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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:
20 February to 13 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 471 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
15 March 2017
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 184939
Purity : 83%
Name of test material (as cited in study report): delta-3-carene
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 06 December 2016

Method

Target gene:
Histidine and tryptophan for Salmonella typhimurium and Escherichia coli, respectively
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
First Test (Plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA (pKM101), with and without S9-mix
Second Test (Pre-incubation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA (pKM101), with and without S9-mix
Additional Second Test (Pre-incubation method):
0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98, TA 100 and TA 1535 strains, without S9-mix
0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 1537 and WP2 uvrA (pKM101) strains, without S9-mix
0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100 and TA 1535 strains, with S9-mix

The highest concentration of test item tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solubility of delta-3-carene was assessed at 50 mg/mL in DMSO, in which it dissolved. DMSO was, therefore, used as the vehicle for this study.
- The highest concentration of delta-3-carene tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log10 intervals. All concentrations cited in this report are expressed in terms of test item as received and containers of the neat test material were used within 7 days of opening for the first time.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM
Strains of S. typhimurium and E. coli were obtained from Moltox Inc.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: Test tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 34-39 °C for 30 minutes with shaking before the addition of the agar overlay.
- Exposure duration: Plates were incubated at 34-39 °C for 48 h

NUMBER OF REPLICATIONS: Triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test item may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.

OTHERS:
- Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), deficiency in DNA excision repair system (uvrA/uvrB mutation), and the pKM101 plasmid that confers resistance to antibiotics. The responses of the strains to a series of reference mutagens were also assessed.
- Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer.
- After incubation period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Evaluation criteria:
If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.
Statistics:
The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY AND MUTAGENICITY:
First Test:
- Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA98, TA100, TA1535 and WP2 uvrA (pKM101) following exposure to the test item at 5000 μg/plate and in strain TA1537 at 1500 μg/plate and above in the absence of S9 mix. In the presence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or with a reduction in revertant colony numbers) was seen in strains TA98, TA100 and TA1535 at 5000 μg/plate and in strains TA1537 and WP2 uvrA (pKM101) at 1500 μg/plate and above following exposure to the test item. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Second Test
- In the absence of S9 mix, toxicity, observed as a thin or absent background lawn of nonrevertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA98, TA100 and TA1535 at 50 μg/plate and above, and in strains TA1537 and WP2 uvrA (pKM101) at 150 μg/plate and above. In the presence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was seen in strain TA1535 at 15 μg/plate and at 500 μg/plate and above, in strains TA98 and TA1537 at 500 μg/plate and above, in strain TA100 at 150 μg/plate and above, and in strain WP2 uvrA (pKM101) at 1500 μg/plate and above. As the required four non-toxic concentrations were not achieved in any of the strains in the absence of S9 mix, or in strains TA100 and TA1535 in presence of S9 mix, an additional test was performed using modified dose concentrations.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Additional Second Test
- In the absence of S9 mix, toxicity, observed as a thin or absent background lawn of nonrevertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA98, TA100 and TA1535 at 50 μg/plate and above, in strain TA1537 at 150 μg/plate and above, and in strain WP2 uvrA (pKM101) at 500 μg/plate. In the presence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was observed in strain TA100 at 150 μg/plate and above, and in strain TA1535 at 500 μg/plate.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 500 μg/plate in either the presence or absence of S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data:
Without S9 mix: 293-2450 (820 ± 351), 180-1232 (689 ± 197), 438-3730 (1944 ± 740), 82-579 (209 ± 80), 72-973 (261 ± 141) for TA 100, TA 1535, WP2 uvrA (pKM101), TA 98 and TA 1537, respectively
With S9 mix: 338-4210 (1995 ± 823), 61-689 (343 ± 125), 378-2083 (1038 ± 331), 90-473 (217 ± 66), 49-411 (128 ± 55) for TA 100, TA 1535, WP2 uvrA (pKM101), TA 98 and TA 1537, respectively
- Negative (solvent/vehicle) historical control data:
Without S9 mix: 97-219 (156 ± 27), 7-44 (21 ± 8), 55-229 (165 ± 33), 19-78 (33 ± 10), 5-41 (17 ± 8) for TA 100, TA 1535, WP2 uvrA (pKM101), TA 98 and TA 1537, respectively
With S9 mix: 91-237 (167 ± 30), 8-47 (19 ± 6), 100-280 (191 ± 34), 18-84 (41 ± 15), 6-50 (24 ± 11) for TA 100, TA 1535, WP2 uvrA (pKM101), TA 98 and TA 1537, respectively

OTHERS:
- The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test item formulation.
- The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA (pKM101) strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to Guideline OECD 471 and in compliance with GLP, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrA (pKM101), were exposed to the test item diluted in DMSO at the concentrations below.

First Test (Plate incorporation method):

5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA (pKM101), with and without S9-mix

Second Test (Pre-incubation method):

5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA (pKM101), with and without S9-mix

Additional Second Test (Pre-incubation method):

0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98, TA 100 and TA 1535 strains, without S9-mix

0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 1537 and WP2 uvrA (pKM101) strains, without S9-mix

0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100 and TA 1535 strains, with S9-mix  

 

Metabolic activation system used in this test is S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

In the first test, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or with a reduction in revertant colony numbers) was seen in strains TA98, TA100, TA1535 and WP2uvrA (pKM101) following exposure to the test item at 5000 μg/plate and in strain TA1537 at 1500 μg/plate and above in the absence of S9 mix. In the presence of S9 mix, toxicity (observed as thinning of the background lawn of non revertant colonies, and/or with a reduction in revertant colony numbers) was seen in strains TA98, TA100 and TA1535 at 5000 μg/plate and in strains TA1537 and WP2uvrA (pKM101) at 1500 μg/plate and above.

 

In the second test in the absence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was seen in strains TA98, TA100 and TA1535 at 50 μg/plate and above, and in strains TA1537 and WP2uvrA (pKM101) at 150 μg/plate and above. In the presence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was seen in strain TA1535 at 15 μg/plate and at 500 μg/plate and above, in strains TA98 and TA1537 at 500 μg/plate and above, in strain TA100 at 150 μg/plate and above, and in strain WP2uvrA (pKM101) at 1500 μg/plate and above. As the required four non-toxic concentrations were not achieved in any of the strains in the absence of S9 mix, or in strains TA100 and TA1535 in presence of S9 mix, an additional test was performed using modified dose concentrations.

 

In the additional test in the absence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was seen in strains TA98, TA100 and TA1535 at 50 μg/plate and above, in strain TA1537 at 150 μg/plate and above, and in strain WP2uvrA (pKM101) at 500 μg/plate. In the presence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was observed in strain TA100 at 150 μg/plate and above, and in strain TA1535 at 500 μg/plate.

 

No evidence of mutagenic activity was seen at any concentration of the test item in any mutation test. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

 

Therefore, the test item is not considered as mutagenic in these bacterial systems.