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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:
12 December 2017 to 15 March 2018
Reliability:
1 (reliable without restriction)

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
Test material form:
liquid
Details on test material:
Appearance: Straw to yellow syrup
Purity/Composition: Not indicated
Test item storage: At room temperature

Additional information
Test Facility test item number: 209099/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Specific details on test material used for the study:
Test Article
Wheat Germ Glycerides, also known as Wickenol 535, (CAS number 84012-44-2, EC Number 281-689-7), batch number P8166, was a yellow semi solid/syrup liquid. It was received on 04 December 2017 and stored at 15-25°C protected from light. Purity was stated as 100% and the retest date was given as 23 October 2020 (3 years from the date of manufacture), see Certificate of Analysis. 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.
P

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 Wheat Germ Glycerides 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). For Experiment 2 the maximum concentration tested was selected on the basis of solubility limitations seen in the assay.
Test article stock solutions were prepared by formulating Wheat Germ Glycerides 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 4.5 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
5 500
16 1600
50 5000

Mutation Experiment 2 - and + 0.2048 10.24
0.512 25.6
1.28 64
3.2 160
8 400
20 1000

0.1 mL volume additions of test article solution were used in Experiment 1 and 0.05 mL volume additions were used in Experiment 2.
Vehicle / solvent:
Vehicle controls comprised treatments with the vehicle Ethanol, using the same addition volumes per plate as the test article treatments, 0.1 mL for all Experiment 1 treatments or 0.05 mL for all Experiment 2 treatments.
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:
Positive controls comprised treatments with the appropriate stock positive control solution. The positive control chemicals were supplied and used according to the following table:

Chemical a Stock b Concentration (µg/mL) Final Concentration (µg/plate) Strain(s) S-9
2-nitrofluorene (2NF) 100 5 TA98 -
Sodium azide (NaN3) 40 2 TA100, TA1535 -
9-aminoacridine (AAC) 1000 50 TA1537 -
Mitomycin C (MMC) 4 0.2 TA102 -
Benzo[a]pyrene (B[a]P) 200 10 TA98 +
2-aminoanthracene (AAN) 100 5 TA100, TA1535, TA1537 +
400 20 TA102 +

a Obtained from Sigma-Aldrich.
b Stock solutions were formulated in purified water (NaN3 and MMC), or in anhydrous analytical grade dimethyl sulphoxide (DMSO) (2NF, AAC, AAN and B[a]P). All stock solutions were stored in aliquots protected from light at 2-8°C, with the exception of B[a]P which was stored in aliquots at <-50ºC and MMC which was prepared freshly on the day of use or stored in aliquots at -50ºC.


Metabolic Activation System
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°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). See Quality Control Statement for S-9.
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. The composition of the MutazymeTM 10% S-9 mix and buffer solution are described in the following table:

Ingredient Final Content per mL in:
10% S-9 mix Buffer Solution
Sodium phosphate buffer pH 7.4 (SPB) 100 µmoles 100 µmoles
Glucose-6-phosphate (disodium) (G-6-P) 5 µmoles -
b-Nicotinamide adenine dinucleotide phosphate (NADP) (disodium) 4 µmoles -
Magnesium chloride (MgCl2) 8 µmoles -
Potassium chloride (KCl) 33 µmoles -
Water To volume To volume
S-9 100 µL -


Supplements
L-histidine HCl (in 250 mM MgCl2) and D-biotin were added at the time of plating, by supplementing the molten top agar. Quantities of each supplement were as follows:
Supplement Final Quantity
L-histidine HCl 20 µg
D-biotin 24.4 µg

Bacteria
Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 108 to 109 cells/mL, based on cell count data from testing of each strain batch. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).
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 (where applicable) between experiments.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity, Solubility and Concentration Selection

Details of all treatment solution concentrations and finalWheat Germ Glyceridesconcentrations are provided in the full report.
Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations ofWheat Germ Glyceridesat 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, marked reductions in revertant numbers observed at 5000 µg/plate in strains TA100 and TA1537 in the absence of S-9 were considered to have been probable evidence of toxicity. Precipitation of test article was observed on all plates treated at 500 µg/plate and above.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9, with the maximum test concentration reduced to 1000 µg/plate, this being an estimate of the lower limit of precipitation. Narrowed concentration intervals were employed covering the range 10.24-1000 µg/plate, in order to examine more closely those concentrations ofWheat Germ Glyceridesapproaching 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, no evidence of toxicity was observed, as would normally be indicated by a thinning of the background bacterial lawn and/or a marked reduction in revertant numbers. Precipitation of test article was again observed, in this experiment being evident on all plates treated at 1000 µg/plate.

Data Acceptability and Validity

The individual mutagenicity plate counts were averaged to give mean values, which are presented in Section 8.From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges, with the exception that one replicate plate count for the vehicle controls in strain TA100 in the presence of S-9 in Experiment 1 fell just outside the laboratory historical control range. As this count was comparable to the range, and the other replicate counts and the mean vehicle count fell within the laboratory historical control range, these data were accepted as being 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 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
FollowingWheat Germ Glyceridestreatments 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 anyWheat Germ Glyceridesmutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:
It was concluded that EC281-689-7 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:

EC281 -689 -7 was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium in accordance with OECD Guideline 471, 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.

It was concluded that EC281 -689 -7 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).