Dicarbonyl(pentane-2,4-dionato-O,O')rhodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity potential of Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium

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

Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium 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:

17 Oct - 29 Nov 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: 40.00% Rh

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 solutions 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 all subsequent experiments, the maximum concentrations tested were selected on the basis of strain specific toxicity.
Toxicity assessed as diminution of background bacterial lawn and/or marke d reduciton in revertant numbers.

Experiment 1 (all strains): 5, 16, 50, 160, 500, 1600,5000 µg/plate (+ and - S9)
Experiment 1 repeat (all strains): 0.16a, 0.5, 1.6, 5, 16, 50, 160, 500b µg/plate (+ and - S9)
Experiment 2 (all strains): 0.2048c, 0.512, 1.28, 3.2, 8, 20, 50, 125d µg/plate (+ and - S9)
Experiment 3 (strains TA100 +S9 and TA1535 +S9 only): 0.16e, 0.5, 1.6, 5, 16, 50, 160, 500f µg/plate (+ and - S9)
a Concentration employed for treatments in the absence of S-9 only
b Concentration employed for treatments in the presence of S-9 only
c Concentration employed for treatments of strains TA98 and TA1535 in the absence of S-9 only
d Concentration employed for treatments of all strains except TA98 and TA1535 in the absence of S-9
e Concentration employed for treatment of strain TA100 in the absence of S-9 only
f Concentration employed for treatment of strain TA1535 in the presence of S-9 only

Vehicle / solvent:

The Sponsor indicated that Dimethyl sulphoxide (DMSO) should not be used as vehicle for this test article. Preliminary solubility data indicated that Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium was soluble in tetrahydrofuran (THF) at concentrations up to at least 50 mg/mL.
Test article stock solutions were prepared by formulating Dicarbonyl(pentane-2,4-
dionato-O,O`)rhodium under subdued lighting in THF with the aid of vortex mixing,
ultrasonication (for up to 15 minutes, as required) 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
approximately 4.5 hours of initial formulation.

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 treatments in
Experiments 1 (and repeat) and 3, and 0.01 mL volume additions were used for all
treatments in Experiment 2. The reduced volume additions were required for the preincubation
methodology treatments (in the presence of S-9) in Experiment 2, but were
also employed for the plate incorporation treatments (in the absence of S-9) in this
experiment for practical ease of conduct.

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 Mutation Experiment 1 repeat were equivocal, treatments in the
presence of S-9 in Experiment 2 included a pre-incubation step. The pre-incubation
mixes were achieved by the following sequence of additions to empty sterile preincubation
tubes:
-0.1 mL of bacterial culture
-0.5 mL of 100 mM Sodium Phosphate Buffer
-0.01 mL of test article solution/vehicle control or 0.05 mL of positive control
-0.5 mL of 10% S-9 mix
The contents of each tube 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 plateincorporation
procedure. In this way, it was hoped to increase the range of mutagenic
chemicals that could be detected in the assay.
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 vehicles, 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 to minimise or eliminate any toxic effects of the vehicle that may
have otherwise occurred. For ease of practical conduct, the plate incorporation
treatments performed in the absence of S-9 in Mutation Experiment 2 were also
performed using 0.01 mL volume additions of vehicle or test article solutions.
Mutation Experiment 3 treatments of strain TA100 in the absence of S-9 and strain
TA1535 in the presence of S-9 were performed using a plate incorporation
methodology (and 0.1 mL vehicle and test article solution additions). Vehicle and
positive control treatments in strain TA1535 in the absence of S-9 were also included,
to confirm the correct strain and assay functioning, but these control data are not
reported.

The background lawns of the plates were examined for signs of toxicity. Revertant
plate count data were also assessed, as a marked reduction in revertants compared to
the concurrent vehicle controls would also be considered as evidence of toxicity.

Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive
Instruments) or manually where confounding factors such as bubbles or splits in the
agar 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:

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.

Data from the Mutation Experiment 1 treatments of strain TA98 were invalidated as
no characteristic positive control response was obtained in the absence of S-9, and
therefore the correct strain functioning could not be confirmed. For all the remaining
strains, fewer than 5 analysable concentrations remained due to extensive toxicity.
These data were therefore used only for toxicity assessment, and all strains were
repeated at lower concentrations in Mutation Experiment 1 repeat to provide mutation
data for each strain.
It should be noted that data from the Experiment 1 repeat treatments of strain TA1535
in the absence and presence of S-9 were invalidated due to unacceptable vehicle
control counts. Further repeat treatments of this strain were therefore performed in
order to provide the Mutation Experiment 1 repeat data for this strain presented in this
report.

Following Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium treatments of all the tester
strains in the absence and presence of S-9, the only notable increases were observed
following the Experiment 1 repeat treatments in strain TA100 in the absence of S-9
and in strain TA1535 in the presence of S-9. The
increase in strain TA100 in the absence of S-9 just exceeded 2-fold the vehicle control
level, and the increase in strain TA1535 in the presence of S-9 fell just below 3-fold
the vehicle control level. However, in neither case were the increases clearly
concentration-related, with the maximum increases occurring at intermediate
concentrations. Neither were these increases reproducible, as no comparable increases
were seen in the corresponding Experiment 2 or Experiment 3 treatments. The observed increases were therefore
not considered to have been true compound-related effects nor biologically relevant,
and therefore were not considered to be evidence of Dicarbonyl(pentane-2,4-dionato-
O,O`)rhodium mutagenic activity.
No other increases in revertant numbers were observed that were ≥1.5-fold (TA102),
≥2-fold (TA98 and TA100) or ≥3-fold (TA1535 and TA1537) the concurrent control.
This study was considered therefore to have provided no clear evidence of any
Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium mutagenic activity in this assay
system.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Following Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium treatments of all the tester

strains in the absence and presence of S-9, the only notable increases were observed

following the Experiment 1 repeat treatments in strain TA100 in the absence of S-9

and in strain TA1535 in the presence of S-9. These increases approached or achieved

the respective 2-fold or 3-fold increases over the vehicle control level for an increase

to be considered as clear evidence of mutagenic activity in these strains. However, in

neither case were the increases clearly concentration-related, with the maximum

increases occurring at intermediate concentrations. Neither were these increases

reproducible, as no comparable increases were seen in the corresponding

Experiment 2 or Experiment 3 treatments. The observed increases were therefore not

considered to have been true compound-related effects nor biologically relevant. This

study was therefore considered to have provided no clear evidence of any

Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium mutagenic activity in this assay

system.

Conclusions:

It was concluded that Dicarbonyl(pentane-2,4-dionato-O,O`)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:

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

initially in two separate experiments. A further experiment was performed in strain

TA100 in the absence of S-9 and in strain TA1535 in the presence of S-9, in order to

investigate equivocal results seen across the first two experiments with these strain

treatments.

All Dicarbonyl(pentane-2,4-dionato-O,O`)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 Dicarbonyl(pentane-

2,4-dionato-O,O`)rhodium at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following

these treatments, the data from strain TA98 were invalidated due to an unacceptable

positive control response in the absence of S-9. Evidence of toxicity was observed at

160 and/or 500 μg/plate and above in all the other strains. As fewer than 5 analysable

concentrations remained for each of these strain treatments, these data were used for

toxicity assessment only, and treatments of all the tester strains were repeated at lower

concentrations to provide the Experiment 1 mutation data.

Mutation Experiment 1 repeat treatments of all the tester strains were performed using

final concentrations of Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium at 0.16, 0.5,

1.6, 5, 16, 50 and 160 μg/plate in the absence of S-9, and at 0.5, 1.6, 5, 16, 50, 160

and 500 μg/plate in the presence of S-9. Following these treatments, evidence of

toxicity was observed extending down to between 16 and 160 μg/plate in each strain

in the absence of S-9, and down to between 160 and 500 μg/plate in each strain in the

presence of S-9. As at least 5 analysable concentrations remained in each strain, these

data were acceptable for mutation assessment.

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 further

reduced to an estimate of the lower limit of toxicity, and narrowed concentration

intervals were employed covering the ranges 0.2048 - 50 μg/plate (strains TA98 and

TA1535 in the absence of S-9 only) or 0.512 - 125 μg/plate (all other strain

treatments), in order to examine more closely those concentrations of

Dicarbonyl(pentane-2,4-dionato-O,O`)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 again observed in all the

tester strains, and extended down to either 20 or 50 μg/plate in each strain in the

absence of S-9, and down to either 50 or 125 μg/plate in each strain in the presence of

S-9. At least 5 analysable concentrations remained in each strain.

In order to investigate further the results seen across the previous experiments in

strain TA100 in the absence of S-9 and in strain TA1535 in the presence of S-9,

further treatments were performed in Mutation Experiment 3. Plate incorporation

treatments were performed at concentration ranges of 0.16 – 160 μg/plate in strain

TA100 in the absence of S-9, and 0.5 – 500 μg/plate in strain TA1535 in the presence

of S-9. Following these treatments, evidence of toxicity was observed at 50 μg/plate

and above in strain TA100 in the absence of S-9, and at the maximum concentration

of 500 μg/plate in strain TA1535 in the presence of S-9.

No precipitation of test article was observed on any of the test plates in any

experiment.

Vehicle and positive control treatments were included for all strains treated in each

experiment. The mean numbers of revertant colonies fell within acceptable ranges for

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

Following Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium treatments of all the tester

strains in the absence and presence of S-9, the only notable increases were observed

following the Experiment 1 repeat treatments in strain TA100 in the absence of S-9

and in strain TA1535 in the presence of S-9. These increases approached or achieved

the respective 2-fold or 3-fold increases over the vehicle control level for an increase

to be considered as clear evidence of mutagenic activity in these strains. However, in

neither case were the increases clearly concentration-related, with the maximum

increases occurring at intermediate concentrations. Neither were these increases

reproducible, as no comparable increases were seen in the corresponding

Experiment 2 or Experiment 3 treatments. The observed increases were therefore not

considered to have been true compound-related effects nor biologically relevant. This

study was therefore considered to have provided no clear evidence of any

Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium 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 Dicarbonyl(pentane-2,4-dionato-O,O`)rhodium

mutagenic activity, and the substance does not require a classification for this endpoint.