Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacteria (OECD 471, GLP): Negative

Mutagenicity in mammal cells in vitro (OECD 476, GLP): Negative

Cytogenicity in mammal cells in vitro (OECD 487, GLP): Negative

Link to relevant study records

Referenceopen allclose all

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.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine ( 5 strains)
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.
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.
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.
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).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 October to 27 December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 476 and in compliance with GLP.
Qualifier:
according to
Guideline:
other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes) (migrated information)
Version / remarks:
Updated and adopted 29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse lymphoma L5178Y systems are statistically more sensitive than CHO and V79 systems (Arlett et al., 1989) and a fluctuation protocol has been developed for use with this assay (Cole et al., 1983).
- Cells: L5178Y tk+/- (3.7.2C) cells were originally obtained from Dr Donald Clive, Burroughs Wellcome Co. Cells. The cells were stored as frozen stocks in liquid nitrogen
- Type and identity of media: RPMI 1640, containing L-glutamine and HEPES. All media may contain antimycotics and antibiotics.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
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:
Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 500 μg/mL (limited by solubility in culture medium): 15.63, 31.25, 62.5,125, 250, 500 µg/mL with and without S9.

In the Mutation Experiment 12 concentrations, ranging from 5 to 100 μg/mL in the absence of S-9 and from 5 to 150 μg/mL in the presence of S-9, were tested: 5, 10, 15, 20, 30, 40, 45, 50, 55, 60, 80, 100 µg/mL withS9 // 5, 10, 20, 40, 50, 60, 70, 80, 90, 100, 120, 150 µg/mL without S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. Amyl Salicylate was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 200.03 mg/mL. The solubility limit in culture medium was in the range of 250.0 to 500.1 μg/mL, as indicated by precipitation at the higher concentration which persisted following 24 hours’ incubation at approximately 37ºC. A maximum concentration of 500 μg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating treatment concentration
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
DMSO
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
in the absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
DMSO
Positive control substance:
benzo(a)pyrene
Remarks:
in the presence of S9-mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Mutation Assay and Range finding: At least 107 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 h incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.

- Expression period: Cultures were maintained in flasks for a period of 7 days during which the hprt-mutation would be expressed. Cultures were selected to be plated for viability (8 cells/mL) and 6TG resistance.
- 6TG resistance: At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10 5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 10 4 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (13 days) and wells containing clones were identified as above and counted.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 experiments in the historical negative control database (mean
MF +/-2 standard deviations.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment.

The historical negative control ranges, based on the last 20 experiments performed in this laboratory, are presented in Section 7.4.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
40 μg/mL in the absence of S-9 and 60 μg/mL in the presence of S-9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolarity: the osmolality of the cultures was acceptable. The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
- Evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation:
The solubility limit in culture medium was in the range of 250.0 to 500.1 μg/mL, as indicated by precipitation at the higher concentration which persisted following 24h incubation at approximately 37ºC. A maximum concentration of 500 μg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating treatment concentration. Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (125 to 500 μg/mL). Following the 3h treatment incubation period, precipitate was observed at the highest concentration tested in the absence of S-9 (500 μg/mL) and the highest two concentrations tested in the presence of S-9 (250 and 500 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the presence of S-9 was retained and the higher concentration was discarded.
In the Mutation Experiment upon addition of the test article to the cultures, precipitate was observed at the highest 3 concentrations tested in the absence of S-9 (60 to100 μg/mL) and the highest 7 concentrations tested in the presence of S-9 (60 to 150 μg/mL). Following the 3h treatment incubation period, no evidence of precipitate was observed.

PRELIMINARY CITOTOXICITY TEST:
The highest concentrations to give >10% RS (Relative Survival) were 31.25 μg/mL in the absence of S-9 and 62.5 μg/mL in the presence of S-9, which gave 75% and 38% RS, respectively
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the Mutation Experiment twelve concentrations, ranging from 5 to 100 μg/mL in the absence of S-9 and from 5 to 150 μg/mL in the presence of S-9, were tested. (45 to 100 μg/mL in the absence of S-9 and 70 to 150 μg/mL in the presence of S-9) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. In addition, an intermediate concentration was rejected in the absence of S-9 (15 μg/mL) due to excessive heterogeneity with respect to survival data. The highest concentrations analysed were 40 μg/mL in the absence of S-9 and 60 μg/mL in the presence of S-9, which gave 21% and 18% RS, respectively.
Conclusions:
It is concluded that Amyl Salicylate was not mutagenic at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3h in the absence and presence of a rat liver metabolic activation system (S-9), under the experimental conditions described.
Executive summary:

Amyl Salicylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3h treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 500 μg/mL (limited by solubility in culture medium). The highest concentrations to give >10% relative survival (RS) were 31.25 μg/mL in the absence of S-9 and 62.5 μg/mL in the presence of S-9, which gave 75% and 38% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 5 to 100 μg/mL in the absence of S-9 and from 5 to 150 μg/mL in the presence of S-9, were tested.

Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40 μg/mL in the absence of S-9 and 60 μg/mL in the presence of S-9 (limited by toxicity), which gave 21% and 18% RS, respectively. It should be noted that no concentration tested in the absence of S-9 gave the desired 10 to 20% RS. The highest concentration analysed (40 μg/mL) gave 21% and this was considered close enough to the 20% limit to be considered acceptable for analysis.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

Following 3h treatment in the absence and presence of S-9 no statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and all concentrations were within the upper limit generated by the last twenty experiments performed in this laboratory (1.14 to 6.46 mutants per 106 viable cells in the absence of S-9 and 1.98 to 6.08 mutants per 106 viable cells in the presence of S-9). A statistically significant linear trend (p≤0.05) was observed in the absence of S-9 but in the absence of any increases in MF this observation was considered not biologically relevant.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Mar 2019 to 01 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 487 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
In vitro Micronucleus in Mammalian cells
Species / strain / cell type:
lymphocytes: peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: peripheral human lymphocytes
- Suitability of cells: Peripheral human lymphocytes are recommended in the international OECD guideline.

For lymphocytes:
- Sex, age and number of blood donors: 6 donors, age: 25, 27, 26, 31, 26, 25 years,
- Whether whole blood or separated lymphocytes were used: separated lymphocytes
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
Culture medium consisted of RPMI 1640 medium (Life Technologies)
Cytokinesis block (if used):
Cytochalasin B (Cyto-B)
Metabolic activation:
with and without
Metabolic activation system:
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:
Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:
Without S9-mix: 5, 10, 15, 20, 24, 27, 30, 33, 36, 39, 42, 45, 50, 60 μg/mL culture medium (3 + 21 Hours treatment + hours recovery, 72 hours harvest time // 24+ 24 Hours treatment + hours recovery, 96 hours harvest time).
and With S9-mix: 10, 30, 40, 45, 50, 60, 65, 70, 75, 80,100 μg/mL culture medium (3 + 21 Hours treatment + hours recovery, 72 hours harvest time).
Vehicle / solvent:
The vehicle for the test item was dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
vinblastine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration : 2
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
Cultures were treated with the test article, vehicle, or positive controls (0.1 mL per culture).
The final culture volume was 10 mL. Cultures were incubated at 37±1°C for the designated exposure time.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3h (with and without S9) / 24h (without S9)
- Harvesting: 72h after 3h of treatment and 96h after 24h of treatment
- Cyto-B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture. The duration after culture initiation were 52h / 73h (for treatment duration of 3/24h respectfully).
- Methods of slide preparation: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried then stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were airdried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.
- Number of cells spread and analysed per concentration: A minimum of one thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. The number of cells containing micronuclei and the number of micronuclei per cell on each slide was recorded.
- Criteria for scoring micronucleated cells: Scoring was carried out using fluorescence microscopy. A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching amain nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.





Rationale for test conditions:
A solubility test was performed based on visual assessment. The test item formed a clear
colourless solution in DMSO.
In order to select the appropriate dose levels cytotoxicity data was obtained in a dose-range finding test in presence and in absence of S9-mix.
The highest tested concentration was the recommended dose level of 1642 μg/mL (= 0.01 M).
Cytotoxicity of GARDENOL in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of
55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the
cytotoxicity of the solvent control.
At least three test concentrations (not including the solvent and positive controls) that meet the acceptability criteria (appropriate cytotoxicity, number of cells, etc) should be evaluated
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Statistics:
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).
The proportion of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.
Species / strain:
lymphocytes: peripheral human lymphocites
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
It is concluded that Amyl Salicylate did not induce biologically relevant increases in MNBN cell frequency in cultured human peripheral blood lymphocytes following treatment up to toxic concentrations in the absence and presence of an Aroclor-induced rat liver metabolic activation system (S-9) under the experimental conditions described.
Executive summary:

Amyl Salicylate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA).

Treatment of cells with Amyl Salicylate in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly higher (at the p≤0.05 level) than those observed in concurrent vehicle controls for all concentrations analysed (3+21 and 24+24 hour treatments) with a single exception. Following 3+21 hour treatment in the absence of S-9, an intermediate concentration (33 µg/mL) demonstrated a statistically significant increase in MNBN cell frequency (p≤0.05). In the absence of any observed increases in actual MNBN cell frequency (that exceeded the normal range) at this concentration, this observation was considered not biologically relevant. The MNBN cell frequency of all Amyl Salicylate treated cultures fell within the normal ranges with a single exception. Following 24+24 hour treatment in the absence of S-9 a single replicate at the lowest concentration analysed (15 µg/mL) exceeded the normal range. As all other treated cultures were within the normal range and the response was not concentration related then this observation was also considered not biologically relevant. A statistically significant linear trend was observed following the 3+21 hour treatment in the presence of S-9 but in the absence of any increases in MNBN cell frequency this observation was also considered not biologically relevant. It should be noted that at the highest concentrations analysed following 3+21 hour treatments in the absence and presence of S-9 (42 and 70 µg/mL, respectively), cytotoxicity was marginally higher than the upper limit of 60% defined by the OECD test guideline. However, no biologically relevant increases in MNBN cell frequency were observed at any concentration analysed for any treatment condition and therefore it was considered that suitable maximum concentrations had been analysed. Cytotoxicity at the maximum concentration tested following 24+24 hour treatment (45 µg/mL) was marginally below the lower limit of 50% defined by the OECD guideline. Due to the steep, concentration-related toxicity observed in this treatment condition (a concentration of 50 µg/mL gave 81% reduction in RI) it was considered that 47% was sufficiently close to the desired cytotoxicity range to be considered acceptable.

It is concluded that Amyl Salicylate did not induce biologically relevant increases in MNBN cell frequency in cultured human peripheral blood lymphocytes following treatment up to toxic concentrations in the absence and presence of an Aroclor-induced rat liver metabolic activation system (S-9) under the experimental conditions described.

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

Genetic toxicity in vivo

Description of key information

There is no available in vivo genotoxicity study on the registered substance, but the weight of evidence from in vitro data is deemed sufficient to show the absence of mutagenicity properties of the registered substance.

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Amyl Salicylate did not show any mutagenic properties in the 3 GLP in vitro studies that were performed (OECD 471, OECD 476 and OECD 487).

The registered substance is not mutagenic, not clastogenic and not aneugenic.

Additional information

Ames test (OECD 471, GLP):

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).

HPRT (OECD 476, GLP):

Amyl Salicylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3h treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 500 μg/mL (limited by solubility in culture medium). The highest concentrations to give >10% relative survival (RS) were 31.25 μg/mL in the absence of S-9 and 62.5 μg/mL in the presence of S-9, which gave 75% and 38% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 5 to 100 μg/mL in the absence of S-9 and from 5 to 150 μg/mL in the presence of S-9, were tested.

Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40 μg/mL in the absence of S-9 and 60 μg/mL in the presence of S-9 (limited by toxicity), which gave 21% and 18% RS, respectively. It should be noted that no concentration tested in the absence of S-9 gave the desired 10 to 20% RS. The highest concentration analysed (40 μg/mL) gave 21% and this was considered close enough to the 20% limit to be considered acceptable for analysis.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

Following 3h treatment in the absence and presence of S-9 no statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and all concentrations were within the upper limit generated by the last twenty experiments performed in this laboratory (1.14 to 6.46 mutants per 106 viable cells in the absence of S-9 and 1.98 to 6.08 mutants per 106 viable cells in

the presence of S-9). A statistically significant linear trend (p≤0.05) was observed in the absence of S-9 but in the absence of any increases in MF this observation was considered not biologically relevant.

Micronucleus in vitro (OECD 487, GLP):

Amyl Salicylate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA).

Treatment of cells with Amyl Salicylate in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly higher (at the p≤0.05 level) than those observed in concurrent vehicle controls for all concentrations analysed (3+21 and 24+24 hour treatments) with a single exception. Following 3+21 hour treatment in the absence of S-9, an intermediate concentration (33 µg/mL) demonstrated a statistically significant increase in MNBN cell frequency (p≤0.05). In the absence of any observed increases in actual MNBN cell frequency (that exceeded the normal range) at this concentration, this observation was considered not biologically relevant. The MNBN cell frequency of all Amyl Salicylate treated cultures fell within the normal ranges with a single exception. Following 24+24 hour treatment in the absence of S-9 a single replicate at the lowest concentration analysed (15 µg/mL) exceeded the normal range. As all other treated cultures were within the normal range and the response was not concentration related then this observation was also considered not biologically relevant. A statistically significant linear trend was observed following the 3+21 hour treatment in the presence of S-9 but in the absence of any increases in MNBN cell frequency this observation was also considered not biologically relevant. It should be noted that at the highest concentrations analysed following 3+21 hour treatments in the absence and presence of S-9 (42 and 70 µg/mL, respectively), cytotoxicity was marginally higher than the upper limit of 60% defined by the OECD test guideline. However, no biologically relevant increases in MNBN cell frequency were observed at any concentration analysed for any treatment condition and therefore it was considered that suitable maximum concentrations had been analysed. Cytotoxicity at the maximum concentration tested following 24+24 hour treatment (45 µg/mL) was marginally below the lower limit of 50% defined by the OECD guideline. Due to the steep, concentration-related toxicity observed in this treatment condition (a concentration of 50 µg/mL gave 81% reduction in RI) it was considered that 47% was sufficiently close to the desired cytotoxicity range to be considered acceptable.

It is concluded that Amyl Salicylate did not induce biologically relevant increases in MNBN cell frequency in cultured human peripheral blood lymphocytes following treatment up to toxic concentrations in the absence and presence of an Aroclor-induced rat liver metabolic activation system (S-9) under the experimental conditions described.

Justification for classification or non-classification

Based on the data available on Amyl Salicylate, no classification for mutagenicity is necessary according to the (EC) No 1272/2008 Regulation (CLP).