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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A negative Ames test is available for hexyl salicylate; a negative mammalian cell mutation assay is available for the read-across substance cyclohexyl salicylate. No study of chromosomal aberration in vitro is available; however a higher tier (in vivo) study is provided to address this data requirement.

Hexyl salicylate was investigated in an Ames test using Salmonella typhimurium strains TA 1535, TA 153, TA 100, TA 98 and TA 102. The study was conducted using the standard plate incorporation method, both in the presence and absence of metabolic activation (S9 fraction). The test substance was tested at concentrations from 5-5000 µg/plate in the presence of metabolic activation and 15-5000 µg per plate in the absence of metabolic activation. Appropriate positive controls were also used to confirm the sensitivity of the assay. Hexyl salicylate did not induce a significant increase in the mutation frequency of the tester strains in the presence or absence of metabolic activation.

 

The read-across (category) substance cyclohexyl salicylate was examined for its ability to induce gene mutations at the HPRT locus when tested in vitro using Chinese Hamster V79 cells. The assay was performed in two independent experiments both in the presence and absence of metabolic activation in the form of S9 mix. Solvent, negative and appropriate positive controls were also used. Based on the results of this study, the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported in this study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 28th to May 30th 2000.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine loci
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable as bacterial cells
Additional strain / cell type characteristics:
other: Strains TA 1535 and TA 100: base pair substitution mutation hisG46. Strain TA 1537: histidine frameshift mutation his3076. Strain TA 98: histidine frameshift mutation his3052.
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable as bacterial cells
Additional strain / cell type characteristics:
other: Strain TA 102 contains the unrevertable deletion hisG8476 in the histidine operonof the bacterial chromosome but offers the revertible mutation hisG428 on the plasmid pAQ1.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver homogenates
Test concentrations with justification for top dose:
Presence of S9 mix: 5 - 5000 µg per plate
Absence of S9 mix: 15 - 5000 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Dissolved in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Dissolved in distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Dissolved in distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Dissolved in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Dissolved in distilled water
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 - 72 hours

NUMBER OF REPLICATIONS:
Three parallel plates were prepared for each experimental point.

DETERMINATION OF CYTOTOXICITY
- Method: other: Number of revertant colonies per plate

OTHER:
To confirm reversion properties and specificity of the bacterial strains as well as the activity of the liver homogenates, positive mutagenesis controls were run simultaneously. The genotypes of the tester strains (histidine requirement, spontaneous reversion frequency, sensitivity to UV light, crystal violet and ampicillin) were checked in each experiment. The Vogel-Bonner minimal plates, the test compound and the S9 mix were checked for sterility.
Evaluation criteria:
When there was a question about the nature of colonies scored as revertants and when positive mutagenic results were obtained, the genotype of revertant colonies was spot-checked by picking and streaking histidine free plates.
Statistics:
No information provided
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed at 1500 and 5000µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of spontaneous revertants observed using each of the 5 strains was similar to those previously established.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of S9 mix, hexyl salicylate was bacteriotoxic towards the strain TA 1537 at 500ug/plate, towards the strains TA 100 and TA 102 at 1500ug/plate and towards the strains TA 98 and TA 1535 at 5000ug/plate. In the absence of S9 mix, hexyl salicylate was bacteriotoxic towards the strain TA 1537 at 500ug/plate and towards the strains TA 98, TA 100 and TA 102 at 5000 ug/plate.
Conclusions:
Under the experimental conditions of this study hexyl salicylate was not considered to be mutagenic to Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of metabolic activation.
Executive summary:

Hexyl salicylate was investigated in an Ames test using Salmonella typhimurium strains TA 1535, TA 153, TA 100, TA 98 and TA 102. The study was conducted using the standard plate incorporation method, both in the presence and absence of metabolic activation (S9 fraction). The test substance was tested at concentrations from 5-5000 µg/plate in the presence of metabolic activation and 15-5000 µg per plate in the absence of metabolic activation. Appropriate positive controls were also used to confirm the sensitivity of the assay. Hexyl salicylate did not induce a significant increase in the mutation frequency of the tester strains in the presence or absence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
February 23rd 1994 to June 29th 1994.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
No further details
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Each batch of cells was screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Thawed stock cultures were propagated at 37°C in 80cm2 plastic flasks. Approximately 5 x 10 E5 cells were seeded into each flask with 15ml of minimal essential medium (MEM) supplemented with 10% foetal calf serum. The cells were subcultured twice weekly and incubated at 37°C in a 4.5% carbon dioxide atmosphere. For the selection of mutants, the medium was supplemented with 11ug/ml thioguanine.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I: without S9 mix - 0.75, 2.5, 5.0, 7.5 and 8.5 µg/ml
with S9 mix - 3.0, 6.0, 10.0, 30.0 and 60.0 µg/ml
Experiment II: without S9 mix - 1.0, 2.5, 5.0, 7.5 and 10.0 µg/ml
with S9 mix - 6.0, 10.0, 30.0 and 60.0 µg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The vehicle was chosen for its solubility properties and its non-toxicity to cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in nutrient medium (MEM without FCS)
Positive control substance:
ethylmethanesulphonate
Remarks:
The solution was prepared on the day of the experiment. Positive control used in the absence of metabolic activation. Migrated to IUCLID6: 0.6 mg/ml (4.8 mM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in Diethylsulfoxide with a final concentration in nutrient medium of 1%
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Positive control used in the presence of metabolic activation. Migrated to IUCLID6: 3.85ug/ml (15.0 µM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Fixation time (start of exposure up to fixation or harvest of cells): 9 days

NUMBER OF REPLICATIONS:
For the determination of toxicity, the test was conducted in duplicate per experimental point.
For the determinaiton of mutation rates, the test was only conducted in one flask per experimental point.

NUMBER OF CELLS EVALUATED:
Stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: other: mutant frequency
Evaluation criteria:
A test article is classified as positive if it induces either a concentration related increase of the mutant frequency or a reproducible and positive response for one of the test points. A test article producing neither a concentration related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
If a significant response is observed, the test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutant frequency that is three times higher than the spontaneous mutation frequency in the experiment. The test article may also be considered to be mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a three-fold increase of the mutant frequency was not observed.
Statistics:
An adequate statistical method was not available.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not documented
- Effects of osmolality: Not documented
- Evaporation from medium: Not documented
- Water solubility: Not documented
- Precipitation: Not documented
- Other confounding effects: Not documented

RANGE-FINDING/SCREENING STUDIES:
Based on the results of the pre-test on toxicty, the concentration ranges were selected to yield concentration-related toxic effects. Without S9 mix, the highest concentration produced strong toxic effects. With S9 mix, no toxicity was observed up to the limit of solubility at 60ug/ml.

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments with and without S9 mix, the range of negative and solvent controls was from 3.5 to 21.9 mutants per 10E6 cells. The range of the groups treated with the test article was from 2.2 to 19.6 mutants per 10E6 cells. The elevated value of 19.6 mutants per 10E6 cells at the concentration of 5.0ug/ml was not considered to be biologically relevant as the obtained value was within the range of the historical control data which was 0 to 45 mutants per 10E6 cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No additional information

Considering the mutation rates found in the groups treated with the test article compared to the negative and solvent controls, it can be concluded that no relevant increase in gene mutations was observed. The test article did not induce a reproducible concentration related increase in mutant colony numbers.

The mutant values of the groups treated with the test article were in the range of the negative controls.

Without metabolic activation, strong toxic effects in the cloning efficiency of cells were observed with concentrations of 7.5 and 8.5 ug/ml in Experiment I. Cytotoxicity was evident at 7.5 and 8.5 ug/ml with reductions in cell density of 54.2 and 31.4% at these concentrations

In experiment II, the cloning efficiency of the cells was strongly reduced at the two highest concentrations, 7.5 and 10 µg/mL, without S9 mix. The cell density at first subcultivation was reduced to 61.2% of the corresponding solvent control whereas 10 µg/ml could not be evaluated at the end of the experiment due to the strong reduction (4%) of the cell density at first subcultivation.

In the presence of metabolic activation, no toxic effects were evident up to the limit of solubility (60 µg/mL).

Pre-test results for toxicity

 

Cloning efficiency assay for negative control (untreated cells)

Concentration per mL

Colonies counted

Mean

Relative CE%

Flask 1

Flask II

Negative control, untreated cells

429

402

415.5

76.7

 

Cloning efficiency assay without metabolic activation

Concentration per mL (µg)

Colonies counted

Mean

Relative CE%

Flask 1

Flask II

Negative control

362

294

328

100

Solvent control, ethanol

346

310

328

100

0.1

310

293

301.5

91.9

0.3

361

358

359.5

109.6

1.0

325

340

332.5

101.4

3.0

301

324

312.5

95.3

6.0

292

353

322.5

98.3

10.0

170

315

242.5

73.9

30.0

0

0

0

0

60.0

0

0

0

0

 

Cloning efficiency assay with metabolic activation

Concentration per mL (µg)

Colonies counted

Mean

Relative CE%

Flask 1

Flask II

Negative control

344

347

345.5

100

Solvent control, ethanol

335

314

324.5

100

0.1

340

315

327.5

100.9

0.3

362

319

340.5

104.9

1.0

375

342

358.5

110.5

3.0

379

342

360.5

111.1

6.0

372

338

355.0

109.4

10.0

447

340

393.5

121.3

30.0

370

375

372.5

114.8

60.0

366

334

350.0

107.9

In the pre-test for toxicity (colony forming ability) the cloning efficiency was reduced following treatment at 10 µg/mL with S9. Based on these results experiment 1 dose selection ranged from 1 to 30 µg/mL. A marked toxic response was evident at the two highest concentrations (10 and 30 µg/mL). The assay was repeated using doses in the range 1-10 µg/mL but 10 µg/ml was very toxic in this experiment also and further testingwas completed at 0.75 to 8.5 µg/mL.

 

In the pre-test no toxicity was evident up to the limit of solubility (60 µg/mL) in the presence of S9. The replicate assessments with S9 were completed using dose concentrations of 6 to 60 µg/mL

 

Conclusions:
Under the experimental conditions reported in this study, cyclohexylsalicylate did not induce gene mutations at the HPRT locus in V79 cells.
Executive summary:

Cyclohexyl salicylate was examined for its ability to induce gene mutations at the HPRT locus when tested in vitro using Chinese Hamster V79 cells. The assay was performed in two independent experiments both in the presence and absence of metabolic activation in the form of S9 mix. Solvent, negative and appropriate positive controls were also used. Based on the results of this study, the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported in this study.

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

Genetic toxicity in vivo

Description of key information

A negative mouse bone marrow micronucleus assay is provided for the read-across (category) substance 2 -ethylhexyl salicylate.

The genetic toxicity of 2 -ethylhexyl salicylate was investigated in a mouse bone marrow micronucleus assay. Groups of five male and five female mice were treated by single oral administration of 2 -ethylhexyl salicylate (in arachis oil) at the limit dose level of 2000 mg/kg bw. The animals were sacrificed and bone marrow smears were prepared at 24, 48 and 72 hours after treatment. A positive (DMBA) and negative (vehicle) control group were also tested. At least 1000 polychromatic erythrocytes (PCEs) from each animal were scored for the incidence of micronuclei. The number of micronucleated normochromatic erythrocytes (NCEs) was also recorded, and the PCE:NCE ratio determined for each animal by counting a total of 1000 erythrocytes. No evidence of micronucleus formation was seen under the conditions of this assay

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
21 April 1989 - 3 July 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
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, D-7964 Kisslegg, F.R.G.
- Age at study initiation: 6 - 10 weeks old
- Weight at study initiation: 25 - 30g
- Assigned to test groups randomly: The animals were separated according to sex, marked for identification and allocated by randomization to cages and groups.
- Fasting period before study: No information provided
- Housing: The animals were group housed, up to 5 per cage in Makrolon cages of size II with Altromin woodshavings. The cages and bedding were changed with clean ones twice a week.
- Diet (e.g. ad libitum): Standard laboratory diet (Altromin 1324) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): Approximately 55%
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark cycle.

IN-LIFE DATES: From: 21 April 1989 To: 3 July 1989
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The compound was dissolved in arachis oil and the solution was freshly prepared just prior to use.

Duration of treatment / exposure:
Single exposure
Frequency of treatment:
The animals were dosed once
Post exposure period:
72 hours
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control group
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Treated group
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
9,10-dimethyl-1,2-benzanthracene dissolved in olive oil.
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
Bone marrow polychromatic erythrocytes (PCEs)
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES
The test animals were killed by cervical dislocation and bone marrow removed from both femora by rinsing with fetal calf serum. Bone marrow cells were centrifuged at 150g for 10 minutes and the supernatant discarded. From the pellet, smears were made on slides and air dired, according to haematological routine.

DETAILS OF SLIDE PREPARATION:
2 slides were made per animal. The preparations were stained by the May-Gruenwald-Giemsa method according to Schmid (1973). The slides were stained for 3 minutes in undiluted May-Gruenwald solution. They were then stained for 2 minutes in May-Gruenwald, diluted with distilled in a 1:1 ratio followed by brief rinsing in distilled water. The slides were stained for 10 minutes in Giemsa, diluted with distilled in a ratio of 1:6 and rinsed thoroughly in distilled water. They were dried in air and the back of the slides was dried with methanol. They were cleared in Xylene for 5 minutes and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides were coded and observed blindly under a microscope with a 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.
Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970).

Evaluation criteria:
No information provided
Statistics:
Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: According to an initial toxicity test, the maximum tolerated dose was fixed to 2000 mg/kg body weight.

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): No increase in the frequency of micronucleated polychromatic erythrocytes in any treatment group at any treatment time. The incidence of micronucleated normochromatic erythrocytes in all treatment groups was within expected levels. The ratio of polychromatic erythrocytes to normochromatic erythrocytes was considered to be normal.

Afte dosing, the animals of all treated groups showed reduced motility.

No additional information

Conclusions:
No evidence of micronucleus formation was seen under the conditions of this assay.
Executive summary:

The genetic toxicity of 2 -ethylhexyl salicylate was investigated in a mouse bone marrow micronucleus assay. Groups of five male and five female mice were treated by single oral administration of 2 -ethylhexyl salicylate (in arachis oil) at the limit dose level of 2000 mg/kg bw. The animals were sacrificed and bone marrow smears were prepared at 24, 48 and 72 hours after treatment. A positive (DMBA) and negative (vehicle) control group were also tested. At least 1000 polychromatic erythrocytes (PCEs) from each animal were scored for the incidence of micronuclei. The number of micronucleated normochromatic erythrocytes (NCEs) was also recorded, and the PCE:NCE ratio determined for each animal by counting a total of 1000 erythrocytes. No evidence of micronucleus formation was seen under the conditions of this assay.

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

Additional information

Justification for classification or non-classification

No evidence of mutagenicity was seen in an Ames test with hexyl salicylate, a mammalian cell mutation assay with the read-across (category) substance cyclohexyl salicylate or in a mouse bone marrow micronucleus assay with the read-across (category) substance 2 -ethylhexyl salicylate. Classification for mutagenicity according to the CLP Regulation is therefore not required.