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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 Mar 2018 to 17Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP.

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)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine ( 5 strains)
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:
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).
Test concentrations with justification for top dose:
Mutation Experiment 1 were performed in the absence and in the presence of S-9, using final concentrations at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate. Following these treatments, evidence of toxicity was observed in all the test strains and extended down to between 50 and 500 µg/plate in each strain in the absence of S-9, and down to between 500 and 1600 µg/plate in each strain in the presence of S-9.
In Experiment 2, narrowed concentration intervals were employed covering the ranges 2.048 – 500 µg/plate (for strains TA100 and TA1537 in the absence of S-9), 5.12 – 1250 µg/plate (all other strains in the absence of S-9) or 2.6214 – 1600 µg/plate (all strains in the presence of S-9). Following these treatments, evidence of toxicity was again observed in all the tester strains, and extended down to either 80 or 200 µg/plate in each strain in the absence of S-9, and down to either 256 or 640 µg/plate in each strain in the presence of S-9.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Amyl Salicylate was soluble in anhydrous analytical dimethyl sulphoxide (DMSO) at concentrations equivalent to at least 100 mg/mL.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 -S9 and TA100 -S9 only
Positive control substance:
9-aminoacridine
Remarks:
TA1537 -S9 only
Positive control substance:
2-nitrofluorene
Remarks:
TA98 -S9 only
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535 +S9, TA 1537 +S9, TA 100 +S9 and TA 102 +S9 only
Positive control substance:
mitomycin C
Remarks:
TA102 -S9
Positive control substance:
benzo(a)pyrene
Remarks:
TA98 +S9
Details on test system and experimental conditions:
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.

METHOD OF APPLICATION: in agar (plate incorporation); • 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

DURATION
- Preincubation period: at 37C protected from light for 2-3 days
- Exposure duration: Quantities of test article, vehicle control, solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix, 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.
The test article solutions were protected from light and used within approximately 5½ hours of initial formulation.

NUMBER OF CELLS EVALUATED:
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as contamination or bubbles or a split in the agar affected the accuracy of the automated counter.

DETERMINATION OF CYTOTOXICITY
-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.

Rationale for test conditions:
According to guideline
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. Any observed response was reproducible under the same treatment conditions.
Statistics:
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Following Amyl Salicylate treatments 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 any Amyl Salicylate mutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:
Amyl Salicylate 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 the lower limit of toxicity in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

Amyl Salicylate 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.

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 Amyl Salicylate at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following these treatments, evidence of toxicity was observed in all the test strains and extended down to between 50 and 500 μg/plate in each strain in the absence of S-9, and down to between 500 and

1600 μg/plate in each strain in the presence of S-9. Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For each strain, the maximum test concentration was reduced based on the extent of the toxicity observed in Experiment 1, in order to test up to an estimate of the lower limit of toxicity in Experiment 2. Narrowed concentration intervals were employed covering the ranges 2.048 – 500 μg/plate (for strains TA100 and TA1537 in the absence of S-9), 5.12 – 1250 μg/plate (all other strains in the absence of S-9) or 2.6214 – 1600 μg/plate (all strains in the presence of S-9), in order to examine more closely those concentrations of Amyl Salicylate approaching the maximum test concentrations 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. Following these treatments, evidence of toxicity was again observed in all the tester strains, and extended down to either 80 or 200 μg/plate in each strain in the absence of S-9, and down to either 256 or 640 μg/plate in each strain in the presence of S-9.

Although quite extensive toxicity was observed in this study, sufficient analysable concentrations remained for each strain in each experiment of this study to provide a thorough and robust assessment of the mutagenicity of the test article in this assay system.

Following Amyl Salicylate treatments of all the test strains in the absence and presence of S-9, no notable or concentration-related increases in revertant numbers were observed, and none 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 evidence of any Amyl Salicylate mutagenic activity in this assay system.

It was concluded that Amyl Salicylate 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 the lower limit of toxicity in the absence and in the presence of a rat liver metabolic activation system (S-9).