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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
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

Method

Target gene:
histidine
Species / strain
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).
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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.

Applicant's summary and conclusion

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.