Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative key studies for Isoburnyl acetate extra are avaialble for reverse bacterial mutation and forward mammalian gene mutation (both in vitro). Supportive information was available for forward mammalian gene mutation (in vitro) from read-across substance and from QSAR (Danish QSAR Database).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
published in the Official Journal of the European Union L 142, dated 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEFM177269
- Expiration date of the lot/batch: 19.09.2022
- Purity test date: 15.10.2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in a tightly closed original container and stored in a dry and well-ventilated place

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Isobornyl acetate extra was completely dissolved in dimethyl sulfoxide (DMSO)


OTHER SPECIFICS: Manufacturiing date: 20.09.2018
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: In addition to the mutation in the histidine operon, these strains contain several other mutations that greatly increase their ability to detect mutagens.
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 treated rats
Test concentrations with justification for top dose:
-Preliminary cytotoxicity test in test strain TA 100 plate incorporation test with and without metabolic activation: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg/plate
Cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at a concentration of 316 µg Isobornyl acetate extra/plate in both experiments. Hence, 316 µg Isobornyl acetate extra/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
-Main study: 1.0, 3.16, 10.0, 31.6, 100 and 316 µg Isobornyl acetate extra per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Isobornyl acetate extra was completely dissolved in dimethyl sulfoxide (DMSO).
Untreated negative controls:
other: The vehicle DMSO served as the negative control
Remarks:
DMSO 100 µL/plate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO 100 µL/plate
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535, TA100 without S9
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 without S9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 without S9
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA98, TA102, TA1537 with S9
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA100, TA1535 with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: 2 independent main experiments
1) in agar (plate incorporation);
2) preincubation.
- Cell density at seeding (if applicable): approximately 10E8 viable cells in the late exponential or early stationary phase

DURATION plate incorporation test
- Preincubation period: No
- Exposure duration: in a dark 37°C incubator for 48 hours and stored after incubation for up to 24 hours at 4°C.
- Expression time (cells in growth medium): in a dark 37°C incubator for 48 hours and stored after incubation for up to 24 hours at 4°C.
- Selection time (if incubation with a selection agent): in a dark 37°C incubator for 48 hours and stored after incubation for up to 24 hours at 4°C.

DURATION preincubation test
- Preincubation period: 20 minutes at 37°C
- Exposure duration: in a dark 37°C incubator for 48 hours and stored after incubation for up to 24 hours at 4°C.
- Expression time (cells in growth medium): in a dark 37°C incubator for 48 hours and stored after incubation for up to 24 hours at 4°C.
- Selection time (if incubation with a selection agent): in a dark 37°C incubator for 48 hours and stored after incubation for up to 24 hours at 4°C.

SELECTION AGENT (mutation assays): histidine

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.
- Any supplementary information relevant to cytotoxicity: In the preliminary test cytotoxicity was noted starting at a concentration of 316 µg Isobornyl acetate extra/plate in both experiments. Hence, 316 µg Isobornyl acetate extra/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test. In the main study cytotoxicity was noted at the top concentration of 316 µg Isobornyl acetate extra/plate in all experiments and all test strains.

Evaluation criteria:
Bacteria colonies were counted employing the Biosys Biocount 5000 system. Print outs of the colony counts were filed with the raw data. Occurrence of test item precipitation was documented after visual inspection of the cultures with the unaided eye. Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.

Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by LPT.
The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 -60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 -35
TA1537: 3 -20

Interpretation of results:
Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p  0.05, U-test according to MANN and WHITNEY compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- Biological relevance of the results should be considered first.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Statistics:
U-test according to MANN and WHITNEY: increased number of revertants compared to the solvent control.
The Spearman's rank correlation coefficient may be applied when a concentration-related increase over the range tested in the number of the revertants per plate is observed.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 316 µg/plate
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:
cytotoxicity
Remarks:
at 316 mg/plate
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:
cytotoxicity
Remarks:
at 316 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 316 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 316 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Prior to the main test, two preliminary cytotoxicity tests (plate incorporation test, without and with metabolic activation) were carried out in test strain TA100.
Isobornyl acetate extra was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 μg/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted starting at a concentration of 316 μg Isobornyl acetate extra/plate in both experiments. Hence, 316 μg Isobornyl acetate extra/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures.
Conclusions:
Under the present test conditions, Isobornyl acetate extra tested up to the cytotoxic concentration of 316 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Executive summary:

The potential of Isobornyl acetate extra to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

Isobornyl acetate extra was completely dissolved in dimethyl sulfoxide (DMSO). A correction factor of 1.057 was used as the purity of the test item was only 94.6% (w/w). The vehicle DMSO served as the negative control.

Preliminary test

Isobornyl acetate extra was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000μg/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted starting at a concentration of 316μg Isobornyl acetate extra/plate in both experiments. Hence, 316μg Isobornyl acetate extra/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations 1.0, 3.16, 10.0, 31.6, 100 and 316μg Isobornyl acetate extra/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 316μg Isobornyl acetate extra/plate in all experiments and all test strains.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for Isobornyl acetate extra, tested up to the cytotoxic concentration of 316μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the historical control range generated by LPT. Hence, all acceptance criteria are met.

In conclusion, under the present test conditions, isobornyl acetate extra tested up to the cytotoxic concentration of 316 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes) (migrated information)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Council Regulation (EC) No. 440/2008 of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEFM177269
- Expiration date of the lot/batch: 19.09.2022
- Purity test date: 15.10.2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in a tightly closed original container and stored in a dry and well-ventilated place

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Isobornyl acetate extra was completely dissolved in dimethyl sulfoxide (DMSO)

OTHER SPECIFICS: Manufacturiing date: 20.09.2018
Target gene:
hprt
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: V79 cells Lot 4; DSMZ, Braunschweig, Germany
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: Yes. The cells were periodically checked for the absence of mycoplasma contamination.
- Number of passages if applicable: 6th passage at start of culturing and 15th or 17th passage at start of the first or second experiment, respectively
- Methods for maintenance in cell culture: V79 cells were maintained in growth medium Dulbecco's modified Eagle-Medium (DMEM) supplemented with 10% foetal calf serum and 1% penicillin/streptomycin solution.
- Cell cycle length, doubling time or proliferation index : Not provided
- Modal number of chromosomes: Not applicable
- Periodically checked for karyotype stability: Not provided
- Periodically ‘cleansed’ of spontaneous mutants: Yes. The spontaneous mutation rate was continuously monitored.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
V79 cells were maintained in growth medium Dulbecco's modified Eagle-Medium (DMEM) supplemented with 10% foetal calf serum and 1% penicillin/streptomycin solution (6th passage at start of culturing and 15th or 17th passage at start of the first or second experiment, respectively). Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2. For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.
DMEM, 10% foetal calf serum and 1% penicillin/streptomycin solution were obtained from GIBCO Invitrogen GmbH, Technologiepark Karlsruhe, 76131 Karlsruhe, Germany.
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium. In this preliminary test pronounced to complete cytotoxicity in form of decreased relative survival compared to the control were noted starting at concentrations of 100 or 316 µg Isobornyl acetate extra/mL medium in the absence or presence of metabolic activation.
Concentrations of 12.5, 25, 50, 100 and 200 µg Isobornyl acetate extra/mL medium were selected for the mutagenicity experiments with or without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Isobornyl acetate extra was completely dissolved in dimethyl sulfoxide (DMSO).
- Justification for percentage of solvent in the final culture medium: Isobornyl acetate extra was completely dissolved in dimethyl sulfoxide (DMSO) and further diluted to the appropriate lower concentrations. A correction factor of 1.057 was used due to the purity of the test item of 94.6% (w/w) only.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
9,10-dimethyl-1,2-benzanthracene (DMBA) dissolved in DMSO: 20 and 30 µg DMBA/m medium
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
EMS dissolved in DMSO: 600 and 700 EMS µg/mL medium
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
MAIN STUDY
- Number of cultures per concentration
Plating efficiency (PE) 1 : 3 replicates
Plating efficiency (PE) 2 : 3 replicates
Mutant frequency: 4 replicates
- Number of independent experiments: 2 (replicate mutagenicity experiment usually performed 1 week delayed to the first experiment)

METHOD OF TREATMENT/ EXPOSURE:
MAIN STUDY
- Cell density at seeding (if applicable):
*Start:
Approximately 1500000 cells were placed in 15 mL DMEM-FCS per 75 cm2 culture flask.
*After exposure period:
• 150 cells per plate in 5 mL growth medium for determination of PE1
• 500000 cells per 100 mm diameter dish in DMEM-FCS containing 6-thioguanine (10 µg/mL) for selection of mutants
• 150 cells per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2)
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No
- Exposure duration/duration of treatment: 4 hour
- Harvest time after the end of treatment (sampling/recovery times):
about 8 days (PE1)
about 14 days (PE2)
about 18 days (mutant plates)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 6 days including one cell passage in between
- Selection time (if incubation with a selective agent): about 12 days (mutant plates)
- Fixation time (start of exposure up to fixation or harvest of cells):
from Day 1-20 (selection of mutants)
from Day 1-16 (PE2 = without 6-thioguanine)
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
6-thioguanine (10 µg/mL) obtained from SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany about 12 days selection period (mutant plates)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Start:
Approximately 1500000 cells were placed in 15 mL DMEM-FCS per 75 cm2 culture flask.
After treatment:
Three replicate plates (60 mm diameter dishes) were used with 150 cells per plate in 5 mL growth medium (PE1)
After the expression period (6 days) the cells were harvested by trypsinisation and replated at a density of 500000 cells per 100 mm diameter dish in DMEM-FCS containing 6-thioguanine (10 µg/mL) for selection of mutants (4 replicate plates), and 150 cells per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2), (3 replicate plates).
Mutation frequency is expressed as number of mutants per number of surviving cells.
Relative Survival (RS) [%] = (PE1 (treated culture)/ PE1 (control culture)) x 100
Plating Efficiency step 1 (PE1) = Mean of the number of clones/culture dish / number of cells plated/culture dish
Mutant Frequency (MF/10E6) = Cloning efficiency (CE) / PE2
Cloning efficiency (CE) = Sum of the number of mutant clones / Sum of the number of cells plated
Plating Efficiency step 2 (PE2) = Mean of the number of clones/culture dish / number of cells plated/culture dish
- Criteria for small (slow growing) and large (fast growing) colonies: /

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative survival (RS) based on plating efficiency
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Evaluation criteria:
Acceptance of a test is based on the following criteria:
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
- Concurrent positive controls induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
- Four tested concentrations are analysable.
The spontaneous mutation frequency may vary from experiment to experiment, but should normally lie within the historical background data obtained at LPT.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
200 µg Isobornyl acetate extra/mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Under the present test conditions, Isobornyl acetate extra tested up to the cytotoxic concentration of 200 µg/mL medium, in the absence and in the presence of metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

Isobornyl acetate extra was tested for its mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix.

Isobornyl acetate extra was completely dissolved in dimethyl sulfoxide. A correction factor of 1.057 was used due to the purity of the test item of 94.6% (w/w) only. The vehicle dimethyl sulfoxide (DMSO) was employed as the negative control.

Preliminary cytotoxicity test

The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium. In this preliminary test pronounced to complete cytotoxicity in form of decreased relative survival compared to the control were noted starting at concentrations of 100 or 316 µg Isobornyl acetate extra/mL medium in the absence or presence of metabolic activation. No relevant changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. Hence, 200 µg Isobornyl acetate extra/mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation.

Main study

Concentrations of 12.5, 25, 50, 100 and 200 µg Isobornyl acetate extra/mL medium were selected for the mutagenicity experiments with or without metabolic activation. The experiments with and without metabolic activation were conducted in duplicates.

Cytotoxicity

Cytotoxicity in form of decreased relative survival compared to the control was noted starting at 50 µg Isobornyl acetate extra/mL medium in the first experiment and at the top concentration of 200 µg/mL medium in the second experiment without metabolic activation. In the presence of metabolic activation cytotoxicity in form of decreased relative survival compared to the control was noted starting at 100 µg/mL medium in the first experiment and at the top concentration of 200 µg/mL medium in the in the second experiment.

Mutagenicity

Experiments without metabolic activation

The mutation frequency of the solvent control DMSO was 24.67 and 23.85 mutant colonies per 106 cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 12.5, 25, 50, 100 and 200 µg Isobornyl acetate extra/mL culture medium ranged from 2.67 to 27.53 mutant colonies per 106 cells. These results are within the normal range of the solvent controls.

Experiments with metabolic activation

The mutation frequency of the solvent control DMSO was 24.40 and 31.50 mutant colonies per 106 cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 12.5, 25, 50, 100 and 200 µg Isobornyl acetate extra/mL culture medium ranged from 4.39 to 34.94 mutant colonies per 106 cells. These results are within the normal range of the solvent controls.

The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 261.89 to 419.90 mutant colonies per 106cells in the case of EMS and ranging from 187.20 to 245.30 mutant colonies per 106 cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.6 to 44.3 mutant colonies per 106 cells without metabolic activation or 2.3 to 40.7 mutant colonies per 106cells with metabolic activationf or the solvent controls. The mutation frequency of the positive controls at LPT ranges from 159.4 to 703.4 mutant colonies per 106 cells for EMS and from 155.7 to 711.8 mutant colonies per 106 cells for DMBA .

Conclusion

Under the present test conditions, Isobornyl acetate extra tested up to the cytotoxic concentration of 200 µg/mL medium, in the absence and in the presence of metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

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

Genetic toxicity in vivo

Description of key information

Negative key study for Isoburnyl acetate extra is available for cytogenetics (in vivo mouse Micronucleus test).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 14th to 17th, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets GLP and guideline requirements.
Qualifier:
according to guideline
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 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 daily

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
Duration of treatment / exposure:
only one administration
Frequency of treatment:
once
Post exposure period:
24, 48 and 72 hours
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
Positive control(s):
Endoxan(R):
Group 3: Cyclophosphamide 50 mg/kg bw (5 males and 5 females) killing time: 24 h post administration (positive control)
- Route of administration: oral, by gavage
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
Polychromatic and normochromatic erytrocytes
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: staining with Giemsa
Statistics:
Comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Uncoordinated gait, increased spontaneous activity. 5-6 hours after application all animals were free of clinical signs of toxicity.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY: Preliminary studies were conducted to determine the highest administrable non lethal dose level.
- Doses: 5000, 4000, 3000 and 2000 mg/kg
- Clinical signs of toxicity in test animals: increased spontaneous activity, impaired gait, impaired general condition, reduced spontaneous activity, etc.
The 2000 mg per kg bodyweight dose level was chosen since it had shown to be the maximum non lethal dose.

Conclusions:
Interpretation of results: negative
Isobornyl acetate was non mutagenic.
Executive summary:

Isobornylacetat - Extra was tested in the micronucleus test, according to OECD guideline 474. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 2000 mg Isobornylacetat - Extra per kg bodyweight.

The 2000 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximum non lethal dose.

The animals were treated once with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

Cyclophosphamide was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

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 with Isobornylacetat - Extra and was statistically not different from the control values.

Cyclophosphamide 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 polychromatic erythrocytes to normocytes was not changed to a significant extent.

The results indicate that, under the conditions of the present study, Isobornylacetat - Extra is not mutagenic in the micronucleus test.

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

Additional information

Key studies:

Ames reverse bacterial mutagenicity study (2019): the potential of Isobornyl acetate extra to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 without and with metabolic activation as a plate incorporation and preincubation test.Six concentrations 1.0, 3.16, 10.0, 31.6, 100 and 316μg Isobornyl acetate extra/plate were tested. No increase in revertant colony numbers as compared with control counts was observed for Isobornyl acetate extra, tested up to the cytotoxic concentration of 316μg/plate.

Forward mammalian gene mutagenicity study (2019): Isobornyl acetate extra was tested for its mutagenic potential in cultured mammalian cells (V79, genetic marker HPRT) both in the absence and presence of metabolic activation. The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix.The concentrations were chosen based on the results of a preliminary cytotoxicity study. Concentrations of 12.5, 25, 50, 100 and 200 µg Isobornyl acetate extra/mL medium were selected for the mutagenicity experiments with or without metabolic activation. Cytotoxicity in form of decreased relative survival compared to the control was noted starting at 50 µg Isobornyl acetate extra/mL medium in the first experiment and at the top concentration of 200 µg/mL medium in the second experiment without metabolic activation. In the presence of metabolic activation cytotoxicity in form of decreased relative survival compared to the control was noted starting at 100 µg/mL medium in the first experiment and at the top concentration of 200 µg/mL medium in the in the second experiment. The mutation frequency of the cultures treated with concentrations of 12.5, 25, 50, 100 and 200 µg Isobornyl acetate extra/mL with and without metabolic activation were within the normal range of the solvent controls. In conclusion, Isobornyl acetate extra tested up to the cytotoxic concentration of 200 µg/mL medium, in the absence and in the presence of metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test.

Isobornylacetate extra was tested in the micronucleus test, by oral gavage in male and female mice at 0 and 2000 mg/kg bw. The animals were treated once and killed 24, 48 or 72 hours after administration. 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 with Isobornylacetat - Extra and was statistically not different from the control values. The results indicate that, under the conditions of the present study, Isobornylacetate extra is not mutagenic in the micronucleus test

Supporting studies:

Experimental results from a study report. Ames test in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538, with and without metabolic activation. It is concluded that IBA is not mutagenic.

Based on published experimental data on the analogue (2-isopropyl-5-methylcyclohexanol, reported under endpoint record 07.06.01_07), which does not induce mutations in human lymphocytes, the substance Isobornyl Acetate is also considered negative for gene mutations under test conditions.

Weight-of-Evidence:

A Danish (Q)SAR prediction with the Multicase model was realized to estimate the mutagenic potencial of Isobornyl acetate on mammalian cells (HGRT (CHO): Chinese hamster ovary cell HGPRT forward mutation assay).

The substance Isobornyl acetate was predicted to be not mutagenic in mammalian cells. This prediction should be used for classification and risk assessment.

A Danish (Q)SAR prediction with the Multicase model was realized to estimate the mutagenic potencial of Isobornyl acetate on mammalian cells (mouse lymphoma).

The substance Isobornyl acetate was predicted to be not mutagenic in mammalian cells. This prediction should be used for classification and risk assessment.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Observations and results show that Isobornyl acetate did not demonstrate mutagenic potential; therefore no classification is needed.