Registration Dossier
Registration Dossier
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EC number: 606-948-7 | CAS number: 2217-02-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue substante DL-Borneol, the test item is considered to be not mutagenic with or without metabolic activation in any of the tested strains.
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue substante DL-Borneol, the test item is determined to be not mutagenic.
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue substante DL-Borneol, the test item is determined to be not mutagenic.
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue substante D-Borneol, the test item is determined to be not mutagenic.
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue isobornyl propionate, the test item is determined to be not mutagenic under the test conditions.
In vitro gene mutation study in mammalian cells. Key study: Read-across approach. Based on the read-across approach from the analogue fenchyl acetate, the test item was determined to be not mutagenic either in the presence or in the absence of metabolic activation system under the conditions of a Mouse Lymphoma Assay.
In vitro cytogenicity in mammalian cells/micronucleus study. Data waiving.According to column 2 of REACH Annex VIII, an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted since adequate data from an in vivo test are available.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- The standard plate incorporation as described by Ames et al. (1975) and modified by Batzinger et al. (1978) and Babish et al. (1983) was used in this research.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 97
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- not specified
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Assay 1: Strains TA 97, TA 98 and TA 100 with preincubation time of 20 min both in the presence and absence of 0.5 ml of S9 mix.
Assay 2: Strain TA 100 with S9 activation and incorporation of another sample preincubated for 60 min.
DURATION
- Preincubation period: 20 min
NUMBER OF REPLICATIONS: 3 - Evaluation criteria:
- Evaluation according to "MR = mean revertants per plate / mean spontaneous revertants per plate" with the following criteria:
Positive: MR greater than 2 - Key result
- Species / strain:
- S. typhimurium TA 97
- 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 98
- 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 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
- Conclusions:
- No evidence of mutagenicity was observed at test conditions for Borneol.
- Executive summary:
A bacterial reverse mutation test (Ames test) was performed on test substance Borneol using Salmonella typhimurium strains TA 97, TA 98 and TA 100 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent at a concentration of 1 mg/ml. Positive and negative/solvent controls were included. No evidence of mutagenicity was observed at test conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: No GLP. Equivalent or similar to OECD 471. Documentation insufficient for assessment.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- up to 5000 μg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: no data
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Plate incorporation method
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- Under the conditions of the study, DL-Borneol was found to be not mutagenic with or without metabolic activation in any of the tested strains.
- Executive summary:
The mutagenic potential of DL-Borneol was assessed in an Ames study following experimental outlines similar to those in OECD Guideline 471 using the plate incorporation method. Salmonella
typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with DL-Borneol at concentrations up to 5000 μg/plate in the presence and absence of metabolic activation (S9 mix). Under the conditions of the study, DL-Borneol is considered not mutagenic in bacteria.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: No data on GLP. Test method not reliable for full assessment of genetic toxicity in bacteria.
- Principles of method if other than guideline:
- A mutation test in Escherichia coli WP 2 uvrA (trp-) was performed in Borneol and some other synthetic flavoring agents widely used in foodstuffs.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- not specified
- Test concentrations with justification for top dose:
- Test concentration range: 0.4 to 3.2 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: no data
- Evaluation criteria:
- Evaluation in mutation test according to "ratio = maximal revertants / spontaneous revertants" with the following criteria:
Positive: when ratio >=2
Negative: when ratio < 2 - Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- No evidence of mutagenicity was observed at test conditions for Borneol.
- Executive summary:
No mutagenic activity was observed for Borneol in the test mutation using E. Coli WP2 uvrA within a concentration range for the test substance of 0.4 -3.2 mg/plate.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 May 2013 - 15 July 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (rat liver)
- Test concentrations with justification for top dose:
- 3h, ± S9 mix: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 µg/mL
3h, + S9 mix: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 µg/mL
24h, - S9 mix. 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was non-soluble in Distilled water or Dimethyl sulfoxide at 500 mg/mL concentration. However, a proper formulation could be made at the same concentration using Acetone or Ethanol (96%) as vehicle. Due to the better biocompatibility to the test system, Ethanol (96%) was selected for vehicle of the study. - Untreated negative controls:
- yes
- Remarks:
- (Untreated)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (Ethanol 96%, DMSO)
- Remarks:
- Test item vehicle: Ethanol (10 µL/mL), positive control vehicle: DMSO (10 µL/mL)
- Positive controls:
- yes
- Remarks:
- (Without metabolic activation)
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- 0.15 µg/mL (3h treatment), 0.10 µg/mL (24h treatment), in DMSO
- Positive controls:
- yes
- Remarks:
- (With metabolic activation)
- Positive control substance:
- cyclophosphamide
- Remarks:
- 4 µg/mL in DMSO
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium (RPMI-5 medium)
DURATION
- Exposure duration: 3h (with and without S9-mix), 24h (without S9-mix)
- Expression time (cells in growth medium, to allow expression of the TK- mutation ): 3 days
- Selection time (plating for -trifluorothymidine (TFT) resistance): Two weeks
SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.
NUMBER OF CELLS EVALUATED:
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by relative survival
OTHER EXAMINATIONS:
- Determination of Survival or Viability:
After the exposure period and the expression time, cells were also diluted to be plated for survival and viability respectively. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.
Parameter calculated:
- Relative survival
- Mutant Frequency
- Small and large colony mutant frequencies
Others:
The number of viable cells in the individual samples was counted manually using a haemocytometer.
Measurement of pH and osmolality was performed after the treatment period. - Evaluation criteria:
- The test item was considered to be mutagenic in this assay if all the following criteria were met (based on M. Moore 2006):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value. - Statistics:
- Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (Assay 1: ±S9mix, 3h: >= 100 µg/L; Assay 3: +S9mix, 3h: >= 95 µg/L; -S9mix, 24h: >= 55 µg/L)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH after treatment.
- Effects of osmolality: There were no large changes in osmolality after treatment.
- Water solubility: No insolubility was detected in the final treatment medium at the end of the treatment (although discoloured medium was detected at some concentrations)
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed to select dose levels for the main assays. During the preliminary test, a 3 hour treatment in the presence and absence of S9-mix and a 24 hour treatment in the absence of S9-mix were performed with a range of test item concentration to determine toxicity. The highest concentration tested in the preliminary experiment was 2500 μg/mL. Insolubility and cytotoxicity were observed in the preliminary experiment. Concentrations for the main experiments were selected to cover the range from cytotoxicity to little or no cytotoxicity according to the instructions of the relevant OECD guideline.
ADDITIONAL INFORMATION:
No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations.
No significant dose-response to the treatment was indicated by the linear trend analysis.
OBSERVATION:
During Assay 2, a contamination was detected in the samples, therefore it was considered invalid and terminated. An additional experiment (Assay 3) was performed using the same experimental conditions as in Assay 2 to provide valid and interpretable data. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- No mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
- Executive summary:
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD 476) to test the potential to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Ethanol was used as vehicle of the test item in this study. The test item was examined up to 2500 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL
Assay 1, 3-hour treatment without metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL
Assay 3, 3-hour treatment with metabolic activation: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 μg/mL
Assay 3, 24-hour treatment without metabolic activation: 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 μg/mL
No insolubility was detected in the final treatment medium at the end of the treatment. There were no large changes in pH or osmolality after treatment. Cytotoxicity was observed in both assays (Assay 1: ± S9mix, 3h: >= 100 µg/L; Assay 3: + S9mix, 3h: >= 95 µg/L; - S9mix, 24h: >= 55 µg/L). No biologically relevant or statistically significant increase in the mutation frequency was observed. No dose response to the treatment was indicated by the linear trend analysis. The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases and the study was considered to be valid. In conclusion, no mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- The standard plate procedure was followed (Ames, McCann and Yamasaki, 1975)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 1535
- Species / strain / cell type:
- S. typhimurium TA 1537
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix of liver rats
- Test concentrations with justification for top dose:
- 5 doses (up to 3.6 mg/plate) in all five tester strains ±S9 mix
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Plate incorporation method:
S-9 liver fractions were prepared from Aroclor-pretreated rats (Aroclor 1254, 500 mg/kg ip) and adjusted to 25 mg protein/ml; 0.5 ml S-9 mix, equivalent to 50 µl S-9, was incorporated into the plates.
Vogel-Bonner medium (Vogel & Bonner, 1956) was used throughout.
Plates were incubated for 48 hr.
- Cell density at seeding (if applicable): Overnight bacterial cultures had cell titres of at least 10^9 cells/ml.
NUMBER OF REPLICATIONS: 2 - Evaluation criteria:
- Criteria regarded as positive: a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency.
Criteria regarded as marginally mutagenic under the experimental conditions: reproducible, dose-related and significant (p<=0.01) but less than two-fold elevations. - Statistics:
- Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- No evidence of mutagenicity was observed at test conditions for isobornyl propionate.
- Executive summary:
A bacterial reverse mutation test (Ames test) was performed on test substance isobornyl propionate using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537 and TA 1538 with and without S9 mix metabolic activation. Five doses up to 3.6 mg/plate of test item were used and 48 h incubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls with sodium azide and benzo(a)pyrene were included. No evidence of mutagenicity was observed at test conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance dextro alpha fenchyl acetate undergoes rapid hydrolysis to acetic acid and dextro alpha fenchyl alcohol which is the target substance.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (Read-across from an analogue)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- (read-across from an analogue determined to be not mutagenic in mammalian cell assay)
- Conclusions:
- Based on the read-across approach from the analogue fenchyl acetate, the test item was determined to be not mutagenic either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
- Executive summary:
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD 476) to test the potential of analogue substance fenchyl acetate to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Ethanol was used as vehicle of the test item in this study. The test item was examined up to 2500 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL
Assay 1, 3-hour treatment without metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL
Assay 3, 3-hour treatment with metabolic activation: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 μg/mL
Assay 3, 24-hour treatment without metabolic activation: 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 μg/mL
No insolubility was detected in the final treatment medium at the end of the treatment. There were no large changes in pH or osmolality after treatment. Cytotoxicity was observed in both assays (Assay 1: ± S9mix, 3h: >= 100 µg/L; Assay 3: + S9mix, 3h: >= 95 µg/L; - S9mix, 24h: >= 55 µg/L). No biologically relevant or statistically significant increase in the mutation frequency was observed. No dose response to the treatment was indicated by the linear trend analysis. The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases and the study was considered to be valid. In conclusion, no mutagenic effect was observed either in the presence or in the absence of metabolic activation system. Based on these results, the read-across was applied and the test item was determined to be not mutagenic under the conditions of this Mouse Lymphoma Assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance isobornyl propionate undergoes rapid hydrolysis to propionic acid and isoborneol which shares the same functional groups with the substance D-alpha fenchol and also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in bacteria
- Conclusions:
- Based on the read-across approach from the analogue isobornyl propionate, the test item is determined to be not mutagenic under the test conditions.
- Executive summary:
A bacterial reverse mutation test (Ames test) was performed on test substance isobornyl propionate using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537 and TA 1538 with and without S9 mix metabolic activation. Five doses up to 3.6 mg/plate of test item were used and 48 h incubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls with sodium azide and benzo(a)pyrene were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across was applied and the test item was determined to be not mutagenic under the test conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance DL-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 97
- 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 98
- 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 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
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in bacteria
- Conclusions:
- Based on the read-across approach from the analogue substante DL-Borneol, the test item is determined to be not mutagenic.
- Executive summary:
A bacterial reverse mutation test (Ames test) was performed on analogue substance Borneol using Salmonella typhimurium strains TA 97, TA 98 and TA 100 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent at a concentration of 1 mg/ml. Positive and negative/solvent controls were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance DL-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in bacteria
- Conclusions:
- Based on the read-across approach from the analogue substante DL-Borneol, the test item is considered to be not mutagenic with or without metabolic activation in any of the tested strains.
- Executive summary:
The mutagenic potential of analogue substance DL-Borneol was assessed in an Ames study following experimental outlines similar to those in OECD Guideline 471 using the plate incorporation method. Salmonella
typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with DL-Borneol at concentrations up to 5000 μg/plate in the presence and absence of metabolic activation (S9 mix). Under the conditions of the study, DL-Borneol is considered not mutagenic in bacteria. Based on these results, the read-across was applied and the test item is considered to be not mutagenic in the Ames test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance DL-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in bacteria
- Conclusions:
- Based on the read-across approach from the analogue substante DL-Borneol, the test item is determined to be not mutagenic.
- Executive summary:
A mutation test was conducted on the analogue substance DL-Borneol with E. Coli WP2 uvrA (trp-) at a dose range of 0.4 -3.2 mg/plate. No evidence of mutagenicity was observed. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- The mutation test was carried out by the preincubation procedure described by Ames et al. (1975). The test chemical was preincubated with S9 mix or phosphate buffer (pH =7.4) for 20 min.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 97
- Species / strain / cell type:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 0 (control), 0.001, 0.005, 0.01, 0.05 and 0.1 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- mitomycin C
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Preincubation procedure
Strains TA 97 and TA 102 with preincubation time of 20 min both in the presence and absence of S9 mix.
DURATION
- Preincubation period: 20 min
NUMBER OF REPLICATIONS: 3 - Key result
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- No evidence of mutagenicity was observed at test conditions for D-borneol.
- Executive summary:
A bacterial reverse mutation test (Ames test) was performed on test substance D-borneol using Salmonella typhimurium strains TA 97 and TA 102 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls were included. No evidence of mutagenicity was observed at test conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance D-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from supporting substance. All data sources agree to consider the test substance as not mutagenic in bacteria and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in bacteria
- Conclusions:
- Based on the read-across approach from the analogue substante D-borneol, no evidence of mutagenicity is observed on the test item.
- Executive summary:
A bacterial reverse mutation test (Ames test) was performed on test substance D-borneol using Salmonella typhimurium strains TA 97 and TA 102 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
- Justification for type of information:
- JUSTIFICATION FOR DATA WAIVING
According to column 2 of REACH Annex VIII, an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted since adequate data from an in vivo test are available - Reason / purpose for cross-reference:
- data waiving: supporting information
Referenceopen allclose all
The results were as follows:
Reagents |
Dose range (mg/plate) |
Ratio |
Conclusion |
Borneol |
0.4-3.2 |
0.9 |
- |
Mutagenicity Results of Assay 1
S9 mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/ total number of wells |
Dn2/var(Dn)♦ |
Mutation frequency |
+ |
3 |
140 µg/mL |
ND |
ND |
ND |
ND |
ND |
130 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
120 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
110 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
100 µg/mL |
NE |
NE |
NE |
NE |
NE |
||
90 μg/mL |
642/768 |
64/768 |
62/768 |
0.047 |
125.0 |
||
80 µg/mL |
640/768 |
71/768 |
57/768 |
0.056 |
124.5 |
||
60 µg/mL |
640/768 |
74/768 |
54/768 |
0.087 |
122.7 |
||
40 µg/mL |
641/768 |
74/768 |
53/768 |
0.009 |
128.8 |
||
20 µg/mL |
652/768 |
68/768 |
48/768 |
0.108 |
143.1 |
||
10 µg/mL |
654/768 |
65/768 |
49/768 |
0.785 |
105.8 |
||
5 µg/mL |
636/768 |
75/768 |
57/768 |
0.003 |
133.5 |
||
Vehicle control |
640/768 |
71/768 |
57/768 |
-- |
131.8 |
||
Vehicle control for CP |
645/768 |
64/768 |
59/768 |
-- |
118.3 |
||
Untreated control |
642/768 |
64/768 |
62/768 |
-- |
135.2 |
||
Positive control |
277/768 |
204/768 |
287/768 |
♦♦ |
1076.7* |
In linear trend analysis β2/var (β) = 0.002, not significant.
S9 mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/ total number of wells |
Dn2/var(Dn) ♦ |
Mutation frequency |
- |
3 |
140 µg/mL |
ND |
ND |
ND |
ND |
ND |
130 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
120 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
110 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
100 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
90 μg/mL |
671/768 |
38/768 |
59/768 |
0.710 |
111.7 |
||
80 µg/mL |
672/768 |
42/768 |
54/768 |
0.229 |
100.8 |
||
60 µg/mL |
652/768 |
28/768 |
88/768 |
0.818 |
112.6 |
||
40 µg/mL |
683/768 |
38/768 |
47/768 |
0.139 |
79.0 |
||
20 µg/mL |
673/768 |
39/768 |
56/768 |
<0.001 |
87.4 |
||
10 µg/mL |
679/768 |
47/768 |
42/768 |
0.094 |
80.6 |
||
5 µg/mL |
672/768 |
36/768 |
60/768 |
0.254 |
101.5 |
||
Vehicle control |
672/768 |
52/768 |
44/768 |
-- |
88.0 |
||
Vehicle control for NQO |
670/768 |
39/768 |
59/768 |
-- |
100.8 |
||
Untreated control |
653/768 |
40/768 |
75/768 |
-- |
119.8 |
||
Positive control |
407/768 |
199/768 |
162/768 |
♦♦ |
749.6* |
In linear trend analysis β2/var (β) =1.199, not significant.
Mutagenicity Results of Assay 3
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/ total number of wells |
Dn2/var(Dn)♦ |
Mutation frequency |
+ |
3 |
110 µg/mL |
ND |
ND |
ND |
ND |
ND |
100 µg/mL |
NE |
NE |
NE |
NE |
NE |
||
95 µg/mL |
NE |
NE |
NE |
NE |
NE |
||
90 μg/mL |
NE |
NE |
NE |
NE |
NE |
||
85 µg/mL |
646/768 |
67/768 |
55/768 |
0.014 |
120.7 |
||
80 µg/mL |
655/768 |
52/768 |
61/768 |
<0.001 |
123.5 |
||
70 µg/mL |
646/768 |
59/768 |
63/768 |
<0.001 |
124.2 |
||
60 µg/mL |
646/768 |
60/768 |
62/768 |
0.276 |
109.1 |
||
40 µg/mL |
661/768 |
50/768 |
57/768 |
0.464 |
103.9 |
||
20 µg/mL |
661/768 |
51/768 |
56/768 |
0.531 |
103.2 |
||
10 µg/mL |
667/768 |
51/768 |
50/768 |
1.148 |
94.2 |
||
62.5 µg/mL |
671/768 |
51/768 |
46/768 |
1.564 |
88.9 |
||
Vehicle control |
649/768 |
57/768 |
62/768 |
-- |
124.4 |
||
Vehicle control for CP |
654/768 |
66/768 |
48/768 |
-- |
105.1 |
||
Untreated control |
658/768 |
57/768 |
53/768 |
-- |
100.4 |
||
Positive control |
183/768 |
240/768 |
345/768 |
♦♦ |
1571.2* |
In linear trend analysis β2/var (β) = 1.619, not significant.
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/ total number of wells |
Dn2/var(Dn) ♦ |
Mutation frequency |
- |
24 |
80 µg/mL |
ND |
ND |
ND |
ND |
ND |
75 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
70 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
65 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
60 µg/mL |
ND |
ND |
ND |
ND |
ND |
||
55 µg/mL |
NE |
NE |
NE |
NE |
NE |
||
50 μg/mL |
684/768 |
44/768 |
40/768 |
0.270 |
80.2 |
||
45 μg/mL |
678/768 |
51/768 |
39/768 |
0.245 |
80.9 |
||
40 μg/mL |
663/768 |
51/768 |
54/768 |
0.043 |
97.5 |
||
30 μg/mL |
672/768 |
68/768 |
28/768 |
0.058 |
86.7 |
||
20 μg/mL |
683/768 |
47/768 |
38/768 |
0.049 |
87.0 |
||
10 μg/mL |
670/768 |
49/768 |
49/768 |
0.001 |
93.2 |
||
5 μg/mL |
668/768 |
65/768 |
35/768 |
<0.001 |
91.9 |
||
Vehicle control |
674/768 |
55/768 |
39/768 |
-- |
92.4 |
||
Vehicle control for NQO |
678/768 |
65/768 |
25/768 |
-- |
87.0 |
||
Untreated control |
681/768 |
53/768 |
34/768 |
-- |
82.1 |
||
Positive control |
384/768 |
269/768 |
115/768 |
♦♦ |
830.8* |
In linear trend analysis β2/var (β) =0.359, not significant.
* = Statistically significant.
♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.48(at p<0.05).
♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05.
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn; β = slope of the curve; var(β) = variance of the slope
+ = in the presence of S9-mix
- = in the absence of S9-mix
Vehicle control = Ethanol
Vehicle control for CP = DMSO
CP = Cyclophosphamide
Vehicle control for NQO = DMSO
NQO = 4-Nitroquinoline-N-oxide
DMSO = Dimethyl sulfoxide
ND = no data (No cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period.)
NE = not evaluated (Due to the high level of cytotoxicity)
Note: Mutation frequency refers to 106viable cells
The overall study was considered to be valid:
The spontaneous mutation frequency of the negative (vehicle) control was in the recommended range (50-170 mutants per 106 viable cells) in each experiment.
The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays.
The plating efficiencies for the negative (vehicle) control at the end of the expression period (PEviability) were within the acceptable range (65-120 %) in all assays.
The number of test concentrations evaluated for each treatment was at least seven in each case,.
The tested concentration range in the study was considered to be adequate as the highest examined concentration produced approximately 80-90% toxicity (i.e. approximately 10-20% relative survival or relative total growth) and lower test concentrations were evenly spaced by a factor of no more than two. For determination of the cytotoxicity, the relative total growth was taken into consideration, as the test item showed late phase cytotoxicity during the expression period.
Table 1. Results of mutation test |
||||||
Chemical |
Solvent |
Dose (mg/plate) |
Nº revertants/plate a) |
|||
TA97 |
TA102 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
|||
D-borneol |
DMSO |
0.1 |
128 |
183 |
222 |
437 |
0.05 |
152 |
181 |
243 |
412 |
||
0.01 |
123 |
194 |
266 |
448 |
||
0.005 |
129 |
179 |
273 |
436 |
||
0.001 |
120 |
191 |
269 |
424 |
||
0 |
135 |
196 |
271 |
467 |
||
9-Aminoacridine |
DMSO |
50 (µg) |
902±257 c) |
|
|
|
Mitomycin C |
DMSO |
0.2 (µg) |
|
|
1993±616 |
|
2-Aminoanthracene |
DMSO |
5 (µg) |
|
1122±365 |
|
799±99 |
a) Mean of three plates.
c) Mean and standard deviation (n=17)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In-vivo micronucleus assay: Key study: Read-across approach. Test method OECD 474, GLP study: Based on read-across approach, the test item was determined to be not mutagenic in mice.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 January 1991 - 17 January 1991
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: 28.7 g (males) and 22.8 g (females)
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 20 % (w/v)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test sample dilutions were prepared fresh each day. 500 mg test item were weight in a 25 mL flask, mixed with sesame oil and topped up to the calibration mark. - Frequency of treatment:
- Once
- Post exposure period:
- 24, 48 and 72 hours (killing times)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 70 animals (35 male and 35 female):
Group 1: 0 mg/kg bw (5 males and 5 females) killing time: 24 h post administration
Group 2: 2000 mg/kg bw (5 males and 5 females) killing time: 24 h post administration
Group 4: 0 mg/kg bw (5 males and 5 females) killing time: 48 h post administration
Group 5: 2000 mg/kg bw (5 males and 5 females) killing time: 48 h post administration
Group 6: 0 mg/kg bw (5 males and 5 females) killing time: 72 h post administration
Group 7: 2000 mg/kg bw (5 males and 5 females) killing time: 72 h post administration - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Endoxan(R). Group 3 (5 males and 5 females) killing time: 24 h post administration
- Route of administration: oral, gavage
- Doses / concentrations: 50 mg/kg bw (0.5% w/v in distilled water , 10 mL/kg bw) - Tissues and cell types examined:
- Bone marrow erythrocytes cells (from both femora)
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Preliminary studies were conducted to determine the highest administrable non lethal dose level. 3 mice per sex and per dose were exposed to 5000, 4000, 3000 and 2000 mg/kg bw test item.
TREATMENT AND SAMPLING:
After treatment, animals were killed by carbon dioxide asphyxiation 24, 48 and 72 hours after application.
For each animal, about 3 mL foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged (5 min, 1200 rpm) and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide and air-dried for 24 hours. The slides were then stained.
METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was determined. - Statistics:
- The number of polychromatic erythrocytes with micronuclei and the number of normocytes with micronuclei were evaluated statistically. The comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase). The results of the treatment groups were compared with the corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). All statistical results were based on a 95% level of significance.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- (see below)
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000, 4000, 3000 and 2000 mg/kg
- Mortality:
At 5000 mg/kg bw: 1/3 males and 2/3 females died;
At 4000 mg/kg bw: 0/3 males and 1/3 females died;
At 3000 mg/kg bw: 0/3 males and 3/3 females died;
At 2000 mg/kg bw: No death were observed.
- Clinical signs of toxicity in test animals: Several clinical signs were observed at 3000-5000 mg/kg bw doses. At 2000 mg/kg bw, uncoordinated gait, increased spontaneous activity and stilted gait was observed.
RESULTS OF DEFINITIVE STUDY
- Mortality: All animals survived.
- Signs of toxicity: uncoordinated gait, increased spontaneous activity. These signs were fully reversible by 5-6 hours after application.
- Induction of micronuclei (for Micronucleus assay): The number of polychromatic (PCE) and normochromatic (NCE) erythrocytes containing micronuclei was not increased.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE in both male and female animals remained unaffected by the treatment, and was statistically not different from the control values.
- Positive control: Endoxan induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. the ratio of PCE/NCE was not changed to a significant extent. - Conclusions:
- Isobornyl acetate was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.
- Executive summary:
An in-vivo micronucleus test was performed with isobornyl acetate according to OECD 474. Five mice per sex and per group (70 in total) were exposed to a single dose of test item at 2000 mg/kg bw, based on preliminary results. Cyclophosphamide was used as a positive control (5 mice per sex). Animals were killed 24, 48 and 72 hours after administration of the test compound. For each animal, bone marrow smears were flushed from both femora and the slides were prepared for erythrocyte micronuclei observation. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values, indicating no mutagenicity. Based on these results, isobornyl acetate was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance isobornyl acetate undergoes rapid hydrolysis to acetic acid and isoborneol which shares the same functional groups with the substance D-alpha fenchol and also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in the mammalian erythrocyte micronucleus test
- Conclusions:
- Based on the read-across approach from the analogue Isobornyl acetate, the test item was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.
- Executive summary:
A mammalian in-vivo erythrocyte micronucleus test was performed with isobornyl acetate according to OECD 474. Five mice per sex and per group (70 in total) were exposed to a single dose of test item at 2000 mg/kg bw, based on preliminary results. Cyclophosphamide was used as a positive control (5 mice per sex). Animals were killed 24, 48 and 72 hours after administration of the test compound. For each animal, bone marrow smears were flushed from both femora and the slides were prepared for erythrocyte micronuclei observation. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values, indicating no mutagenicity. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.
Referenceopen allclose all
Summary of findings in bone marrow erythrocytes:
Sex |
Dose (mg/kg) |
Sample time |
No. animals |
Erythrocytes |
Erythrocytes with micronuclei |
|||||||||
Poly mean |
Normo mean |
mean |
No |
% |
|
Mut I |
No |
% |
|
Mut I |
||||
Male |
0 |
24 h |
5 |
1000 |
1000 |
0.92 |
2 |
0.16 |
I |
1.0 |
1 |
0.08 |
I |
1.0 |
2000 |
5 |
1000 |
1000 |
1.07 |
2 |
0.18 |
-I |
1.1 |
1 |
0.08 |
-I |
1.0 |
||
Endoxan |
5 |
1000 |
1000 |
0.82 |
23 |
2.32 |
*A |
14.5 |
1 |
0.14 |
-I |
1.7 |
||
Female |
0 |
5 |
1000 |
1000 |
1.03 |
1 |
0.12 |
I |
1.0 |
1 |
0.08 |
I |
1.0 |
|
2000 |
5 |
1000 |
1000 |
0.86 |
1 |
0.08 |
-I |
0.7 |
0 |
0.04 |
-I |
0.5 |
||
Endoxan |
5 |
1000 |
1000 |
0.79 |
21 |
2.10 |
*A |
17.5 |
2 |
0.22 |
*A |
2.8 |
||
Male |
0 |
48 h |
5 |
1000 |
1000 |
0.90 |
2 |
0.20 |
I |
1.0 |
2 |
0.16 |
I |
1.0 |
2000 |
5 |
1000 |
1000 |
0.78 |
1 |
0.08 |
-I |
0.4 |
0 |
0.04 |
-I |
0.2 |
||
Female |
0 |
5 |
1000 |
1000 |
0.96 |
1 |
0.12 |
I |
1.0 |
1 |
0.08 |
I |
1.0 |
|
2000 |
5 |
1000 |
1000 |
0.86 |
1 |
0.14 |
-I |
1.2 |
0 |
0.04 |
-I |
0.5 |
||
Male |
0 |
72 h |
5 |
1000 |
1000 |
1.06 |
2 |
0.18 |
I |
1.0 |
0 |
0.04 |
I |
1.0 |
2000 |
5 |
1000 |
1000 |
1.08 |
1 |
0.10 |
-I |
0.6 |
1 |
0.10 |
-I |
2.5 |
||
Female |
0 |
5 |
1000 |
1000 |
1.05 |
2 |
0.20 |
I |
1.0 |
1 |
0.08 |
I |
1.0 |
|
2000 |
5 |
1000 |
1000 |
1.20 |
1 |
0.14 |
-I |
0.7 |
0 |
0.04 |
-I |
0.5 |
||
Mut. I = Mutagenic index
- = No difference from control (P>0.05
I = within the normal range
* = Significantly different from control (P<0.05)
A = Outside the normal range
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. The mutagenic potential of analogue substance DL-Borneol was assessed in an Ames study following experimental outlines similar to those in OECD Guideline 471 using the plate incorporation method. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with DL-Borneol at concentrations up to 5000 μg/plate in the presence and absence of metabolic activation (S9 mix). Under the conditions of the study, DL-Borneol is considered not mutagenic in bacteria. Based on these results, the read-across was applied and the test item is considered to be not mutagenic in the Ames test.
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. A mutation test was conducted on the analogue substance DL-Borneol with E. Coli WP2 uvrA (trp-) at a dose range of 0.4 -3.2 mg/plate. No evidence of mutagenicity was observed. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. A bacterial reverse mutation test (Ames test) was performed on analogue substance Borneol using Salmonella typhimurium strains TA 97, TA 98 and TA 100 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent at a concentration of 1 mg/ml. Positive and negative/solvent controls were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. A bacterial reverse mutation test (Ames test) was performed on test substance D-borneol using Salmonella typhimurium strains TA 97 and TA 102 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic.
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. A bacterial reverse mutation test (Ames test) was performed on test substance isobornyl propionate using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537 and TA 1538 with and without S9 mix metabolic activation. Five doses up to 3.6 mg/plate of test item were used and 48 h incubation time. Dimethylsulfoxide (DMSO) was used as solvent. Positive controls with sodium azide and benzo(a)pyrene were included. No evidence of mutagenicity was observed at test conditions. Based on these results, the read-across was applied and the test item was determined to be not mutagenic under the test conditions.
In vitro gene mutation study in mammalian cells. Key study: Read-across approach. An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD 476) to test the potential of analogue substance fenchyl acetate to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). No mutagenic effect was observed either in the presence or in the absence of metabolic activation system. Based on these results, the read-across was applied and the test item was determined to be not mutagenic under the conditions of this Mouse Lymphoma Assay.
In vitro cytogenicity in mammalian cells/micronucleus study. Data waiving.According to column 2 of REACH Annex VIII, an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted since adequate data from an in vivo test are available.
In-vivo micronucleus assay: Key study: Read-across approach. A mammalian in-vivo erythrocyte micronucleus test was performed with isobornyl acetate according to OECD 474. Five mice per sex and per group (70 in total) were exposed to a single dose of test item at 2000 mg/kg bw, based on preliminary results. Cyclophosphamide was used as a positive control (5 mice per sex). Animals were killed 24, 48 and 72 hours after administration of the test compound. For each animal, bone marrow smears were flushed from both femora and the slides were prepared for erythrocyte micronuclei observation. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values, indicating no mutagenicity. Based on these results, the read-across approach was applied and the test item was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.
Justification for classification or non-classification
Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
