Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

End point summary:

OECD 471_Ames test (Benzyl salicylate): non-mutagenic with and without S9.
OECD 473_In vitrocytogenicity evaluated with chromosome aberration (CHL/IU cells): non-clastogenic with and without S9.
OECD 476_In vitrogene mutation in mammalian cells (HPRT): non-mutagenic with and without S9.

Benzyl Salicylate has no mutagenic or clastogenic effects (negative).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 14, 2016 to March 27, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Requested by ECHA: In vitro cytogenicity study in mammalian cells (Annex VIII, Section 8.4.2., test method: OECD 473).
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
OECD Council: July 29, 2016
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 84OJN
- Purity test date: 99.9%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In cool and dark place (actual temperature: 4.1 to 6.0°C from October 4, 2016 to February 13, 2017), in well-closed containers
- Stability under test conditions: stable (confirmed after the end of the experiment)


Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Chinese hamster lung fibroblast (CHL/IU) from the National Institute of Biomedical Innovation, JCRB Cell Bank
- Suitability of cells: according the guideline
- Normal cell cycle time (negative control): DMSO, used as the vehicle, was used as the negative control article.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 30. The passage number of the cells at the time of use was 18 in the cell-growth inhibition test, and 28 in the chromosomal aberration test.
- Methods for maintenance in cell culture: The cells were cultured in a carbon dioxide gas incubator under conditions of 5% CO2 at 37°C and at high humidity. Subcultivation was carried out every 1 to 4 days.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: carbon dioxide gas incubator under conditions of 5% CO2 at 37°C and at high humidity
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- The S9 and cofactor (S9/cofactor C set, Lot numbers: C160902061 and C161118091) were mixed to prepare S9 mix.
Test concentrations with justification for top dose:
9 dose concentrations, 17.0, 14.0, 12.0, 11.0, 10.0, 8.00, 6.00 and 4.00 mg/mL.
Vehicle / solvent:
DMSO (lot DSH0997 and DSR0111)
DMSO was selected as the vehicle since the test article was not dissolved in water but dissolved in DMSO at 200 mg/mL in the examination for the vehicle.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
Judgment was done using the number of cells with structural / numerical aberrations in chromosomes. The total incidence of cells with structural aberrations was calculated in 2 ways, one including gaps (TAG) and the other excluding gaps (TA), and the latter was used for the final evaluation based on statistical analysis.
Statistics:
For statistical significance of the difference in the incidence of cells with abnormalities, the numbers of the cells with chromosome structural aberrations and numerical aberrations between the negative control group and the test article treatment group were analyzed by Fisher’s exact test (level of significance: 5%, one-tailed) 2) and Cochran-Armitage trend test (level of significance: 5%, one-tailed) 3), while the numbers of the cells with chromosome structural aberrations between the negative control group and the positive control group were analyzed by Fisher’s exact test (level of significance: 5%, one-tailed) 2).
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
It was concluded that benzyl salicylate had neither chromosome structural aberration inducibility nor chromosome numerical aberration inducibility under the conditions of this study.
Executive summary:

In order to evaluate the clastogenic potential of Benzyl Salicylate, a chromosomal aberration test using cultured Chinese hamster (CHL/IU) cells was conducted.

 

As a preliminary study to select dose levels for the chromosomal aberration test, a cell-growth inhibition test was conducted setting the highest dose level at 2000 μg/mL, and this dose diluted using a common ratio of 2 to prepare a total of 8 concentrations. In the results, cell growth inhibition effects of more than 50% were recorded at the dose levels of 125 μg/mL and above for the short-term treatment method without metabolic activation and for the continuous treatment method and at the dose levels of 250 μg/mL and above for the short-term treatment method with metabolic activation, and thus the 50% cell growth inhibition concentration (approximate value) was calculated to be 92 μg/mL for the short-term treatment method without metabolic activation, 178 μg/mL for the short-term treatment method with metabolic activation, and  97 μg/mL for the continuous treatment method. Based on these results, chromosome aberration study was conducted setting the maximum dose concentration at 120 μg/mL and using 5 dose concentrations with common difference of 20 μg/mL for the short-term treatment method without metabolic activation and for the continuous treatment method, and setting the maximum dose concentration at 200 μg/mL and using 5 dose concentrations with common difference of 30 μg/mL for the short-term treatment method with metabolic activation.

 

In the results of the chromosome aberration study, the incidence of the occurrence of cells with chromosomal aberrations not containing gaps, an index for the chromosome structural aberration (TA value), and the incidence of the occurrence of cells with polyploidy (poly value) were not higher than those in the negative control group with statistical significance for any treatment method, and the values for the negative control group within the range of 95% probability distribution of the negative control values in the historical background data of the test facility. Therefore, the test article was judged to be negative for chromosome aberration effects.

 

For all the treatment methods, the incidence of the occurrence of cells with chromosome structural aberrations and the incidence of the occurrence of polyploidy in the negative control group were within the range of 95% probability distribution of the historical background data of the test facility. In the positive control group, a statistically significant increase in the incidence of cells with chromosome structural aberration was recorded in comparison with that of the negative control group. Therefore, it was judged that the study was conducted appropriately.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 december 2018 to 09 april 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 476
Justification for type of information:
Requested by ECHA: In vitro gene mutation study in mammalian cells (Annex VIII, Section 8.4.3.; OECD 476 or 490)
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
updated and adopted 29 july 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signature 09 april 2019
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: VE00577653
- Expiration date of the lot/batch: 01 November 2019
- Purity ≥99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under storage conditions: considered stable through expiration date
- Solubility and stability of the test substance in the vehicle: soluble at 500 mg/mL in DMSO

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item diluted in DMSO. Serial dilutions prepared from stock.
- Final dilution of a dissolved solid, stock liquid or gel: 125, 250, 500, 1000 and 2000 μg/mL

Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO-K1-BH4 cell line obtained from Dr. Abraham W. Hsie, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN
- Suitability of cells:
- Normal cell cycle time (negative control): doubling time of 12-14 hours

For cell lines:
- Absence of Mycoplasma contamination: Yes frozen stock cultures tested to confirm the absence of mycoplasma
- Number of passages if applicable: Cells used in the mutation assay did not exceed 15 passages from frozen stock
- Cell cycle length, doubling time or proliferation index : doubling time of 12-14 hours
- Modal number of chromosomes: 20 chromosomes
- Periodically checked for karyotype stability: yes , frozen stock cultures were tested for karyotype stability
- Periodically ‘cleansed’ of spontaneous mutants: yes, the cells were cleansed in medium supplemented with hypoxanthin, aminopterin and thymidine (HAT).


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: CHO cells were maintained in Ham's F12 medium supplemented with 3 mM L-glutamine and 5% (v/v) heat-inactivated and dialyzed fetal bovine serum (Complete Ham’s F12) under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air). All media contained antimycotics and antibiotics.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3998, Expiration Date: 28 Aug 2020) was purchased commercially from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.
Test concentrations with justification for top dose:
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated approximated the limit dose for this assay. Lower concentrations were prepared by 2-fold dilutions. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. The osmolality of the solvent control, highest dose level, lowest precipitating dose level and the highest soluble dose level in treatment medium also was measured at the beginning of treatment. Precipitation was assessed at the beginning and end of
treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival.

Preliminary Toxicity Assay
Benzyl Salicylate (CAS# 118-58-1) was evaluated at concentrations of 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum
concentration evaluatedapproximated the limit dose for this assay.
The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
Adjusted relative survival was 44.74 and 64.94% at a concentration of 2000 μg/mL with and without S9, respectively.

Definitive Mutagenicity Assay
Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 μg/mL with and without S9.



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- 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. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
Preliminary toxicity test: 1 x 10^6 cells / 5 mL; determination of initial survival (after 5 hours treatment): 200 cells/60-mm dish in 5 mL;

Definitive test: 5 x 10^6 cells / 10 mL;
Subculture for phenotypic expression (after 5 hours treatment and each 2-3 days): 2,4 x 10^6 cells / 225-cm^2 flask in 30 mL; culture for initial survival: 200 cells/ 60-mm plate in 5 mL
Mutant selection: 6 x 10^5 cells / 150 mm plate in 30 mL; cloning efficiency 200 cells / plate in 5 mL

- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- Selective agent: 6-thioguanine (TG) at 10 μM, 7 days of cell exposure at the end of the phenotypic expression period
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.4 x 10^6 cells are plated at a density of 6 x 10^5 cells/150-mm plate (4 plates total) in 30 mL Complete Ham’s F12-Hx containing 10 μM TG (selective agent).
Three 60-mm plates also were plated, at 200 cells/plate in 5 mL Complete Ham’s F12-Hx in triplicate (without selective agent), to determine the cloning efficiency at the time of selection.
The colonies were fixed with methanol, stained with crystal violet and counted. Mutant frequencies were expressed as the number of TGr mutants/10^6 clonable cells. The number of clonable cells was determined from the triplicate 60-mm plates.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method : the cytotoxicity was expressed as the adjusted relative survival (ARS)

Adjusted Relative Survival (ARS) = [(CE (treated) x cell density (treated)) / (CE (control) x cell density (control))] x 100%

Cloning Efficiency (CE) = (number of colonies / number of cells plated) x 100%
Rationale for test conditions:
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated approximated the limit dose for this assay. Lower concentrations were prepared by 2-fold dilutions. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. The osmolality of the solvent control, highest dose level, lowest precipitating dose level and the highest soluble dose level in treatment medium also was measured at the beginning of treatment. Precipitation was assessed at the beginning and end of
treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival,calculated as described below.

Cloning Efficiency* (CE) = (number of colonies / number of cells plated ) x 100%

Adjusted Relative Survival (ARS) = [(CE (treated) x cell density (treated)) / (CE (control) x cell density (control))] x 100%

*CE is equivalent to absolute cloning efficiency
Evaluation criteria:
The test substance was considered to have produced a positive response if it induces a doserelated increase in mutation frequency and an increase exceeding 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the result were considered equivocal.
If none of these criteria were met, the results were considered to be negative.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
Statistics:
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Strain CHO-K1-BH4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The average adjusted relative survival was 21.80 and 78.14% at a concentration of 2000 μg/mL with and without S9, respectively
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the conditions of the assay, Benzyl Salicylate (CAS# 118-58-1) was concluded to be negative for the induction of forward mutations at the hypoxanthineguanine
phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.
Executive summary:

The test substance, Benzyl Salicylate (CAS# 118-58-1), was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥31.3μg/mL at the beginning of treatment and at concentration 2000 μg/mL by the end of treatment with S9. Adjusted relative survival was 44.74 and 64.94% at a concentration of 2000 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 μg/mL with and without S9.

In the definitive mutagenicity assay, visible precipitate was observed at all concentrations at the beginning of treatment and at concentrations ≥1000 μg/mL by the end of treatment with S9. The average adjusted relative survival was 21.80 and 78.14% at a concentration of 2000 μg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 were chosen for mutant selection. No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p < 0.01).

These results indicate Benzyl Salicylate (CAS# 118-58-1) was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with good scientific principles, suitable for use in classification/ labelling and risk assessment purposes.
Qualifier:
equivalent or similar to
Guideline:
other: Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983): Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen S [Suppl 1]:3-142
Principles of method if other than guideline:
Ames test.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mixes from rat (RLI) and hamster (HLI), tested separately.
Test concentrations with justification for top dose:
EGG: 0, 0.3, 1, 3.3, 10, 20, 33, 100, 333 and 666 µg/plate.
SRI: 0, 3.3, 10, 33.3, 100 and 333 µg/plate.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Assessed by each individual laboratory from a choice of distilled water, DMSO, 95 % ethanol, acetone, in that order of preference.
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:
other: 4-nitro-o-phenylenediamine
Remarks:
TA98 -S9 only
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535 +S9, TA 1537 +S9, TA 98 +S9 and TA 100 +S9 only
Details on test system and experimental conditions:
The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37"C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel- Bonner medium [Vogel and Bonner, 19561. The histidine-revertant (his') colonies arising on these plates were counted following 2 days incubation at 37°C.
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
As per Haworth et al. 1983:

An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose-related increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen.

The distinctions between a weak mutagenic response and a mutagenic response, or between a weak mutagenic response and a questionable mutagenic response were considered highly subjective.

A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his⁺ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable (?) if a reproducible increase of hist revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his⁺ revertants in repeat trials. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Positive controls validity:
valid
Additional information on results:
For full results please refer to the attached supporting information.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information): negative with and without metabolic activation

Zeiger et al. screened 255 chemicals using Ames tests, including benzyl salicylate, which was screened in two separate tests in two different laboratories. Both test found benzyl salicylate to be negative for genotoxicity.
Executive summary:

As discussed as part of the category of salicylate substances (RAAF Document).

Seven of the category substances including the “target” substance (benzyl salicylate, have negative experimental Ames data, this indicates that although benzyl salicylate has an alert for DNA binding (OECD [Q]SAR Toolbox profile), this alert is not predictive of the actual DNA interaction within anin vitrotest system. Especially when taking into account the lack of DNA alerts based on the OASIS profiling. Therefore this indicates that all the category members would be negative in thein vitroAmes test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Non-GLP study according to method proposed by Ishidate and Odashima (1977). To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance) it is proposed to read-across to Methyl Salicylate (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore, read across is justified.
Principles of method if other than guideline:
Tests were performed according to Ishidate and Odashima (1977)
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung fibroblast cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum Essential Medium supplemented with 10% calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
without
Test concentrations with justification for top dose:
Three different doses up to 0.25 mg/mL were tested
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: no justification provided
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa solution (1.5% at pH 6.8)

NUMBER OF REPLICATIONS: one for each exposure duration

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases were observed

DETERMINATION OF CYTOTOXICITY
- Method: was determined in a pre-test in which the dose leading to 50% cell-growth inhibition was determined (this dose was used as maximum dose in the test)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
The results were considered to be negative if the incidence of chromosome aberrations were less than 4.9%.
Statistics:
Not reported
Species / strain:
other: Chinese hamster lung fibroblast cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: the maximum dose was established in a pre-test and defined as the dose leading to 50% growth inhibition
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
The incidence of polyploid cells was 4% and the incidence of structural aberrations was 1% after 48 hours of exposure.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance) it is proposed to read-across to Methyl Salicylate (Source Substance).

The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible.

The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From this profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific.

See Section 13, document Read across justification_Methyl salicylate.

Conclusions:
Interpretation of results (migrated information):
negative

The substance was negative in an in vitro mammalian chromosomal aberration assay using Chinese hamster lung fibroblast cells in the absence of metabolic activation.
Executive summary:

As discussed as part of the category of salicylate substances (RAAF Document).

Four of the category substances have negative experimentalmammalian chromosome aberrationdata, this data alongside the BlueScreen experimental data, the Ames experimental data and the lack of DNA alerts (OECD [Q]SAR Toolbox - Ames, MN and CA by OASIS v.1.3.) for the 11 category members, this indicates that benzyl salicylate would also be predicted to negative in this in vitro test system.

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

Genetic toxicity in vivo

Description of key information

No in vivo study available for Benzyl Salicylate itself, however information was available for similar structure used for Read Across (Cyclo Hexyl Salicylate and 2 -ethylhexyl salicylate)

in the previous submission.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from supporting substance (structural analogue or surrogate)
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CF-1
Sex:
male/female
Details on test animals and environmental conditions:
Male mice were individually housed and females were housed 4/cage and given free access to rodent diet and drinking water except for 16 hours prior and 3 hours post dosing of the high-dose group and 4-6 prior to 3 hours post dosing of the other test groups. The animal room had a light photoperiod of 12 hours.
Route of administration:
oral: gavage
Vehicle:
Peanut Oil
Details on exposure:
8-week-old CFW-1 (Winkelmann) albino mice (7/sex/group) weighing 20-30 g were gavaged with 3000 mg test substance/kg body weight in peanut oil at a dose volume of 10 ml/kg body weight and euthanized at 24, 48, or 72 hours post dosing.
Duration of treatment / exposure:
dose volume of 10 ml/kg body weight and euthanized at 24, 48, or 72 hours post dosing.
Frequency of treatment:
Once
Post exposure period:
24, 48, or 72 hours
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1500 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
3000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
7/sex/group
Positive control(s):
Positive control (20 mg/kg body weight of Endoxan)
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
The smears were fixed in methanol, rinsed with distilled water 2x, stained in 10% Giemsa, rinsed with distilled water, and air-dried. The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes.
Evaluation criteria:
The proportion of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes and the number of micronucleated normochromatic erythrocytes was recorded.
Statistics:
Statistical analyses were conducted using the tables of Kastenbaum and Bowman.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Dose: 300 mg/kg-Effects:no effects. Results:No mortalities; no statistically significant change in number of micronucleated polychromatic erythrocytes or micronucleated normochromatic erythrocytes; no statistically significant change in the ratio of polychromatic to normochromatic erythrocytes compared to vehicle control.
Dose: 1500 mg/kg-Effects:no effects. Results:No mortalities; no statistically significant change in number of micronucleated polychromatic erythrocytes or micronucleated normochromatic erythrocytes; no statistically significant change in the ratio of polychromatic to normochromatic erythrocytes compared to vehicle control.
Dose: 3000 mg/kg-Effects:no effects. Results:No mortalities; no statistically significant change in number of micronucleated polychromatic erythrocytes or micronucleated normochromatic erythrocytes at any of the 3 sampling times; no statistically significant change in the ratio of polychromatic to normochromatic erythrocytes compared to vehicle control.
Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, the test substance did not show any evidence of mutagenic potential when administered orally.
Executive summary:
Under the conditions of this study, the test substance did not show any evidence of mutagenic potential when administered orally.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1989-06-07 to 1989-06-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study according to principles similar to those of OECD TG 474, but only 1000 instead of 2000 erythrocytes evaluated. To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance) it is proposed to read-across to Ethylhexyl Salicylate (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore, read across is justified.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
1000 instead of 2000 erythrocytes were evaluated
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SAVO GmbH, Kisslegg, Germany
- Age at study initiation: 6 to 10 weeks old
- Weight at study initiation: 25 to 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: in groups of up to five animals in Makrolon cages of size II with Altromin woodshaving
- Diet (e.g. ad libitum): Altromin 1324 standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: no justification provided
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the solutions were prepared freshly directly before application
Duration of treatment / exposure:
Groups were treated with the substance and sampling was performed after 24, 48 and 72 hours
Frequency of treatment:
One treatment at the beginning of the test.
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Five males and five females
Control animals:
yes, concurrent vehicle
Positive control(s):
Five males and five females treated with 50 mg/kg 9,10-dimethyl-1,2-benzanthracene (DMBA) by oral gavage with sampling time after 48 hours
Tissues and cell types examined:
Bone marrow smears and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: a limit test was performed showing that a dose of 2000 mg/kg bw was the maximum tolerated dose not leading to cytotoxicity.
DETAILS OF SLIDE PREPARATION: animals were killed by cervical dislocation and bone marrow was removed from both femora by rinsing with foetal calf serum. Bone marrow cells were centrifuged at 150 g for 10 minutes and the supernatant was discarded. From the pellet, smears were made on slides and air dried. Two slides were made per animal. The slides were stained by the May-Gruenwald-Giemsa method according to Schmid (1973) by staining for 3 minutes in undiluted May-Gruenwald solution, then staining for 2 minutes in May-Gruenwald diluted with distilled water to a ratio 1:1, rinsing briefly in distilled water, staining for 10 minutes in Giemsa diluted with distilled water to a ratio 1:6, rinsing thoroughly in distilled water, drying in air, cleaning the back side of the slides with methanol, cleaning in xylene for 5 minutes and mounting in Eukitt.

METHOD OF ANALYSIS: slides were coded and observed blindly under a microscope with 100x oil immersion objective lens at 1250 fold magnification. At least 1000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The number of micronucleated normochromatic erythrocytes was also recorded. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.
Evaluation criteria:
An increased frequency of micronucleated polychromatic erythrocytes among treated animals compared to control animal values is taken as an indication of treatment-induced genetic damage.
Statistics:
Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
According to an initial toxicity test the maximum tolerated dose was 2000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Both negative and positive control frequencies of micronucleated polychromatic erythrocytes agreed with the values previously established in the testing laboratory.

To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance) it is proposed to read-across to Ethylhexyl Salicylate (Source Substance).

The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible.

The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific.

Therefore, read across is justified.

Table: Detailed results of analysis for single animals used in the micronucleus test

Dose and sampling time

Sex

Animal identification

PE examined

PE with MN

NE with MN (‰)

Ratio PE/NE

Total

Vehicle control, 24 hours after oral application by gavage

female

A1

1086

2

1.84

0.00

1.76

A2

1071

2

1.87

1.00

2.05

A3

1010

0

0.00

0.98

1.88

A4

1016

2

1.97

3.00

2.34

A5

1004

3

2.99

2.98

2.24

male

B1

1010

1

0.99

1.94

1.52

B2

1019

1

0.98

0.00

2.08

B3

1087

1

0.92

1.98

1.85

B4

1002

2

2.00

1.97

1.56

B5

1020

2

1.96

0.00

2.07

Test substance at 2000 mg/kg bw, 24 hours after oral application by gavage

female

C1

1032

1

0.97

0.99

2.24

C2

1018

2

1.96

0.00

2.15

C3

1002

2

2.00

0.00

1.83

C4

1014

1

0.99

1.99

1.32

C5

1006

2

1.99

0.00

1.89

male

D1

1005

2

1.99

0.98

1.16

D2

1003

2

1.99

1.98

1.10

D3

1004

1

1.00

1.99

1.96

D4

1003

0

0.00

1.90

1.29

D5

1003

1

1.00

2.00

1.50

Test substance at 2000 mg/kg bw, 48 hours after oral application by gavage

female

E1

1006

1

0.99

1.96

1.15

E2

1001

1

1.00

0.00

1.45

E3

1032

2

1.94

0.98

1.62

E4

1010

3

2.97

0.00

1.20

E5

1002

0

0.00

0.99

1.69

male

G1

1012

3

2.96

2.96

1.27

G2

1003

1

1.00

1.00

1.56

G3

1030

2

1.94

2.94

1.17

G4

1013

1

0.99

2.00

2.02

G5

1006

1

0.99

0.99

1.96

Test substance at 2000 mg/kg bw, 72 hours after oral application by gavage

female

H1

1006

1

1.00

0.00

1.22

H2

1005

2

1.99

2.96

1.16

H3

1046

2

1.91

1.00

1.48

H4

1033

3

2.90

1.99

1.74

H5

1009

1

0.99

0.99

1.76

male

I1

1008

2

1.98

1.92

1.85

I2

1075

1

0.93

0.99

1.67

I3

1043

1

0.96

2.97

1.20

I4

1029

2

1.94

1.99

1.80

I5

1009

1

0.99

0.00

1.77

DMBA at 50 mg/kg bw, 24 hours after oral application by gavage

female

L1

1013

10

9.87

5.81

1.27

L2

1011

12

11.87

4.80

1.28

L3

1000

8

8.00

6.56

1.19

L4

1005

10

9.95

4.96

1.33

L5

1009

13

12.88

4.69

1.30

male

M1

1019

12

11.78

9.79

1.00

M2

1001

9

8.99

11.93

1.32

M3

1003

11

10.97

4.73

0.95

M4

1017

13

12.78

5.66

1.06

M5

1009

10

9.91

5.00

1.37

PE = polychromatic erythrocytes; NE = normochromatic erythrocytes; PE with MN = micronucleated polychromatic erythrocytes; NE with MN = micronucleated normochromatic erythrocytes; DMBA = 9,10-dimethyl-1,2-benzanthracene

Conclusions:
Interpretation of results (migrated information): negative
The substance did not induce chromosomal damage or damage to the mitotic apparatus in bone marrow cells of mice after a single oral application at a dose of 2000 mg/kg bw.
Executive summary:

As discussed as part of the category of salicylate substances (RAAF Document).

Only 2 members of this category have in vivo mutagenicity micronucleus data. However, all 11 of these substances have in vivo mutagenicity micronucleus alerts for H-acceptor-path3-H-acceptor interaction which indicates a possiblechemical interaction with DNA and/or proteinsvianon-covalent binding, such as DNA intercalation or groove-binding.

However, the results of the 2 main source substances both have negative experimental data for this endpoint and therefore it can be assumed that when taken into account with the in vitro data and the experimental data consistencies (negative data) within the category and the total lack of any DNA interactions (in vitro and in vivo), that the target substance benzyl salicylate would also be negative in such a genetox in vivo micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

OECD 471_Ames test (Benzyl salicylate): non-mutagenic with and without S9.
OECD 473_In vitrocytogenicity evaluated with chromosome aberration (CHL/IU cells): non-clastogenic with and without S9.
OECD 476_In vitrogene mutation in mammalian cells (HPRT): non-mutagenic with and without S9.

Benzyl Salicylate has no mutagenic or clastogenic effects (negative).

Additional information

In the previous dossier submission of Benzyl Salicylate, a read-across to Cyclohexyl Salicylate, Ethylhexyl Salicylate and Methyl Salicylate was originaly proposed and presented to ECHA. The target substance (Benzyl Salicylate) and Source Substances have been caracterised using the categories and databases present in the OECD (Q)SAR Toolbox, where similarities on structure and mode of action were observed. Benzyl Salicylate was predicted and non-mutagenic and non-clastogenic based on Read-Across from the above susbtances, however this approach was not accepetd and an in vitro cytogenicity study in mammalian cells (OECD 473 or OECD 487) and an in vitro gene mutation in mammalian cells were requested, after ECHA evaluation.

New data was generated to evaluate the genotoxicity of Benzyl Salicylate and are summarized below:

Short description of key information:
OECD 471_Ames test (Benzyl salicylate): non-mutagenic with and without S9.
OECD 473_In vitro cytogenicity evaluated with chromosome aberration (CHL/IU cells): non-clastogenic with and without S9.
OECD 476_In vitro gene mutation in mammalian cells (HPRT): non-mutagenic with and without S9


Endpoint Conclusion: Benzyl Salicylate has no mutagenic or clastogenic effects (negative)

Justification for classification or non-classification

No mutagenic effects were observed in two in vitro mutagenicity studies and one in vivo micronucleus studies on the registration substance (Ames, in vitro Chrom. ab. and in vitro gene mutation in mammalian cells. The substance is therefore not considered to be mutagenic and does not warrant classification in accordance with Regulation (EC) No. 1272/2008.