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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 December 2017 to 5 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
Glyceryl Triacetyl Ricinoleate (CAS number 101-34-8; EC Number 202-935-1), was a clear yellow liquid. It was received on 31 October 2017 and stored at 15-25°C protected from light. The test article was assigned a nominal 100% purity and had a 1,2,3-propanetriyl tris [(R)-12-(acetoxy)oleate] content of 92.11%. 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.
Preliminary solubility data indicated that the test article 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.

Method

Species / strain
Species / strain:
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 the test article 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 the test article 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 5.5 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
0.16 16
0.5 50
1.6 160
5.0 500
16 1600
50 5000

Mutation Experiment 2 - and + 1.563* 156.3
3.125* 312.5
6.25* 625
12.5* 1250
25* 2500
50* 5000
*Concentration of treatment solutions used for the Experiment 2 pre-incubation treatments were twice that stated above, in order to permit treatments at the final concentration stated, whilst volume additions were reduced to 0.05 mL.

0.1 mL volume additions of test article solution were used for all plate-incorporation treatments, 0.05 mL volume additions were used for all pre-incubation treatments.
Vehicle:
Ethanol
Controls
Negative controls:
yes
Solvent 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 conditions:
Preliminary solubility data indicated that the test article 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.

The following bacterial strains were used in this study:
Organism Strain Type of Mutation Mutant Gene
S. typhimurium TA98 frame-shift histidine
S. typhimurium TA100 base-pair substitution histidine
S. typhimurium TA1535 base-pair substitution histidine
S. typhimurium TA1537 frame-shift histidine
S. typhimurium TA102 base-pair substitution histidine

Test article stock solutions were prepared by formulating Dermol GTR 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 5.5 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
0.16 16
0.5 50
1.6 160
5.0 500
16 1600
50 5000

Mutation Experiment 2 - and + 1.563* 156.3
3.125* 312.5
6.25* 625
12.5* 1250
25* 2500
50* 5000
*Concentration of treatment solutions used for the Experiment 2 pre-incubation treatments were twice that stated above, in order to permit treatments at the final concentration stated, whilst volume additions were reduced to 0.05 mL.

0.1 mL volume additions of test article solution were used for all plate-incorporation treatments, 0.05 mL volume additions were used for all pre-incubation treatments.

Formulations Analysis
In accordance with the regulatory test guidelines applicable for this study (see Section 2.4), no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparation of test article were employed.
Following discussions with the Sponsor, analyses for achieved concentration and homogeneity of test article formulations were not conducted as part of this study, as this is not a requirement of the regulatory test guidelines.
The absence of such analyses is noted in the Study Director’s statement of GLP compliance.
Evaluation criteria:
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 between experiments.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes

Any other information on results incl. tables

Results

Toxicity, Solubility and Concentration Selection

Details of all treatment solution concentrations and final test article concentrations are provided below.

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test article at 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 usually be manifest by a diminution 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 the test article approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. 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. Additional plate incorporation treatments of strain TA1537 in the presence of S-9 were also performed. Following all the treatments in Experiment 2 there was no clear evidence of toxicity observed, but a small reduction in revertant numbers at 5000 µg/plate in strains TA1535 and TA1537 in the absence of S-9 may have been the result of a weak toxic effect.

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

Data Acceptability and Validity

The individual mutagenicity plate counts were averaged to give mean values. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges, with the exception of a single vehicle control replicate plate count in the Experiment 1 treatments of strain TA100 in the absence of S-9 and the Experiment 2 treatments of strain TA100 in the presence of S-9 and strain TA1537 (plate incorporation methodology) in the presence of S-9. In each case these replicate plate counts were sufficiently comparable to the laboratory historical control range to be accepted as characteristic and valid. 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 controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

No data were obtained for one replicate plate treatment of strain TA1537 in the presence of S-9 (pre-incubation methodology) at 2500 and 5000 µg/plate in Experiment 2. Sufficient other data points remained such that the absence of these data points was not considered to have adversely affected the mutagenicity assessment of the test article or the validity of this study.

Mutation

Following the test article treatments of all the test strains in the absence and presence of S-9, the only notable increase in revertant numbers occurred following the Experiment 1 treatments of strain TA1537 in the presence of S-9. This increase appeared to be concentration-related, but the maximum increase was 2.4-fold the concurrent vehicle control level, and therefore fell below the 3-fold threshold for an increase to be considered as clear evidence of mutagenic activity. This increase was also not reproducible following comparable treatments in Experiment 2, using both plate incorporation and pre-incubation treatment methodologies. The increase seen in Experiment 1 was therefore not considered to have been a true compound-related effect. As no 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 test article related mutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:
It was concluded that the test article 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:

The test article 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 test article treatments in this study were performed using formulations prepared in Ethanol.

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

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 range156.3-5000 µg/plate, in order to examine more closely those concentrations of the test article approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. 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. Additional plate incorporation methodology treatments of strain TA1537 in the presence of S-9 were also performed in order to investigate the reproducibility of a small increase in revertant numbers observed in Experiment 1 using this treatment methodology. Following all the Experiment 2 treatments there was no clear evidence of toxicity observed, but a small reduction in revertant numbers at 5000 µg/plate in strains TA1535 and TA1537 in the absence of S-9 may have been due to a weak toxic effect. Precipitation of test article was observed on all the test plates treated at concentrations of 1250 µg/plate and above.

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

Following test article treatments of all the test strains in the absence and presence of S-9, the only notable increase in revertant numbers occurred following the Experiment 1 treatments of strain TA1537 in the presence of S-9. This increase appeared concentration-related, but fell below the 3-fold the concurrent vehicle control level for an increase to be considered as clear evidence of mutagenic activity. This increase was also not reproducible following comparable treatments in Experiment 2, using both plate incorporation and pre-incubation treatment methodologies. 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 therefore to have provided no clear evidence of any test article related mutagenic activity in this assay system.

It was concluded that the test article did not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) ofSalmonella typhimuriumwhen 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).