Trihexadecyl citrate

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:

7 December 2017 to 15 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 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:

Trihexadecyl citrate

EC Number:

224-931-9

EC Name:

Trihexadecyl citrate

Cas Number:

4560-68-3

Molecular formula:

C55H106O6

IUPAC Name:

trihexadecyl citrate

Test material form:

liquid

Specific details on test material used for the study:

Triisocetyl citrate (CAS number 93385-14-9), was a clear liquid. It was received on 04 December 2017 and stored at 15-25°C protected from light. A nominal 100% purity has been assigned. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

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)

Test concentrations with justification for top dose:

Preliminary solubility data indicated that Triisocetyl citrate was soluble in ethanol at concentrations equivalent to at least 100 mg/mL. A maximum concentration of 5000 µg/plate was selected for Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). A maximum concentration of 5000 µg/plate was also selected for Experiment 2.
Test article stock solutions were prepared by formulating Triisocetyl citrate under subdued lighting in ethanol with the aid of vortex mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using ethanol. The test article solutions were protected from light and used within approximately 5 hours of initial formulation. The following concentrations were tested:


Experiment S-9 Concentration of Treatment Solution (mg/mL) Final Concentration (µg/plate)
Mutation Experiment 1
- and + 0.05 5
- and + 0.16 16
- and + 0.5 50
- and + 1.6 160
- and + 5 500
- and + 16 1600
- and + 50 5000

Mutation Experiment 1
- and + 3.125 156.3
- and + 6.25 312.5
- and + 12.5 625
- and + 25 1250
- and + 50 2500
- and + 100 5000

0.1 mL volume additions of test article solution were used for all treatments in Experiment 1 and 0.05 mL volume additions were used for all treatments in Experiment 2.

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

- Test System:
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls,in the absence or presence of S-9 mix.

- Mutation Experiments :
Triisocetyl citratewas tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to 2 mL of supplemented molten agar at 45±1°C:
·      0.1 mL bacterial culture
·      0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
·      0.5 mL 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 at 37±1°C protected from light for 3 days. 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 equivocal, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) 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. 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 0.05 mL due to the vehicle (ethanol) 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 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.
Additional treatments of strain TA102 in the presence of S-9 were also performed using the same plate incorporation methodology as employed in Experiment 1 (with the exception that the test article and vehicle control volume additions were reduced to 0.05 mL, as for all the other Experiment 2 treatments) in order to assess the reproducibility of a small increase in revertant numbers observed following these strain treatments in the initial experiment.

- Toxicity Assessment:
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls.

- Colony Enumeration:
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as contamination or precipitation affected the accuracy of the automated counter.

Evaluation criteria:

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. The positive trend/effects described above were reproducible.
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 neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Toxicity, Solubility and Concentration Selection

Experiment 1 treatments of all thetester strains were performed in the absence and in the presence of S-9, using final concentrations ofTriisocetyl citrateat 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no clear 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.

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. Narrowed concentration intervals were employed covering the range 156.3-5000 µg/plate, in order to examine more closely those concentrations ofTriisocetyl citrateapproaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. All strains were treated in the presence of S-9 with a pre-incubation step included. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Additional treatments of strain TA102 were also performed in the presence of S-9 using the same plate incorporation methodology employed in Experiment 1. Following all these Experiment 2 treatments, there was again no evidence of toxicity observed.

Precipitation of test article was observed on the all the test plates treated at 5000 µg/plate in Experiment 1, and all those treated at concentrations of 1250 µg/plate and above in Experiment 2.

Data Acceptability and Validity

From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3‑fold (in strains TA1535 and TA1537) the concurrent vehicle controlsconfirming discrimination between different strains, and an active S‑9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Mutation

Following Triisocetyl citratetreatments of all the test strains in the absence and presence of S-9, the only notable increase in revertant numbers occurred in strain TA102 in the presence of S-9 in Experiment 1. The maximum increase was 1.6 -fold over the concurrent control level, which just exceeded the 1.5-fold threshold level required for an increase to be considered as indicative of mutagenic activity in this strain. However, this increase was not concentration-related, with similar revertant numbers occurring over the entire treatment concentration range, and was not reproduced in Experiment 2, despite comparable treatments being conducted using both plate incorporation and pre-incubation treatment methodologies. The increase observed in Experiment 1 was therefore not considered to have been a true compound-related effect or biologically relevant, nor sufficient to be considered as clear evidence of mutagenic activity. As no other increases in revertant numbers were observed 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 to have provided no clear evidence of anyTriisocetyl citratemutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:

It was concluded that Triisocetyl citrate 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 a precipitating concentration), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Triisocetyl citrate 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 Triisocetyl citratetreatments in this study were performed using formulations prepared inethanol.

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Triisocetyl citrateat 5, 16, 50, 160, 500, 1600and 5000 µg/plate. Following these treatments, no clear evidence of toxicity was observed, as would normally manifest as a thinning of the background bacterial lawn and/or a marked reduction in revertant numbers.

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. Narrowed concentration intervals were employed covering the range 156.3-5000 µg/plate, in order to examine more closely those concentrations of Triisocetyl citrate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. All strains were treated in the presence of S-9 with a pre-incubation step included. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Additional treatments of strain TA102 were also performed in the presence of S-9 using the same plate incorporation methodology employed in Experiment 1. Following all these Experiment 2 treatments, there was again no evidence of toxicity observed.

Precipitation of test article was observed on the all the test plates treated at 5000 µg/plate in Experiment 1, and all those treated at concentrations of 1250 µg/plate and above in Experiment 2.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell withinacceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Triisocetyl citrate treatments of all the test strains in the absence and presence of S-9, the only notable increase in revertant numbers occurred in strain TA102 in the presence of S-9 in Experiment 1. This increase just exceeded the 1.5‑fold over the concurrent control level required for an increase to be considered as indicative of mutagenic activity in this strain. However, this increase was not concentration-related, with similar revertant numbers occurring over the entire treatment concentration range, and was not reproduced in Experiment 2, despite comparable treatments being conducted using both plate incorporation and pre-incubation treatment methodologies. The increase observed in Experiment 1 was therefore not considered to have been a true compound-related effect, nor a biologically relevant observation. As no other increases in revertant numbers were observed 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 to have provided no clear evidence of any Triisocetyl citrate mutagenic activity in this assay system.

It was concluded that Triisocetyl citrate 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 a precipitating concentration), in the absence and in the presence of a rat liver metabolic activation system (S-9).