Carbonyl(pentane-2,4-dionato-O,O')(triphenylphosphine)rhodium

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:

11 Sept 2019 - 18 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Carbonyl(pentane-2,4-dionato-O.O')(triphenylphosphine)rhodium

IUPAC Name:

Carbonyl(pentane-2,4-dionato-O.O')(triphenylphosphine)rhodium

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Acetylacetonatocarbonyl(triphenylphosphine) Rhodium (I)
- Molecular weight (if other than submission substance): 492.32 g/mol
- Physical state: Yellow solid
- Analytical purity: Not reported

Specific details on test material used for the study:

Rh content: 20.59% (corresponding to 100% substance purity)

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Treatments in this study were performed using suspensions of test item in vehicle up to a maximum concentration of 5000 μg/plate in Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997).
For Experiment 2 the maximum concentration tested was selected on the basis of toxicity seen in Experiment 1.
Toxicity assessed as diminution of background bacterial lawn and/or marke d reduciton in revertant numbers.
Experiment 1: 5, 16, 50, 160, 500,1600, 5000 µg/plate (+ and - S9)
Experiment 2: 2.048, 5.12, 12.8, 32, 80, 200, 500 µg/plate (+ and - S9), treatments +S9 further modified by inclusion of pre-incubation step.

Vehicle / solvent:

Preliminary solubility data indicated that Carbonyl(pentane-2,4-dionato-O,O`)
(triphenylphosphine) rhodium was soluble in tetrahydrofuran (THF) at concentrations
equivalent to at least 50 mg/mL.
Test article stock solutions were prepared by formulating Carbonyl(pentane-2,4-
dionato-O,O`) (triphenylphosphine) rhodium under subdued lighting in THF with the
aid of vortex mixing, ultrasonication (for approximately 5-10 minutes) and warming
at 37°C (as required) to give the maximum required treatment concentration.
Subsequent dilutions were made using THF. The test article solutions were protected
from light and used within 5 hours of initial formulation.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

0.1 mL THF

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 solution were used for all plate-incorporationtreatments, 0.01 mL volume additions were used for all pre-incubation 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,
followed 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 (reduced to 0.01 mL) or positive control, bacteria and S-9 mix detailed
above, plus an additional 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4), were
mixed together and placed in an orbital incubator set to 37°C for 20 minutes, before
the addition of 2 mL of supplemented molten agar at 45±1°C. Plating of these
treatments then proceeded as for the normal plate-incorporation procedure.

Volume additions for the Experiment 2 pre-incubation treatments were reduced to
0.01 mL, and 0.5 mL of 100 mM Sodium Phosphate Buffer was added to the preincubation
mixes, due to the vehicle (THF) employed in this study. This, and some
other organic solvents, are known to be near to toxic levels when added at volumes of
0.1 mL in this assay system when employing the pre-incubation methodology. By
reducing the addition volume to 0.01 mL per plate and adding additional phosphate
buffer, it was hoped that this would minimise or eliminate any toxic effects of the
solvent that may have otherwise occurred. In order to ‘correct’ for the additional
volume in the pre-incubation mix, these were plated out using 2 mL of 1.125%
supplemented soft agar, therefore the additions to each plate were comparable to that
of the plate incorporation treatments.

It may be noted that Experiment 2 data for treatments in the presence of S-9 are from
repeat treatments, as data from the initial treatments and some subsequent repetitions
were invalidated due to vehicle-related toxicity. Initial pre-incubation treatments in
the presence of S-9 in Experiment 2, and a subsequent repeat treatment, were
performed using 0.05 mL volume additions, but data from these treatments were
invalidated due to extreme vehicle-related toxicity, as were data from a subsequent
repeat treatment where volume additions were further reduced to 0.02 mL, and
0.5 mL of 100 mM Sodium Phosphate Buffer was added to the pre-incubation mixes.
Only when further repeat treatments were performed using volume additions further
reduced to 0.01 mL, and with 0.5 mL of 100 mM Sodium Phosphate Buffer also
added, was the vehicle-related toxicity eliminated and valid mutation data obtained.
Unacceptable vehicle control counts were obtained following these treatments of
strains TA100 and TA1535, and so the data from these strains were again invalidated,
and the Experiment 2 data presented for those strains in the presence of S-9 are from a
further repeat treatment using the same treatment volumes and methodology. For each
strain treated in each of these Experiment 2 repeat treatments in the presence of S-9,
control treatments were also included in the absence of S-9, in order to confirm the
correct strain and assay functioning, although these control data are not reported.

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:

As the vehicle for this study was THF, which is incompatible with the pH probe, no
pH readings could be taken from any test article formulations or treatment solutions
used in this study.

Precipitation of test article was observed on all the test plates treated at concentrations
of 1600 μg/plate and above in Mutation Experiment 1, and at 200 μg/plate and above
in Mutation Experiment 2, but only in strains TA100 and TA1535 in the presence of
S-9.

Due to toxicity, mutation data from only 4 concentrations were obtained from all
strains in the absence of S-9 in Mutation Experiment 1. However, as these treatments
spanned greater than a log range of treatment concentrations (5–160 μg/plate), and
mutation data from at least 5 concentrations were obtained from all strain treatments
in the absence of S-9 in Mutation Experiment 2, there are considered to be mutation
data obtained from sufficient treatment concentrations across this study to provide a
through and robust assessment of the mutagenicity of the test article in this assay
system

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Any other information on results incl. tables

Following Carbonyl(pentane-2,4-dionato-O,O`) (triphenylphosphine) rhodium

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 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 Carbonyl(pentane-2,4-

dionato-O,O`) (triphenylphosphine) rhodium mutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:

It was concluded that Carbonyl(pentane-2,4-dionato-O,O`) (triphenylphosphine)
rhodium 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
toxic levels, in the absence and in the presence of a rat liver metabolic activation
system (S-9).

Executive summary:

Carbonyl(pentane-2,4-dionato-O,O`) (triphenylphosphine) rhodium 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 Carbonyl(pentane-2,4-dionato-O,O`) (triphenylphosphine) rhodium treatments in

this study were performed using formulations prepared in tetrahydrofuran (THF).

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 Carbonyl(pentane-

2,4-dionato-O,O`) (triphenylphosphine) rhodium at 5, 16, 50, 160, 500, 1600 and

5000 μg/plate. Following these treatments, evidence of toxicity was observed

extending down to 500 μg/plate in all strains in the absence and presence of S-9, with

toxicity also observed at 160 μg/plate in strain TA1537 in the absence 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 was reduced to

500 μg/plate based on the toxicity observed in Experiment 1. Narrowed concentration

intervals were employed covering the range 2.048-500 μg/plate, in order to examine

more closely those concentrations of Carbonyl(pentane-2,4-dionato-O,O`)

(triphenylphosphine) rhodium 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. In this way, it was hoped to increase the range of mutagenic

chemicals that could be detected using this assay system. Following these treatments,

evidence of toxicity was observed at either 200 μg/plate and above or at 500 μg/plate

in all strains in the absence of S-9, and at 500 μg/plate in all strains except TA100 and

TA1535 in the presence of S-9. In strain TA1537 in the absence of S-9, marked

reductions in revertant numbers were also seen at 32 and 80 μg/plate.

Precipitation of test article was observed on all the test plates treated at concentrations

of 1600 μg/plate and above in Mutation Experiment 1, and at 200 μg/plate and above

in Mutation Experiment 2, but only in strains TA100 and TA1535 in the presence of

S-9.

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 Carbonyl(pentane-2,4-dionato-O,O`) (triphenylphosphine) rhodium

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 Carbonyl(pentane-2,4-dionato-O,O`)

(triphenylphosphine) rhodium mutagenic activity in this assay system.