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Genetic toxicity in vitro

Description of key information

Studies conducted to internationally recognised testing guidelines with GLP certification.

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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
Reference:
Composition 0
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 information:
Composition 1
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.
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
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.
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

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.

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).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 December 2010 - 03 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
no
Remarks:
The study was reported throughly and the lack of GLP certification is considered not to effect the reliability of the study result.
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
Storage conditions: Room temperature
CPTC ID No.: MID-4853.01
Target gene:
Histidine locus
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Test concentrations with justification for top dose:
Test sample: 5.0, 1.0, 0.5, 0.1, 0.05 mg/plate (with and without metabolic activation).
Vehicle:
- Solvent: 2-Propanol
- Justification: The solubility ofthe test sample was tested in different solvents at 50 mg/mL concentration and found to
be soluble in 2-Propanol and was the solvent used to dissolve this test sample in the study.
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
mitomycin C
other: 2-aminoanthracene (w/S9), ICR 191 Acridine, Daunomycin
Details on test system and conditions:
The bacterial reverse mutation assay was used to evaluate the mutagenic potential of the test sample at 5 concentrations of the test sample: 5.0, 1.0, 0.5, 0.1 and 0.05 mg. Testing was done with the appropriate solvent control and positive controls were plated with overnight cultures of the test systems (TA 97a, TA 98, TA 100, TA102, TA 1535) on selective minimal agar in the presence and absence ofAroclor-induced rat liver S9. All dose levels of the test sample, solvent controls and positive controls were plated in triplicate. (Refer to attachment A: Protocol M1 0-4853 for detailed test procedure).
Rationale for test conditions:
The Bacterial Reverse Mutation Assay is widely used to evaluate the mutagenic properties of chemicals.The test is based on the work of Dr. Bruce Ames and his coworkers and is commonly referred to as the Ames Test. Their studies involved the development of select histidine auxotrophs of S. typhimurium that are normally growth arrested due to mutations in a gene needed to produce the essential amino acid Histidine. in the absence of an external histidine source, the cells cannot grow to form colonies unless a reversion of the mutation occurs which allows the production of histidine to be resumed. As might be expected, Spontaneous reversions occur with each of the strains. However, chemical agents can induce a mutagenic response so that the number of revertant colonies is substantially higher than the spontaneous background reversion level. The test involves the analysis of the number of revertant colonies that are obtained with each strain in the presence and absence of the test chemical. Since the mutagenic response of a formulation could vary with the concentration, test articles are routinely dosed over an appropriate concentration range. in this-protocol, a complete set of positive and negative controls is included with each assay, and is plated routinely with all of the tester strains. AroclorTM 1254 induced rat liver microsomes are included to mimic the in viva activity of the liver enzymes in activating some pro-mutagens to mutagenic status.
Evaluation criteria:
Negative (solvent) Control Counts
The colonies that grew on the Minimal Glucose Agar plates developed from single cells that had regained their ability to grow in the absence of added histidine. The genetic reversion, from histidine auxotrophy to prototrophy, that enabled those cells to grow in the absence of exogenous histidine might have arisen spontaneously or as the result of a mutation induced by the treatments (see Maron & Ames, p. 181). It is important to realize that some of the colonies that arose in the positive control plates would have grown in the absence of treatment; they arose spontaneously. Accordingly, the negative (solvent) control colony counts constitute an important baseline in your evaluation of the test results. Unfortunately, the spontaneous reversion frequencies for the various tester strains can be quite variable - nevertheless, large deviations form the "normal" range of spontaneous reversion values may signal systematic problems with the assay.
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
The results in Tables 1 and 2 show that the test strains were sensitive to the positive control mutagens and had a spontaneous reversion rate well within the accepted values of each strain, indicating that under the test conditions, the strains were sensitive to the detection of potentially genotoxic agents. Test sample M10-4853.01 was not cytotoxic to the test system. The metabolic activation using the S9 activation mixture shows an active microsomal preparation.
Using the same test conditions, there was no detectable genotoxic activity associated with the five concentrations (5.0, 1.0, 0.5, 0.1, 0.05 mg) of test sample M10-4853.01 (Hetester HCA Lot #: G30074 Tables 1 and 2) either in the presence or absence of S9 enzyme activation.
Conclusions:
The test material failed to induce reverse mutation in any of the test concentrations and cell strains (Salmonella typhimurium) tested with and without metabolic activation.
Executive summary:

In this guideline (OECD 471) study, conducted without GLP certification, the test material did not induce mutagenic effects in any of the test concentrations or cell strains used, with and without metabolic activation. The test was conducted at 0.05, 0.1, 0.5, 1.0, and 5.0 mg/plate. Salmonella typhimurium was chosen as the model using strains 97, 98, 100, 102, 1535 with and without metabolic activation (Aroclor induced rat liver S9 mix).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The registered substance failed to induce statistically significant genetic mutations in either of the key studies. The registered substance was therefore considered not to fulfill the requirements for classification as a germ cell mutagen under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).