using suspensions of test item in vehicle up to a maximum concentration of 5000 μg/plate in Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997).
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: 25, 50, 100, 200, 400, 800, 1600, 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.

Controls

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.

Results and discussion

Test results

Key result

Species / strain:

other: all tester strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Although in each experiment a concentration-related reduction in pH resulted in a pH change of >1 across the concentration range, this effect is considered to be
related to hydrolysis of the hexachloroiridate. Since the assay system can tolerate low pH, treatments proceeded as planned. It should also be noted that the test article was
treated as a suspension rather than a solution, and therefore the significance of these pH values is unclear.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Any other information on results incl. tables

Mutation Experiment 1:

Evidence of toxicity ranging from a slight thinning of the bacterial background lawn to a complete killing of the test

bacteria was observed at 1600 and/or 5000 μg/plate in all strains except TA102 in the absence of S-9 and in strain TA100 alone in the presence of S-9.

Mutation Experiment 2:

The maximum test concentration of 5000 μg/plate was retained for all strains except TA98 in the absence of S-9, where the maximum

test concentration was reduced to 1600 μg/plate based on strain specific toxicity

observed in Experiment 1.

Narrowed concentration intervals were employed covering the ranges 50-5000 μg/plate or 25-1600 μg/plate.

Evidence of toxicity manifest as either a slight thinning of the background bacterial lawn or a marked reduction in revertant numbers,

was observed at 1600 μg/plate in strain TA98 in the absence of S-9 and at 5000 μg/plate in strains TA1535 and TA1537 in the absence of S-9.

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.

Following Diammonium Hexachloroiridate 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.

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Diammonium Hexachloroiridate 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 Diammonium Hexachloroiridate 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 of Diammonium

Hexachloroiridate at 5 - 5000 μg/plate. Following these

treatments, evidence of toxicity was observed at 1600 and/or 5000 μg/plate in all

strains except TA102 in the absence of S-9 and in strain TA100 alone 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 test concentration of 5000 μg/plate

was retained for all strains except those in strain TA98 in the absence of S-9, where

the maximum test concentration was reduced to 1600 μg/plate based on strain specific

toxicity observed in Experiment 1. Narrowed concentration intervals were employed

covering the ranges 50-5000 μg/plate or 25-1600 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, evidence of toxicity was observed at

1600 μg/plate in strain TA98 in the absence of S-9, and at 5000 μg/plate in strains

TA1535 and TA1537 in the absence of S-9.

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 fell within acceptable ranges

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

Following Diammonium Hexachloroiridate treatments of all the test 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 Diammonium Hexachloroiridate mutagenic activity in this assay

system.