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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:
weight of evidence
Study period:
September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
2016
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : FAB-29062016
Purity : 96.2% (sum of the two main constituents)
Name of test material (as cited in study report): alpha-pinene multiconstituent
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 28 June 2017

Method

Target gene:
His+ for S. typhimurium; trp+ for E. coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Phenobarbital sodium/5,6-benzoflavone.
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains
Experiment 2 (pre-incubation method): 0,05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains
Vehicle / solvent:
DMSO
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Strains of S. typhimurium and E. coli were obtained from

METHOD OF APPLICATION:
Experiment 1: In agar (direct plate incorporation)
Experiment 2: preincubation method

DURATION
- Preincubation period: Exp. 2, 30 minutes at 37 °C
- Incubation period: Approximately 48-72 h at 37 °C for both direct plate incorporation and preincubation methods

NUMBER OF REPLICATIONS:
-3 plates/dose for treatment, vehicle and positive controls

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

OTHER: After 72 h of incubation at 37 °C, 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 substance produces a reproducible increase in 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 substance does not produce a reproducible increase in 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 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 judgement.
Statistics:
- Statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
hinning of background lawn observed at 5000 µg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Remarks:
Plate incorporation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
: Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning of background lawn observed at 15 µg per plate, without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning of background lawn observed at 15 µg per plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning of background lawn observed at 15µg per plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not valid
Species / strain:
S. typhimurium TA 100
Remarks:
Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning of background lawn observed at 15 µg per plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Remarks:
Pre-incubation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Positive control results (Plate incorporation assay, without metabolic activation) :

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts (Sorcerer)

TA98

2NF

2 µg

238.3

57.3

11.7

174, 284, 257

TA100

NaN3

2 µg

554.0

19.9

3.1

531, 565, 566

TA1535

NaN3

2 µg

635.7

38.3

38.1

647, 593, 667

TA1537

AAC

50 µg

214.7

186.2

16.5

71, 148, 425

WP2 uvrA (pKM101)

NQO

2 µg

2692.7

110.4

16.2

2682, 2808, 2588

Table 2: Positive control results (Plate incorporation assay, with metabolic activation) :

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts (Sorcerer)

TA98

B[a]P

5 µg

173.7

17.2

5.3

177, 155, 189

TA100

AAN

5 µg

3118.0

192.7

16.1

3269, 2901, 3184

TA1535

AAN

5 µg

210.3

24.4

16.6

205, 237, 189

TA1537

B[a]P

5 µg

167.7

34.1

14.4

147, 207, 149

WP2 uvrA (pKM101)

AAN

10 µg

1432.0

66.6

7.0

1435, 1497, 1364

Table 3: Positive control results (Pre-incubation assay, without metabolic activation):

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts (Sorcerer)

TA98

2NF

2 µg

295.7

40.0

12.5

308, 328, 251

TA100

NaN3

2 µg

624.3

9.3

4.1

614, 627, 632

TA1535

NaN3

2 µg

632.3

49.2

41.2

577, 649, 671

TA1537

AAC

50 µg

226.3

66.3

33.9

282, 244, 153

WP2 uvrA (pKM101)

NQO

2 µg

2622.3

12.9

16.7

2613, 2617, 2637

Table 4: Positive control results (Pre-incubation assay, with metabolic activation):

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts (Sorcerer)

TA98

B[a]P

5 µg

236.0

22.1

8.6

244, 253, 211

TA100

AAN

5 µg

912.0

327.7

5.9

1286, 775, 675

TA1535

AAN

5 µg

314.3

22.1

24.8

335, 291, 317

TA1537

B[a]P

5 µg

157.3

3.2

9.4

155, 156, 161

WP2 uvrA (pKM101)

AAN

10 µg

1661.0

48.1

9.1

1716, 1627, 1640

Applicant's summary and conclusion

Conclusions:
alpha-Pinene multiconstituent is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD 471 Guideline and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to alpha-pinene multiconstituent at the following concentrations:

Experiment 1 (plate incorporation method) 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains;

Experiment 2 (pre-incubation method) 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains.

 

Metabolic activation system used in this test was10 % (v/v) S9 mix;S9 fraction prepared from liver homogenates of rats induced with Phenobarbital sodium/5,6-benzoflavone.Vehicle and positive control groups were also included in mutagenicity tests.

 

No signs of toxicity towards the tester strains were observed in the first experiment following exposure to test item. Toxicity, observed as a reduction in revertant colony numbers, ans slight to severe thinning of the background lawn, was obtained in all strains in the second experiment following exposure to test item at 15 µg/plate in the absence of S9 mix. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to alpha-pinene multiconstituent, at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Therefore, alpha-pinene multiconsituent is not considered as mutagenic in these bacterial systems